Steroid Biosynthesis

Question 1

What is the precursor of all steroids ?

Answer 1

Cholesterol

Question 2

Describe and draw the chemical structure of cholesterol.

Answer 2

Cyclopentano- perhydro- phenanthrene nucleus
+ 8-carbon aliphatic side chain

Refer to slide 2 in lecture slides.

Question 3

Where in the body is cholesterol distributed ?

Answer 3

1) Membrane lipid (regulator of membrane fluidity)
2) In plasma associated with apoproteins, B triacylglycerols and phospholipids in various types of micellular structures called lipoproteins
3) Cytosolic lipid droplets as cholesterol esters
(mainly in steroid-secreting endocrine cells)

Question 4

Describe cholesterol biosynthesis.

Answer 4

The liver synthesizes cholesterol de novo from acetyl CoA in a multi-step process that occurs in the SER and cytosol. The rate-limiting step is the conversion of 3-hydroxy-3- methylglutaryl (HMG-CoA) to mevalonate by HMG-CoA reductase.
Once cholesterol is produced, it produces a negative feedback inhibitory effect on the rate limiting step of its biosynthesis.

Question 5

Identify the main processes of cholesterol metabolism.

Answer 5

Endogenous and exogenous pathways

Question 6

Describe the main features of the endogenous pathway for cholesterol transport.

Answer 6

-Basically: Taking cholesterol (also triacyl glycerols, free FAs) that is synthesised in liver and distributing it in body or returning it from sites of body to the liver

• Specifically:
  1) VLDL transport cholesterol and newly synthesised TG to tissues (TG > cholesterol)
  2) VLDL transports TG and cholesterol from liver to tissues (they are split through hydrolysis by lipoprotein lipase to release free fatty acids (FFAs. FFAs taken up by muscle and adipose tissue)
  3) TGs removed from VLDL leaving LDL with a high cholesterol (taken up by liver through endocytosis using LDL receptors in liver or by tissues)
  4) HDL absorbs cholesterol from cell breakdown (from tissues) and transfer it to VLDL and LDL for return back to the liver

Question 7

Describe the main features of the exogenous pathway for cholesterol transport.

Answer 7

-Basically: Taking cholesterol (also triacyl glycerols, free FAs) from diet (from GI system) and take it to liver for distribution and processing.

• Specifically :
  1) Cholesterol, triglycerides uptaken from GI system into chylomicrons (TG > cholesterol)
  2) Chylomicrons transport TG and cholesterol esters from the GI (diet) to tissues (they are split through hydrolysis by lipoprotein lipase to release free fatty acids (FFAs. FFAs taken up by muscle and adipose tissue)
  3) Now cholesterol > TG in the chylomicron. Hence, chylomicron remnants taken up in the liver (cholesterol stored, oxidised to bile acids or released to VLDL)

Question 8

Identify the main classes of plasma liporoteins. What is their significance ?

Answer 8

Chylomicrons (very large, and least dense because contain high level of TGs and low levels of surface proteins. Particles made in epithelial cells in gut, and then sent into systemic circulation for distributing fats to parts of body)
VLDL
IDL
LDL
HDL

Cholesterol is carried around in plasma associated with a number of lipoproteins

Question 9

Which type of lipoprotein is most of cholesterol esters found in ? Are these rich or depleted of TGs ?

Answer 9

In IDL and LDL (but depleted of TGs)

Question 10

Where on the lipoprotein particles are cholesterol esters found ? Free cholesterol ?

Answer 10

Cholesterol esters are found in core of particle, free cholesterol on surface

Question 11

What is the main function of lipoproteins ?

Answer 11

Transporting fats, lipids, and cholesterol in blood

Question 12

Which classes of cholesterol are associated with which pathway ?

Answer 12

ENDOGENOUS PATHWAY: 
High density lipoproteins (HDL) 
Intermediate density lipoproteins 
Low density lipoproteins (LDL) 
Very low density lipoproteins (VLDL) 

EXOGENOUS PATHWAY:
Chylomicrons

Question 13

Rank all classes of cholesterol by size, smallest to largest.

