principal investigator dr. sunil kumar khare, paper

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Biochemistry METABOLISM OF LIPIDS

Lipids: Biosynthesis Vs Oxidation

Dr. Vijaya Khader Dr. MC Varadaraj

Paper : 05 Metabolism of Lipids

Module: 26 Lipids: Biosynthesis Vs Oxidation

Principal Investigator

Paper Coordinator and

Content Writer

Dr. Sunil Kumar Khare, Professor,

Department of Chemistry, IIT-Delhi

Dr. Suaib Luqman, Scientist (CSIR-CIMAP)

& Assistant Professor (AcSIR)

CSIR-CIMAP, Lucknow

Content Reviewer Prof. Prashant Mishra, Professor,

Department of Biochemical Engineering

and Biotechnology, IIT-Delhi

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DESCRIPTION OF MODULE

Subject Name Biochemistry

Paper Name 05 Metabolism of Lipids

Module Name/Title 26 Lipids: Biosynthesis Vs Oxidation

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1. Objectives

To understand the regulation of fatty acid

How breakdown of the lipids is dilapidated

What is the fate of lipid metabolism

2. Concept Map

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3. Description

The metabolism of lipids including biosynthesis and oxidation imparts the course of action which rivets the

intercourse and degradation. One may foresee that the pathway for the breakdown of the lipids would be the

swap of the biosynthesis pathway. Nevertheless, this would not allocate discrete regulation of both the pathways

to crop up yet specified the verity that the pathways are alienated within diverse cellular compartments.

The biosynthesis of lipids and its related pathway primarily takes place in cytoplasm whereas the oxidation

related pathway for lipid breakdown arises in the mitochondria. The utilization of nucleotide co-factors is the

additional key difference as the biosynthesis of lipids involves NADPH oxidation while the lipids breakdown

engrosses the reduction of NAD+ and FADH

+. Conversely, the fundamental chemistry of both the pathways

(Biosynthesis & Oxidation) are transposing to apiece. Both the biosynthesis and oxidation of lipids exploits

Acetyl CoA (two carbon activated intermediate). Though, in lipid biosynthesis, the activated form of Acetyl

CoA subsists provisionally vault to the enzyme complex as Malonyl CoA. The Malonyl CoA synthesis is the

initial unswerving tread of the fatty acid biosynthesis and Acetyl CoA carboxylase (ACC) enzyme catalyzes the

reaction. It is the foremost site of fatty acid biosynthesis regulation. Like other carboxylases, ACC requires

biotin as a co-factor which transfers carbon dioxide to the substrates. In addition to biotin and carbon dioxide

ACC also require ATP for the reaction hence sometimes the enzyme refer to as ABC (ATP, Biotin, Carbon

dioxide) enzyme.

As compare to distinct macronutrient classes (proteins and carbohydrates), lipids or fats acquiesce a good

amount of energy (in terms of number of adenosine triphosphate molecules) on per gram basis. In addition,

lipids or fats are vital for energy storage, membrane (phospholipid) formation and signaling pathways. The

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metabolism of lipids comprise of anabolic route which generates biologically significant molecules from dietary

sources and catabolic procedure that engender primary metabolites from fatty acids and energy.

A detailed distinction and similarity between the biosynthesis and oxidation of lipids are described below.

Biosynthesis Oxidation

Reaction Type Anabolic & Ordered Catabolic & Non-ordered Process Reductive Oxidative

Site Cytoplasm Mitochondria Bonding Covalent Covalent

Nature of Enzyme Multifunctional Biosynthetic enzymes on one polypeptide chain known as Fatty Acid Synthase (FAS)

Separate Enzymes are degradative in nature and not associated with each other

Co-factor NADPH NAD, FAD

Energy 49 ATP equivalent required 33 ATP equivalent yield Regulation Acetyl CoA carboxylase Availability of Acetyl CoA

Starting Point Methyl End (CH3CH2-) Carboxyl End (CH3COO-)

Precursor Acetyl CoA Fatty Acid

Product Fatty Acid Acetyl CoA Thioester Formation With Acyl Carrier Proteins (ACP-SH) With CoA-SH

Configuration D β-Hydroxyacyl intermediates L β-Hydroxyacyl intermediates Biotin Requirement Required Not Required

