6. Cholesterol and Steroid Metabolism

8/3/2019 6. Cholesterol and Steroid Metabolism

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CHOLESTEROL ANDSTEROID METABOLISM

I

Structure, Function,

Biosynthesis and Elimination



Cholesterol is a lipid

Review:

Definition of lipid

Examples of lipid molecules

Amphipathic

Hydrophobic



Structure of free cholesterol

an amphipathic lipid



Ester bond formation

alcohol + acid = ester + watercan be organic or mineral acid



Structure of cholesteryl ester

a hydrophobic lipid



Enzymology of cholesterol esterification

In plasma lecithin: cholesterol acyltransferase(LCAT) forms CE (dienoic) (LCAT deficiency/fisheye disease)

In cells acylcoenzyme A: cholesterolacyltransferase (ACAT) forms CE (monenoic)

In pancreas, intestine, liver and kidneycholesterol esterase hydrolyzes CE

In all cells CE from LDL hydrolyzed by lysosomalacid lipase deficiency + Wolman disease (CE

storage disease)



Distribution

In plasma, 70 72 % of total cholesterolis CE

In cells, nearly all cholesterol is freecholesterol

CE is the transport or storage form of cholesterol



Lipids in the body

In cells Amphipathic lipids in

membranes

Hydrophobic lipids in oildroplets

In extracellular fluid Amphipathic lipids inlipoprotein surface coat

Hydrophobic lipids inlipoprotein core



Essential cholesterol!

Despite its bad press, cholesterol is absolutelyessential for human life

Obtained in diet and synthesized endogenously Cholesterol synthesis requires molecular O2 -

ancient organisms like Tetrahymena lackcholesterol synthetic pathway use

tetrahymenol in membranes but usecholesterol if available in culture medium



Tetrahymenol looks like cholesterol

and

..its synthesis is blocked by cholesterol



Biomembrane structure

Membranes contain amphipathic lipids and proteinswith some carbohydrate (from glycoproteins andglycolipids)



Cholesterol intercalates between fatty acylchains in cell membranes and reduces

membrane fluidity



Free cholesterol in membranes

Often measured as C/PL molar ratio - C/PL is veryvariable erythrocyte = 0.8,Hepatocyte = 0.2

Most FC is in plasma membrane very little in

mitochondrial inner membrane In plasma membrane FC is evenly distributed

between the 2 leaflets contrast PL and glycolipids

Within a single leaflet, FC may be concentrated in lipid rafts with low fluidity/permeability leavingFC-free areas of high fluidity/permeability



Free cholesterol in membranes

FC is not covalently bound in membrane

Free to exchange with FC of lp particles

Important for removal of excess cellularcholesterol

Also provides a receptor-free pathway for

cholesterol uptake by cells In disease, low LCAT activity leads to

increase in membrane FC (RBC C/PL >1 )



Biosynthesis of cholesterol

From 2 carbons to 27 carbons a medicalstudents worst nightmare!

Even biochemists dont understand all that it entails!

Breakdown into sections for easier generalunderstanding



Overview

Acetyl CoA (C2) Mevalonate (C6)

(Isoprenyl pyrophosphates) (C5, C10, C15)

Squalene (C30) Lanosterol (C30)

Cholesterol (C27)



Biosynthesis of mevalonate



HMGCoA reductase is the rate-

limiting enzyme

Metabolic regulation of any pathway is usually

achieved by modulation of the activity of onekey enzyme:

The rate-limiting enzyme

Catalyzes the committed step

For cholesterol biosynthesis, control occurs earlyin the pathway at HMGCoA reductase



...of cholesterol biosynthesis

Fasting (+)

Increased dietary intake of cholesterol (-)

HMGCoA reductase is controlled by

covalent modification induced bycholesterol feedback and by varioushormones



. and the target for statins

Rings

Partially reduced naphthalene lovastatin, simvastatin, pravastatinIndole fluvostatin Pyrrole atorvastatin

Pyrimidinine rosuvastatin Pyridine glenvastatin

Quinoline - pitavastatin



Statins

Competitive inhibitors of HMGCoAreductase

Natural products from Penicillium citrinum(mevastatin), Aspergillus, Monascus oranalogues

Effective at lowering cholesterol, but mayhave other effects as well impotence bone-building etc.