Answer 13

HDL (smallest) < LDL < IDL < VLDL < Chylomicron

Question 14

What does a high HDL and a high LDL suggest respectively, wrt to levels of cholesterol in the liver, and in the circulation.

Answer 14

High levels of LDL suggests high levels of circulating cholesterol
High levels of HDL suggests cholesterol is being taken back to liver

Question 15

Describe the structure of lipoproteins.

Answer 15

• Central core of hydrophobic lipid (triglycerides or cholesterol esters)
• Hydrophilic coat of polar substances (phospholipids, free cholesterol, associated proteins)

Question 16

Where do steroid secreting cells get their cholesterol from ?

Answer 16

The cholesterol needed as the starting material in the synthesis of steroid hormones comes from two sources. Approximately 80% is taken up as LDL particles via receptor-mediated endocytosis (either from diet i.e. exogenous pathway of cholesterol metabolism, or made in liver, i.e. endogenous pathway of cholesterol metabolism). The cell synthesizes the remaining cholesterol de novo from acetyl coenzyme A (Acetyl CoA).

Question 17

Describe the process of uptake of cholesterol from the diet/produced by the liver by cells.

Answer 17

UPTAKE OF CHOLESTEROL BY RECEPTOR-MEDIATED ENDOCYTOSIS:

-LDL particles have this Apo B-100 proteins, embedded in outer layer between phospholipids and free cholesterol
-These proteins are sensed by LDL receptor in tissues that take up LDL particles (e.g. on steroid secreting cells)
-LDL receptors bind to Apo B-100 protein, and then because of binding, invagination of particle into cell occurs. This invagination is fueled by Clathrin protein.
LDL particle is therefore engulfed in coated vesicle (coated because clathrin molecules polymerise in cage like structure around it)
-This forces the absorption of LDL particle into the cell
-Can then depolymerise clathrin, and receptors are then budded off from uncoated vesicle to form another vesicle which is devoid of LDL particle (while LDL particle stays in main part of uncoated vesicle)
-These receptors are recycled back to to cell surface for rebinding
-Uncoated vesicle devoid of receptors but containing LDL particle, fuse with lysosomes which contain
proteases, lipases, nucleotidases, thereby yielding AAs (used for protein synthesis), FAs (used for metabolism), free cholesterol (taken out of particle and usually stored esterified (cholesterol esters) to fatty acids in cytosolic lipid droplets)

Question 18

Identify the main physiological roles of cholesterol.

Answer 18

1) component of cell (plasma) membranes
- decreases membrane fluidity
- decreases physical permeability to charged/ polar compounds associated with the formation of lipid rafts

2) precursor for the production of bile salts
(for uptake of fats and fat-soluble vitamins in GI tract)

3) precursor for all steroid hormones glucocorticoids, mineralocorticoids and sex steroids

Question 19

Describe the initial steps in the biosynthesis of steroid hormones.

Answer 19

-In steroid hormone secreting cells, most of cholesterol
is stored as cholesterol esters
-Cholesterol ester hydrolase removes FAs from OH group,
to form free cholesterol (this enzymes is regulated by hormones which can increase activity to induce higher levels of free cholesterol in cells)
-Desmolase (AKA side chain cleavage enzyme) (found in mitochondria), first
enzyme in pathway to making steroid hormone (also regulated by number of hormones), removes side chain from cholesterol and forms pregnenolone, which
is the precusor to the synthesis of all steroid hormones in the
body
-Pregnenolone is then processed by enzymes in different types of cells (and
these enzymes are found in mitochondria or SER, so Pregnenolone is shuttled between SER and mitonchonria and enzymes process it into final product)

Question 20

Identify the main steroid hormones in the body, stating where in the body they are produced.

Answer 20

GONADS

• Progestins (main one is progesterone)
• Estradiol
• Testosterone (can be converted into estradiol)

ADRENAL GLANDS

• Cortisol
• Aldosterone
• Certain amount of testosterone

Question 21

What is the main structural feature of estradiol ?

Answer 21

A ring is aromatised (conjugated double bond all the way around)

Question 22

What are the main structural similarities between the final steroid hormones produced ?

Answer 22

Keeping same structure of the four rings. Only difference are in substituent groups
of each of these.