Malonyl CoA Source of 2C unit Not Involved Elongation Stops at C16, Longer chains require other

enzymes. Reaction driven by Carbon dioxide release

Co-ordinated Regulation of Fatty Acid Synthesis (FAS) and Breakdown

In diet, when carbohydrate affords as an equipped source of energy and fuel, β-oxidation of lipids (fatty acids) is

redundant and is therefore down regulated. Two key enzymes (Acetyl CoA carboxylase and Carnitine acyl

transferase I) are crucial to the synchronization of lipid (fatty acids) metabolism. Acetyl CoA carboxylase

(ACC) is the first and regulatory enzyme of the lipid (fatty acids) biosynthesis and Carnitine acyl transferase I

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(CATI) is another regulatory enzyme that check the transport of lipids (fatty acids) into the mitochondrial matrix

for β-oxidation.

Intake of an excessive carbohydrate feast elevates the level of blood glucose and thus affects the following

phenomenon:

1. Elicits the insulin discharge.

2. Dephosphorylation of ACC by Insulin-dependent protein phosphatase through activation.

3. Formation of Malonyl CoA with the aid of ACC.

4. Inhibition of CATI by Malonyl CoA thereby averting the entry of fatty acid into the matrix of

mitochondria.

When the level of blood glucose plummet between the meals, then following events takes place:

5. Activation of cAMP-dependent Protein kinase A (PKA) by the release of glucagon.

6. Inactivation and phosphorylation of ACC.

7. Descendence of Malonyl CoA concentration.

8. Alleviation of the inhibition of fatty acid entry into the mitochondria.

9. Entry of the fatty acids into the matrix of the mitochondria.

10. Triggering of the fatty acid mobilization by glucagon in the adipose tissue.

11. Arrival and supply of fatty acid begins in the blood.

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The pace of the oxidation of fatty acid is a function of the concentration of un-esterified fatty acids (UeFAs) in

plasma. UeFAs or free fatty acids (FFAs) are either produced by lipoprotein lipase or are discharged from the

adipose tissues into the circulatory system that cart them to discrete tissues or organs. In adipose tissues, insulin

and glucagon hormones control the recuperation of TAGs. The deployment of fatty acids for either biosynthesis

or oxidation relies on the nutritional status of the organism and more exclusively on the accessibility of the

carbohydrates. Due to the close association amid carbohydrate metabolism, lipid metabolism and ketogenesis,

the decree of fatty acid breakdown in the liver vary from that of the tissues present in skeletal and heart muscle.

The heart and skeletal muscle have an irresistible catabolic utility. In animals, the trend of lipid or fat

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metabolism in the liver relies on the nutritional state, for example, in fed animal, the liver change carbohydrates

to fats or lipids, whereas in fasted animals, oxidation or breakdown of lipids or fats, gluconeogenesis and

ketogenesis are the supplementary vigorous procedures.

Undoubtedly, there is a reciprocal (give and take) relationship exists between the biosynthesis of lipids (fatty

acids) and oxidation or breakdown. Although, it is well-established fact that lipid metabolism and carbohydrate

metabolism are under the control of hormones but it has been further intricate to recognize the exact mechanism

that control the biosynthesis and oxidation of lipids (fatty acids).

McGarry and Foster (1970s) while studying the oxidation or breakdown of lipids have proposed that the

Malonyl CoA concentration (the primary unswerving intermediary of fatty acid biosynthesis) established the

rate of lipid or fat oxidation. The fundamental traits of their hypothesis are as under:

In the fed (nourished) animal:

1. The Malonyl CoA concentration or level has been found elevated due to the active conversion of

glucose to fatty acids.

2. At a micromolar concentration, Malonyl CoA restrains hepatic CPT I thus diminishing the repositioning

of fatty acyl residues from CoA to carnitine and their translocation into the mitochondria.

3. A depressed β-oxidation is reported.

In the fasting (starved) animals:

4. The hepatic metabolism move from the breakdown of the glucose to gluconeogenesis with a

consequential decline in the biosynthesis of fatty acid.

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5. The Malonyl CoA concentration decreases and the reticence of CPT I is reassured.