Biosynthesis of farnesyl pyrophosphate



Transmethylglutaconate shunt

Until recently, it was thought that mevalonateonce formed was destined to become farnesyl

pyrophosphate and hence cholesterol

Now known that dimethylallyl pyrophosphatecan escape this fate and can be

dephosphorylated and converted back toacetoacetate and acetyl CoA



Biosynthesis of squalene



From squalene to sterols



Regulation of cholesterol

biosynthesisSophisticated process because synthesisrepresents a large investment of energy

Diet inverse relationship between dietaryintake and cholesterol synthesis liver

Daily cholesterol availability is fairly constant dietary intake has only weak effect on plasmacholesterol



Intracellular cholesterol pools

In liver FC used for: Bile acid synthesis

Bile cholesterol

VLDL cholesterol

HDL cholesterol

In adrenals & gonads Steroid hormones

In other tissues Repair & growth



HMGCoA reductase

If intracellular FC rises, regulation occurs by:

reduction in activity of HMGCoA reductase

reduction of synthesis of HMGCoA reductase

downregulation of LDL receptors

increase in esterification of FC by ACAT increase in FC removal from plasma

membrane by HDL promoted by LCAT



Factors influencing HMGCoA

reductase activity Intracellular [HMGCoA]

Intracellular [cholesterol]

Hormones

Insulin (+)

Tri-iodothyronine (+)

Glucagon (-)

Cortisol (-)



Factors influencing cholesterol

regulationIncrease [FC] Decrease

All cells

de novo synthesis inhibition of synthesishydrolysis of stored CE esterification by ACAT

receptor-mediated LDL uptake downregulation of receptors

direct uptake of FC from lipoproteins release of FC to lipoproteins

Liver and gonads only

receptor-mediated HDL uptake synthesis of bile acids/steroidsLiver only

uptake of chylomicron remnants secretion in bile



Circadian rhythm

Hepatic cholesterol synthesis peaks 6hours after dark

At a minimum, 6 hours after light

Changes in HMGCoA reductase activity mechanism unclear



BBRREE A AKKTTIIMMEE



Cholesterol is not broken down

in the human body

Once formed cholesterol is hard to degrade

Only micro-organisms can break down steroidring for energy

Instead in humans, cholesterol is converted intoother compounds bile salts, hormones, VitaminD



Bile acids are quantitativelythe most important

cholesterol-derived products

Bile acids - C24 compounds differ by position and numberof E-OH groups

Methyl groups are in F-configuration

Amphipathic in that one face of the nucleus is polar andthe other non-polar good emulsifiers

However, pK a of carboxyl group is about 6 not fullyionized at physiological pH



Primary bile acids

Synthesis in liver

Multi-step, multi-organelleprocess

OH groups are inserted at specific positions of ring

Double bond in B ring isreduced

Chain shortened by 3 carbonsand ends in a carboxyl group



Cholesterol 7-E-hydroxylase

Rate-limiting step inbile acid synthesis

Cytochrome P450enzyme

Only in ER of liver



Bile salts enhanced

amphipathic compounds Before leaving liver

bile acids are

conjugated witheither glycine ortaurine by amidebond formation

New acidic groups arefully ionized at physiological pH



Bile salt function

Act as detergents

Emulsify dietary lipids toenable enzymic digestion.Products form into smallmicelles