Question 23

Describe the process of biosynthesis of aldosterone, starting at pregnenolone, naming each step along the way along with the enzyme involved and the location in the cell of this enzyme.

Answer 23

ALDOSTERONE SYNTHESIS

• Pregnenolone converted into Progesterone using 3β hydroxysteroid dehydrogenase (in SER)
• Progesterone converted into 11 Deoxy-corticosterone using 21α-hydroxylase (in SER)
• 11 Deoxy-corticosterone is converted into Corticosterone using 11β-hydroxylase (mitonchondria), which is a glucocorticosteroid
• Corticosterone is then converted into Aldosterone (a mineralocorticosteroid) by Aldosterone Synthase (mitochondria), but exclusively in Glomerulosa cells (enzyme not present in other cells)

Question 24

Describe the process of biosynthesis of aldosterone, starting at cortisol, naming each step along the way along with the enzyme involved and the location in the cell of this enzyme.

Answer 24

Refer to diagram in slide 9 of lecture slides for easier understanding.

CORTISOL SYNTHESIS (2 routes): 
ROUTE 1
-Pregnenolone converted into Progesterone using 3β hydroxysteroid dehydrogenase (in SER)
-Progesterone converted into 11 Deoxy-corticosterone using 21α-hydroxylase (in SER)
-11 Deoxy-corticosterone is converted into Corticosterone using 11β-hydroxylase (mitonchondria), which is a glucocorticosteroid
-Corticosterone then converted into cortisol (also a glucocorticosteroid) by 17α-hydroxylase (in SER)

ROUTE 2

• Pregnenolone converted into 17α-hydroxypregnenolone by 17α-hydroxylase (in SER)
• 17α-hydroxypregnenolone converted into 17α-hydroxyprogesterone by 3β hydroxysteroid dehydrogenase (in SER)
• 17α-hydroxyprogesterone converted into 11 Deocycortisol by 21α-hydroxylase (in SER)
• 11 Deocycortisol converted into Cortisol (glucocorticosteroid) by 11β-hydroxylase (mitonchondria)

Question 25

What is the consequence of a block in the 21 alpha- hydroxylase ?

Answer 25

A block in the 21 alpha- hydroxylase will diminish production of both cortisol and aldosterone and increase production of the sex steroids because if the synthesis of cortisol is prevented by any one of several dysfunctional enzymes, other steroid products might be produced in excess

Question 26

How come androgens are produced (albeit in low levels) in adrenal glands ?

Answer 26

Because the enzyme necessary for testosterone production (17, 20 desmolase) is only present at low levels, but still present, so can eventually produce testosterone or Androstenediole

Question 27

Identify the main enzymes involved in steroidogenesis, including the gene involved in their production.

Answer 27

All of them are cytochrome P450 class of mixed functional oxidases: - Cholesterol side chain cleavage, AKA P-450SSC - 11β-Hydroxylase AKA P-450c11 - 17α-Hydroxylase AKA P-450c17 - 17,20-Desmolase AKA P-450c17 - 21α-Hydroxylase AKA P-450c21 - Aldosterone synthase AKA P-450aldo - Aromatase AKA P-450arom These process cholesterol into steroids, including steroid hormones.

Question 28

What is the significance of Aromatase ?

Answer 28

Catalyzes a reaction essential for the production of estrogens

Question 29

Describe the anatomy of the adrenal gland.

Answer 29

- An adrenal gland sits upon each kidney. - The adrenal gland is actually two glands-the cortex (on the outside) and the medulla (in the center) - The adrenal cortex comprises three layers that surround the medulla. The outermost layer contains the glomerulosa cells that secrete aldosterone (due to presence of aldosterone synthase), and the two inner layers of cortex, fasciculata (secretes cortisol), and reticularis (secretes cortisol and androgens) (as they lack aldosterone synthase) - The blood supply enters the cortex in the subcapsular region and flows through anastomotic capillary beds while coursing through both the cortex and the medulla. - The adrenal medulla contains chromaffin cells that secrete epinephrine and a small amount of norepinephrine.

Question 30

Define androgen.