6. The total CPT I activity increases and the sensitivity of CPT I en route for Malonyl CoA decreases.

7. Rapid formation of Acyl carnitines and translocation into the mitochondria.

8. Stimulation of β-oxidation and ketogenesis.

It becomes perceptible that the cellular Malonyl CoA concentration or level is unswervingly allied to the

commotion of ACC which is hormonally synchronized. The short term ACC control entails both the

phosphorylation and dephosphorylation phenomenon of the enzyme. In the starved (fasting) animal, a high ratio

of insulin : glucagon causes the inactivation and phosphorylation of ACC. As an upshot, the Malonyl CoA

concentration or level and the biosynthesis rate decreases, even as the pace of β-oxidation enhances. A lessen

ration of insulin and glucagon annul these effects. Accordingly, both the biosynthesis of lipids (fatty acid) and

oxidation or breakdown processes are synchronized by the insulin: glucagon ratio.

In non-hepatic tissues like skeletal and heart muscle, Malonyl CoA also controls the oxidation or breakdown of

fatty acid. In these tissues, the Malonyl CoA formation is catalyzed by ACC1 (a 265 kDa prime form of

lipogenic tissues) and ACC2 (a 280 kDa isoform). The Malonyl CoA removal is done by the cytosolic Malonyl

CoA decarboxylase and the concentration or level is indomitable by the actions of both the carboxylase and

decarboxylase. In retort to stress caused by hypoxia, ischemia and exercise, ACC2 is triggered allosterically by

citrate whereas it is inactivated and phosphorylated by AMP-dependent kinase.

In skeletal and heart muscle, an apprehension exists about the regulation of breakdown or oxidation of fatty acid

emphasizing the incongruity amid the tissue concentration of Malonyl CoA (µM) and the Ki of muscle CPT I

(nM) for Malonyl CoA. As a consequence, the enzyme ought to be utterly inhibited at all times except during

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the lower level or concentration of Malonyl CoA owing to the intracellular compartmentation or binding of

distinct proteins. The rate of breakdown or oxidation of fatty acid in heart, and probably in other tissues, is

refrained to the energy demand of the cell in addition to being dependent on the FFAs concentration of plasma.

Adequately at a high concentration (>0.6 mM) of FFAs, the oxidation or breakdown is merely a utility of the

energy demand of the cell. Investigations with the perfuse hearts and the isolated mitochondria have revealed

that a reduction in the demand of energy results in the eminent concentrations of NADH and Acetyl CoA and in

lesser concentrations of NAD + and CoA. The consequential increase in the Acetyl CoA:CoA ratio and

NADH:NAD+ ration in the mitochondrial matrix may possibly be the reason for the diminished β-oxidation

rate. It is the ratio of Acetyl CoA:CoA that regulates the β-oxidation rate and not the NADH:NAD+ ratio.

Although the site of this control has not been recognized explicitly, it is likely that the Acetyl CoA:CoA ratio

adjust the β ketoacyl-CoA thiolase activity and thus regulate the fatty acid flux towards the β-oxidation.

Regulation of Oxidation or Breakdown of Fatty Acid in Mitochondria

During 1970s, McGarry & Foster revealed the foremost hegemony on β-oxidation along with intersect amid

carbohydrate oxidation and lipid or fat metabolism. When the supply of carbohydrate is in abundance, it

increases its oxidation inside mitochondria causing high accretion of citrate which ought to be exported to

cytosol where it is cleaved by ATP bound citrate lyase into Acetyl CoA and malate. The high concentration of

citrate activates ACC enzyme to transform Acetyl CoA to Malonyl CoA (substrate for biosynthesis of fatty acid)

which is being referred to as ‘Signal of Plenty’.

In an effort to restrain that biosynthesis of the fatty acid does not occur concurrently with the oxidation or

breakdown, Malonyl CoA inhibits the action of CPTI, and accordingly prevent the entry of fatty acid into the

mitochondria for β-oxidation. CPTI controls the high flux coefficient and has been revealed to be the rate

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limiting (feedback control) step for β-oxidation. In addition to the regulation arbitrated by Malonyl CoA, it has

also been verified that the AMP-activated Protein kinase (AMPK) kindle CPTI. Consequently, AMPK amplify

CPTI activity by releasing the inhibition, inactivates ACC, lower Malonyl CoA concentrations and intercedes a

strenuous response to metabolic stress by invigorating the oxidation or breakdown of fatty acid. The mechanism

of increasing the activity of CPTI by AMPK involves the phosphorylation of the cytoskeleton (cytokeratin 18, 8)

element. Recycling of cofactors at a restricted concentration is considered as Intra-mitochondrial control on β-

oxidation flux.