Micelles transport fatty

acids, cholesterol, M AGs,Lysolipids to mucosal cellsurface for absorption



Effect of intestinal flora onEffect of intestinal flora on

bile saltsbile salts Bacteria remove

glycine or taurine to

regenerate primarybile salts

May also remove one

hydroxyl group toyield secondary bileacids



Enterohepatic circulation of

bile acids and salts



Cholestyramine interruptseneterohepatic circulation of bile salts

and lowers plasma cholesterol

Cholestyramine is a positively chargedresin

In the gut, it binds negatively charged bileacids

The bile acids are excreted rather thanreturned to liver

Liver uses cholesterol to make updeficiency of bile acids



Cholelithiasis cholesterol

gallstone disease Bile contains cholesterol

which is solubilized bybile salts and

phospholipids

If balance is disturbed,then cholesterol may

precipitate out to formgallstones



Cholesterol solubility dependsupon phospholipids and bile

salts



Causes of gallstones

Decrease in bile salt content of bile from:

Gross malabsorption of bile acids as in ileal disease

Bile duct obstruction Severe hepatic dysfunction leading to decreased

synthesis

Excessive feedback suppression of de novo bile salt

synthesis Increased bile acid excretion with fibrates orcholestyramine



Steroid hormones

Cholesterol is precursor of all steroid hormones glucocorticoids, mineralocorticoids(corticosteroid hormones) and sex hormones

Groups of different hormones differ in carbonNo. C-21, C-19 and C-18

Within each group, individual hormones maydiffer by a C=C double bond or orientation of an

OH group

Systematic nomenclature necessary



Steroid hormone groups

C-21 (pregnane ring)

C-19 (androstane ring)

C-18 (estrane ring)

Only 2-C left in side chain

Side chain completely lost

Loss of methyl C-19 as ring A is aromatized



Steroidogenic tissues

Adrenal cortex cortisol, aldosterone andandrogens

Ovaries (and placenta) estrogens andprogestins

Testes - testosterone



Steroid transport

Steroid hormones are hydrophobic

Complexed to protein for transport

Albumin non-specific carrier transports

aldosterone. Specific transporters transcortin for corticosteroids and sex-hormone-binding protein



Steroidogenesis

Formation of Pregnenolone

Progesterone

Cortisol Aldosterone Testosterone

Estradiol



Formation of progesterone

Rate-limiting stepcatalyzed by desmolase

(Cytochrome P450 mixed

function oxidase in innermitochondrial membrane usesO2 and N ADPH) followed byisomerization - pregnenolone

Oxidation catalyzed by3- F-hydroxysteroiddehydrogenase



3- F-hydroxysteroid

dehydrogenase deficiency A congenital adrenal hyperplasia (CAH)

No formation of glucocorticoids,mineralocorticoids or sex hormones

Marked salt excretion in urine

All patients have female genitalia

i f



Conversion ofprogesterone to

active hormones

Other CAHs involveother mixed functionoxidases on thesepathways

Enzyme deficiencieslead to deficiency of product hormone andexcess of substratehormone/metabolite



Cholesterol (C27)

Pregnenolone (C21)

Progesterone (C21)

17-a-Hydroxyprogesterone

11-Deoxycorticosterone (C21) 11-Deoxycortisol (C19) Androstenedione (C19)

Corticosterone Testosterone (C19)

Aldosterone Cortisol (C21) Estradiol (C18)

ENZYME BLOCKSLEADING TO CAH

X

X

X

X

X

X

X

3 F-hydroxysteroiddehydrogenase

17-E-hydroxylase

21-a-hydroxylase

11-E-hydroxylase



17-E-hydroxylase deficiency

Lack of enzyme blocks conversion of

progesterone to 17--hydroxyprogesterone onpathway to sex hormones and cortisol

Increased production of mineralocorticoids leads

to sodium and fluid retention and hypertension

All patients have female genitalia



Cholesterol (C27)

Pregnenolone (C21)

Progesterone (C21)






X

X

X

X

X

X

X


17-E-hydroxylase

21-a-hydroxylase

11-E-hydroxylase



21-E-hydroxylase deficiency

Conversion of both progesterone to 11-deoxycorticosterone and 17-E-hydroxyprogesterone to 11-deoxycortisol

Commonest CAH - total or partial absence of corticosteroids

Androgen overproduction masculinization of external genitalia in females and precociousvirilization in males



Cholesterol (C27)

Pregnenolone (C21)