Answer 30

Any steroid hormone that promotes male secondary sex characters. The two main androgens are androsterone and testosterone.

Question 31

What happens once the end product is reached in the steroid biosynthesis pathway ?

Answer 31

No more other enzymes that will bind to them and modify them so concentrations build up inside the cell, they are lipid soluble so can diffuse across to the circulation where they are carried away, usually by plasma binding proteins (helps prevent first pass metabolism and excretion).

Question 32

Describe the biosynthesis of testosterone. (and based on this, explain how you can also get other androgens).

Answer 32

There are four possible pathways from pregnenolone to testosterone: 1) the preferred pathway in the human testis appears to be: - pregnenolone into 17α hydroxypregnenolone using 17α Hydroxylase (SER) - 17α hydroxypregnenolone into dehydroepiandrosterone (DHEA) using 17, 20 Desmolase (SER). - DHEA into Androstenediol using 17β Hydroxysteroid dehydrogenase (SER) - Androstenediol into Testosterone (through oxidation of A ring) using 3β hydroxysteroid dehydrogenase (in SER) 2) Pregnenolone into progesterone using 3β hydroxysteroid dehydrogenase (in SER) - Progesterone into 17α hydroxyprogesterone using 17α Hydroxylase (SER) - 17α hydroxyprogesterone into Andristenedione using 17, 20 Desmolase (SER) - Androstenedione into Testosterone using 17β Hydroxysteroid dehydrogenase (SER) 3) -pregnenolone into 17α hydroxypregnenolone using 17α Hydroxylase (SER) -17α hydroxypregnenolone into 17α hydroxyprogesterone using 3β hydroxysteroid dehydrogenase (in SER) 7α hydroxyprogesterone into Andristenedione using 17, 20 Desmolase (SER) -Androstenedione into Testosterone using 17β Hydroxysteroid dehydrogenase (SER) 4) - pregnenolone into 17α hydroxypregnenolone using 17α Hydroxylase (SER) - 17α hydroxypregnenolone into dehydroepiandrosterone (DHEA) using 17, 20 Desmolase (SER) - DHEA into Androstenedione using 3β hydroxysteroid dehydrogenase (in SER) - Androstenedione into Testosterone using 17β Hydroxysteroid dehydrogenase (SER) In the circulation, some cells are very sensitive to androgens. These cells (testosterone target cells) contain 5 alpha reductase (SER) which convert testosterone into dihydrotestosterone, which has a higher binding affinity to testosterone receptor, so larger response. Using the route for testosterone biosynthesis in which androstenediol is involved, the body may also convert androstendiol into androsterone using 5 alpha reductase (SER), giving you an end product of androsterone. Furthermore, using the other three routes for testosterone biosynthesis, involving androstenedione, androstenedione and testosterone are in equilibium (both reactions catalysed by 17β Hydroxysteroid dehydrogenase (SER)) so you will also get a certain amount of androstenedione