NAD+ is evidently required by 3-hydroxyacyl CoA dehydrogenases (3HCD), Acyl CoA dehydrogenases

(ACD), CPTII and 3-oxoacyl CoA thiolases (3OCT). If re-decomposition of NADH is weakening, β-oxidation

itself will be subdued at the levels of the 3HCD and ACD correspondingly. Amassing of esters of 3-hydroxyacyl

CoA ensuing to reticence of the 3HCD will instigate feedback inhibition of the 2-enoyl CoA hydratases

followed by ACDs. A redox regulation of β-oxidation has been revealed in normal liver mitochondria or where

the respiratory chain is prejudiced, either by anoxia, high ATP/ADP ratio, and inhibition of enzyme or

deficiency of enzyme. The mitochondrial CoA pool (Acyl CoA esters) also restrain β-oxidation at 3OCT step

and direct to the accretion of 3-oxoacyl CoA esters, which inhibit 3HCDs, 2-enoyl CoA hydratases and ACDs

and are therefore prospective powerful feedback inhibitors of β-oxidation. As Acetyl CoA is an inhibitor of

3OCTs, an enhancement in the ratio of Acetyl CoA:CoA would guide to the reticence of 3OCTs activity

followed by the increase of 3-oxoacyl CoA esters and β-oxidation inhibition. Furthermore, CPTII is also

inhibited by deficit of unesterified CoA prior to 3OCT, hence averting the entry of additional acyl groups to the

mitochondrion. The kinetic distinctiveness of CPTII and the CAT would support the dissemination of acyl

groups under these conditions.

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ChREBP: An Adept Regulator of Lipid in Liver

In liver, when glycogen storage exceeds its limit, surplus of glucose is sidetracked into the lipid metabolic

pathway. Glucose is broken down to Acetyl CoA through pyruvate which is used for the de novo biosynthesis of

fatty acid. The so formed fatty acids are subsequently integrated into TAGs and exported from hepatocytes as

VLDL which eventually stored in adipose tissues. A carbohydrate rich diet motivates both the lipogenic and

glycolytic pathways. Genes encoding FAS and ATP bound citrate lyase of lipogenesis and liver pyruvate kinase

and glucokinase of glycolysis are synchronized by the intonation of their transcription rates. Subsequently, genes

encoding these enzymes are subjected to allosteric and post-translational regulation. These genes enclose

carbohydrate or glucose-response elements liable for their transcriptional control.

SREBP (Sterol Response Element Binding Protein), in instance, SREBP1c is one of the most important

transcription factors that wield its power over lipid and glucose homeostasis. In lipogenesis, SREBP regulates

the expression of numerous genes and its own transcription is subdued by glucagon and enhances by insulin.

Conversely, in addition to SREBP activity, a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription

factor was also recognized as ChREBP (Carbohydrate Responsive Element Binding Protein). ChREBP was

acknowledged as the foremost Glucose Responsive Transcription Factor (GRTF) and it is essential for glucose-

induced expression of liver pyruvate kinase and the lipogenic genes (ACC1 and FAS).

In liver, the ChREBP gene expression is tempted in retort to the augmented glucose uptake. Moreover, ChREBP

activity is controlled by post-translational modifications together with sub-cellular localization. Both AMPK and

PKA phosphorylate ChREBP depicting it immobile as a transcriptional activator. AMPK is known to

phosphorylates ChREBP at position Serine 568 while PKA on Serine 196 and Threonine 666. ChREBP resides

in the cytosol in a phosphorylated state during low glucose concentration but as soon as the level increases

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protein phosphatase 2A delta (PP2Aδ) is triggered by xylulose 5 phosphate (an intermediary of the pentose

phosphate pathway). PP2Aδ dephosphorylates Serine 196 ensuing in ChREBP translocation in the nucleus and

subsequent dephosphorylation of Threonine 666 in the nucleus allocate ChREBP binding to specific sequence

elements in the target genes. ChREBP also intermingle with an additional bHLH protein recognized as MLX

(MAX like protein X). MLX is a part of the MAD/MAX/ MYC family of transcription factors that dole out as

related allies in the network of transcription factors. ChREBP binding to MLX takes place inside a realm

positioned in the C-terminal portion of the ChREBP. This interface between MLX and ChREBP is indispensable

for the binding of DNA.