Progesterone (C21)






X

X

X

X

X

X

X


17-E-hydroxylase

21-a-hydroxylase

11-E-hydroxylase



11- F-hydroxylase deficiency

Conversion of both 11-deoxycorticosterone tocorticosterone and 11-deoxycortisol to cortisol isblocked

Increased 11-deoxycorticosterone causes fluidretention and supresses the renin/angiotensinsystem hypertension

Masculinization and virilization



Secretion of steroid hormones

Steroid Tissue Control

Cortisol Middle layer of adrenal cortex

Hypothalmus via Cortictropin RH which stimulates

Adrenocorticotropic H secretionin pituitary via cAMP

Aldosterone Outer layer of adrenal cortex

Decrease in plasma Na+/K+ andby Angiotensin II - via PIP2

Androgens -

androstenedioneetc.

Both layers Weak androgens converted in

peripheral tissues totestosterone and estradiol

Sex hormones Gonads Hypothalmus via gonadotropin-releasing hormone stimulates LH and FSH secretion by pituitary

via cAMP



Steroid hormone action

Diffuse into cells in cytosol or nucleus, bind to specificsteroid receptor

Receptor-ligand complex accumulate in nucleus and

dimerize Bind to hormone response elements (specific regulatory

DN A sequences) in conjunction with co-activator proteins

Increase transcription by promotor activation

HRE are found in enhancer elements near genes that respond to steroid hormones co-ordinated regulation

Hormone-receptor complexes can also inhibit transcription in association with co-repressor proteins



Vitamin D3

Vitamin D3 or Cholecalciferolis derived fromcholesterol

Dietary sources include (fishliver oil, egg yolk) orergocalciferol invegetables

Majority is made in theMalpighian layer of theepidermis of skin byphotolysis of 7-dehydrocholesterol

Cholecalciferol is biologically inactive but is



Cholecalciferol is biologically inactive, but isconverted into the active form 1,25 dihydroxycholecalciferol by sequential

hydoxylationsCholecalciferolCholecalciferol



Regulation Transport of cholecalciferol from skin to liver and of 25-

hydroxycholecalciferol from liver to kidney requires aspecific Vit D-binding protein

Formation of 25-hydroxycholecalciferol is unregulated

Formation of 1,25-dihydroxycholecalciferol by 1-E-hydroxylase enzyme is the rate-limiting step

Low calcium diets and hypocalcaemia markedly increaseactivity effect requires parathyroid hormone (PTH) low phosphorus diets and hypophosphatemia weaklystimulate

1,25-dihydroxycholecalciferol also regulates its ownproduction inhibiting the 1-E-hydroxylase and causing

inactive 24,25-dihydroxycholecalciferol to accumulate



1,25 dihydroxycholecalciferol orcalcitriol is an important calciotropic

hormone It diffuses into its target cell and binds to a

nuclear receptor

Intestine increased transcription of proteinsincluding calcium-binding protein effect is toincrease transfer of calcium and phosphate ionsfrom intestinal lumen across the mucosal cell

and into the circulation Bone in presence of PTH, stimulates

mobilization of calcium and phosphate ions



Vitamin D deficiency

Demineralization of bone:

Rickets in infants

Osteomalacia in

adults



Signs of RicketsSigns of Rickets



Vitamin D - summary



Summary I

Hydrophobic and amphipathic lipids

Cholesteryl ester and free cholesterol

Enzymology of cholesteryl esters

Lipids in cells and extracellular fliud

Functions of cholesterol

Cholesterol in biomembranes

Cholesterol biosynthesis



Summary II Bile acids

Primary bile acids bile salts function

Secondary bile acids enterohepatic circulation Questran

Cholesterol gallstones Steroid hormones

Steroidogenic tissues and steroidogenesis

Different hormones action and control

Congenital adrenal hyperplasias -21-E-hydroxylasedeficiency etc.

Vitamin D Dietary sources sunlight formation of calcitriol

mode of action - deficiency

6. Cholesterol and Steroid Metabolism

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