Question 33

``` What class of molecules do pregnenolone, progesterone, 7α hydroxyprogesterone or 17α hydroxypregnenolone belong to ? ```

Answer 33

Progestins

Question 34

What class of molecules do Androsterone, Testerone, Andristenediol, Androstendione, DHEA, and Dihydrotestosterone belong to ?

Answer 34

Androgens

Question 35

Describe the main differences between gonads and adrenal glands in terms of activity of some enzymes, describing the implications of these.

Answer 35

Main difference with adrenal glands is elevated activity of 17 alpha desmolase in gonads so 17α hydroxyprogesterone and 17α hydroxypregnenolone can be converted into androgens + lack of 20α hydroxylase so cannot process progesterone molecules into glucocortiocosteroids

Question 36

Identify the cells responsible for development and secretion of androgens in the male.

Answer 36

Leydig and Sertoli

Question 37

Describe the role of Leydig and Sertoli cells.

Answer 37

In testes in male, two main cell types involved in allowing spermatozoa to mature: 1) Leydig cell -LH binding through LH receptors, increases testosterone synthesis (through cAMP mechanism which activates protein kinase A to stimulate expression of of enyzmes that will convert cholesterol into testosterone -Testosterone released from Leydig cells and carried out into all cells in body, giving masculinisation + acts on Sertoli cells + is required for maturation of spermatogonia into mature sperm. 2) Sertoli: -Release various factors into lumen which stimulate development of primordial spermatogonia into mature sperm which is released into duct. -FSH binds to FSH receptors on Sertoli cell (through cAMP mechanism which activates protein kinase A to stimulate synthesis of proteins) and thereby promotes the synthesis of androgen-binding protein (ABP) (secreted into lumen), aromatase, growth factors (which act on Leydig cells), and inhibin. -Upon reception of Testosterone from Leydig cells, Sertoli cells release some of this Testosterone into lumen, and convert some of testosterone to estradiol (because of the presence of some aromatase), which can act on the Leydig cells (estradiol has actions directly on Leydig cells, allowing expression of enzymes that make testosterone, similar to the way PKA does)

Question 38

What are the female sex steroids ?

Answer 38

Estrogens (estrone, estradiol, and estriol)

Question 39

Describe the biosynthesis of ovarian steroids.

Answer 39

Same pathway as androgens, but then androgens are converted into estrogens by aromatase (the ovary differs from the testis in having much more aromatase, which converts androgens to estrogens). - Androstenedione into estrone (E1) using Aromatase - Testerone into estradiol (E2) using aromatase - Estrone and estradiol can be converted into each other (equilibium) using 17beta Hydroxysteroid Dehydrogenase - Estrone and Estradiol then converted into Estriol (E3) in the liver

Question 40

Which cells are estrogen and progesterone produced by ?

Answer 40

Ovarian thecal and granulosa cells

Question 41

Describe the estrogen and progesterone production by ovarian thecal and granulosa cells.

Answer 41

The final steroid products of the ovaries depend on the different phases of the menstrual cycle: 1) During the follicular phase, the major product of the follicle is estradiol. LH primes the theca cell to convert cholesterol to androstenedione (and potentially some testosterone) (through a cAMP mechanism, LH stimulates enzymes involved in converting cholesterol into final end product). Because the theca cell lacks aromatase, it cannot generate estradiol from this androstenedione. Instead, the androstenedione (or testosterone) diffuses to the granulosa cell, whose aromatase activity has been stimulated by FSH. The aromatase converts the androstenedione (or testosterone) to estradiol (hence get estrogens in early stages of menstrual cycle). ---- During ovulation, there is a surge in LH, ovum is expelled and moves towards myometrium of the ovary, and what’s left, the follicular remnants containing granulosa cells and thecal cells, turns into corpus luteum. This tissue is heavily vascularised and so there is an increase delivery of LDL, providing cholesterol to both thecal cells and granulosa cells. ---- 2) During the luteal phase, the major products of the corpus luteum are the progestins, although estradiol synthesis is still substantial. In this phase, the vascularization of the corpus luteum makes LDL available to the granulosa - lutein cells. Thus, both the theca-lutein and the granulosa-lutein cells can produce progesterone, the major product of the corpus luteum. HOWEVER, the granulosa cells do not have the 17 alpha dehydroxylase and 17, 20 desmolase required to convert progesterone into an androgen. Consequently, progesterone is the main steroid that is released post second half of the menstrual cycle. However, small amounts of progesterone can be taken into thecal cells and made into androstenedione and testosterone, both of which can then be taken back into granulosa cells and converted into estrogens (but lower levels of estrogens only).

Question 42

Which cells do LDL and LH bind to in the production of estrogen and progesterone ?

Answer 42

LDL and LH bind | to receptors on both thecal cells and granulosa cells (in the process of producing estrogen and progesterone)

Question 43

Which genes are responsible for making cytochrome P450 class of mixed functional oxidases ?

Answer 43

CYP genes

Question 44

Identify the main progestins, glucocorticosteroids, mineralocorticoids and the sex steroids.

Answer 44

Progestins: pregnenolone, progesterone, 7α hydroxyprogesterone or 17α hydroxypregnenolone Glucocorticosteroids: Corticosterone, Cortisol Mineralocorticoids: Aldosterone Sex steroids: Estrone, Estradiol, Estriol (estrogens) Testosterone, Androstenedione, Androstenediol, DHEA (androgens)