The N terminal portion of ChREBP contains glucose sensing domain (GSM) which is in fact poised of two

discrete sub-domains acknowledged as LID (low glucose inhibitory domain) and GRACE (glucose responsive

activation conserved element). LID restrains transcriptional trans-activation by the GRACE domain during low

glucose concentration whereas the inhibition is overturned by the availability of either glucose or a glucose

metabolite.

In the adipose tissue, a novel mechanism of ChREBP activity has been revealed involving a tissue specific

transcription (exon 1b) and representing a persuasive means for glucose-arbitrated intonation of fatty acid

biosynthesis and sensitivity to insulin. Now the ChREBP is accredited as ChREBP α and ChREBP β (novel

alternative splice form). Both the α and β form of ChREBP toil in recital to relentlessly modify the lipogenic

gene expression.

ACC1, FAS and Liver Protein kinase are the genes whose expression patterns are under the control of ChREBP

activity and α and β form of ChREBP augments the transcription of lipogenic genes in adipose tissue. Further, it

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has also been revealed that the expression levels and activity of SCD1 (Δ9-stearoly CoA Desaturase 1) and

GPAT (Glycerol 3 Phosphate Acyltransferase) gets reduced when ChREBP expression is curtailed. SCD1 is the

rate-limiting enzyme implicated in the biosynthesis of palmitoleic acid (16:1) and oleic acid (18:1), the foremost

mono-unsaturated fatty acids while GPAT esterifies Glycerol 3 Phospate producing lysophosphatidic acid (the

first step of TAGs Biosynthesis).

Liver X Receptors: LXRs

Being the members of the steroid/thyroid hormone superfamily of cytosolic ligand binding receptors, LXRs

wander to the nucleus for binding and expression of specific target sequences. LXR-α and LXR-β are the two

forms of LXRs and they articulate heterodimers with RXRs (retinoid X receptors) and control the expression of

genes consequent to the binding of 9 cis retinoic acid or oxysterols. LXRs are also vital switch of the lipogenic

pathway and ChREBP gene is an unswerving target of LXRs which is activated by glucose.

GPAT: Glycerol 3 Phosphate Acyltransferase, SCD1: Stearoyl CoA Desaturase, LPK: Liver Pyruvate kinase, ACC1: Acetyl CoA Carboxylase 1, FAS: Fatty Acid Synthase, MUFA: Monounsaturated Fatty Acid , S: Serine, T: Threonine. RED T-lines indicate inhibition.

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Modulation of Glucose and Lipid Homeostasis by ChREBP

In the cells, when glucose entry is high it results in enhanced activity of the pentose phosphate pathway ensuing

high accumulation of xylulose 5 phosphate. It activates PP2Aδ that dephosphorylates ChREBP both in nucleus

and cytosol. In liver, the active ChREBP roll on the expression of many genes involved in the homeostasis of

lipid and glucose metabolism. Also the activation of LXR-α in liver by lipid ligands results in upsurge ChREBP

expression that lead to further regulation of glucose and lipid homeostasis. In both the liver and adipose tissue,

ChREBP is an adept regulator of glucose-mediated lipid homeostasis. Overall, ChREBP regulates 50% of

lipogenesis in liver throughout its intensive performance on the expression of glycolytic and lipogenic genes.

4. Summary

In this lecture we learnt about:

Biosynthesis Vs Oxidation

Co-ordinated Regulation of Fatty Acid Synthesis (FAS) and Breakdown

Regulation of Oxidation or Breakdown of Fatty Acid in Mitochondria

ChREBP: An Adept Regulator of Lipid in Liver

Liver X Receptors: LXRs

Modulation of Glucose and Lipid Homeostasis by ChREBP

principal investigator dr. sunil kumar khare, paper

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