Leydig cells convert cholesterol to testosterone
Cholesterol is the obligate precursor for androgens and other testicular steroids. The Leydig cell can synthesize cholesterol de novo from acetyl coenzyme A or can take it up as low-density lipoproteins from the extracellular fluid by receptor-mediated endocytosis (see p. 42). The two sources appear to be equally important in humans.
Preceding the metabolism of cholesterol is the translocation of this precursor to the mitochondrial inner membrane, which requires two proteins. The first is sterol-carrier protein 2 (SCP-2), a 13.5-kDa protein that translocates cholesterol from the plasma or organellar membranes to other organellar membranes, including the outer mitochondrial membrane. The second protein is the steroidogenic acute regulatory protein (StAR), which belongs to a large family of proteins involved in lipid trafficking and metabolism. The 37-kDa pro-StAR protein—the precursor to StAR—ferries cholesterol from the endoplasmic reticulum to the outer mitochondrial membrane. The 30-kDa mature StAR protein resides in the mitochondrial intermembrane space and extracts cholesterol from the mitochondrial outer membrane and ferries it across to the mitochondrial inner membrane.
The Leydig cell uses a series of five enzymes to convert cholesterol to testosterone. Three of these enzymes are P-450 enzymes (see Table 50-2). N54-4 As summarized in Figure 54-6, because 3β-hydroxysteroid dehydrogenase (3β-HSD) can oxidize the A ring of four intermediates, testosterone synthesis from cholesterol can take four pathways. The following is the “preferred” pathway:
FIGURE 54-6 Biosynthesis of testosterone. This scheme summarizes the synthesis of the androgens from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; they are located in either the SER or the mitochondria. The side-chain-cleavage enzyme that produces pregnenolone is also known as 20,22-desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. There are four possible pathways from pregnenolone to testosterone; the preferred pathway in the human testis appears to be the delta-5 pathway, the one along the left edge of the figure to androstenediol, followed by oxidation of the A ring to testosterone. Some of these pathways are shared in the biosynthesis of the glucocorticoids and mineralocorticoids (see Fig. 50-2) as well as the estrogens (see Fig. 55-8).
Cytochrome P-450 Enzymes
Contributed by Emile Boulpaep, Walter Boron
The term cytochrome P-450 enzymes refers to a family of several hundred heme-containing enzymes that are located primarily in the SER. See Table 50-2 for some examples of these enzymes that play a role in steroidogenesis. We discuss the roles of these P-450 enzymes for the adrenal gland on page 1021, for the testis on page 1097, and for the ovary on page 1117. On page 64, we discuss the role of a P-450 enzyme (i.e., epoxygenase) in the metabolism of arachidonic acid (see also Fig. 3-11).
These enzymes are monooxygenases.* That is, they transfer one atom of molecular oxygen to an organic substrate RH to form ROH, whereas the other oxygen atom accepts two protons from the reduced form of the enzyme to form water:
This monooxygenation reaction is also referred to as a hydroxylation reaction because the enzyme hydroxylates RH to form ROH. Note that at the end of the reaction, the P-450 monooxygenase is in its reduced form. Another enzyme—a cytochrome P-450 reductase—recycles the P-450 monooxygenase to its reduced form; in the process, this P-450 reductase becomes oxidized. Finally, the oxidized P-450 reductase recycles to its reduced form by oxidizing the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+ or the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+ or one of the flavin nucleotides (reduced flavin adenine dinucleotide [FADH2] or reduced flavin mononucleotide [FMNH2]).
The P-450 enzymes are so named because when the reduced forms of the enzymes bind carbon monoxide, they absorb light strongly at 450 nm.
Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 3rd ed. Worth Publishers: New York; 2000 [pp; 782; 783].
*Enzymes that transfer both oxygen atoms of molecular oxygen to an organic substrate are termed dioxygenases. In contrast, oxidases (e.g., cytochrome oxidase in the electron transport chain of mitochondria) are enzymes that catalyze oxidations in which neither of the atoms of molecular oxygen becomes part of the oxidized product. Instead, the molecular oxygen acts as an electron acceptor to form a molecule such as H2O or H2O2.
1. Cholesterol conversion to pregnenolone. The pathway for testosterone synthesis begins in the mitochondrial inner membrane, where the cytochrome P-450 side-chain-cleavage enzyme (P-450SCC, also called 20,22-desmolase) N54-4 removes the long side chain (carbons 22 to 27) from the carbon at position 20 of the cholesterol molecule (27 carbon atoms), yielding pregnenolone (21 carbon atoms). This reaction is the rate-limiting step in the biosynthesis of testosterone, as it is for other steroid hormones. LH stimulates this reaction in the Leydig cell in two ways. First, LH increases the affinity of P-450SCC for cholesterol. Second, LH has long-term actions of increasing the levels of SCP-2, StAR, and P-450SCC via PKA-stimulated gene transcription.
2. Pregnenolone conversion to 17α-hydroxypregnenolone. In the smooth endoplasmic reticulum (SER), 17α-hydroxylase (P-450c17) N54-4 then adds a hydroxyl group at position 17 to form 17α-hydroxypregnenolone. P-450c17, a key branch-point enzyme in the steroidogenic pathway, also converts progesterone to 17α-hydroxyprogesterone (see Fig. 54-6, middle column).
3. 17α-hydroxypregnenolone conversion to dehydroepiandrosterone. In the SER, the 17,20-desmolase (a different activity of the same P-450c17 whose 17α-hydroxylase activity catalyzes the previous step) removes the position-20 side chain from position 17 of 17α-hydroxypregnenolone, producing a 19-carbon steroid called dehydroepiandrosterone (DHEA). This so-called delta-5 pathway on the left of Figure 54-6 is the preferred route in Leydig cells to yield DHEA, the precursor for all sex steroids.
4. DHEA conversion to androstenediol. In the SER of the Leydig cell, a 17β-hydroxysteroid dehydrogenase (17β-HSD, which is not a P-450 enzyme) converts the ketone at position 17 of DHEA to a hydroxyl group to form androstenediol.
5. Androstenediol conversion to testosterone. Finally, in the SER, 3β-HSD (not a P-450 enzyme) oxidizes the hydroxyl group of androstenediol at position 3 of the A ring to a ketone, forming testosterone. N54-5
Delta-5 and Delta-4 Steroids
Contributed by Sam Mesiano
Pregnenolone is called P5. Progesterone is P4. This is where the terms delta-5 and delta-4 come from.
3β-HSD is a major branch-point enzyme in the steroidogenic pathway (see Fig. 54-6). It converts all delta-5 steroids to delta-4 steroids via an isomerase activity and therefore is essential for the production of mineralocorticoids and glucocorticoids. The competition between 17α-hydroxylase/17,20-desmolase (two enzymatic activities mediated in the same protein, also known as P-450c17) and 3β-HSD for pregnenolone and 17α-hydroxypregnenolone is a major determinant of whether a steroidogenic cell will produce mineralocorticoids, glucocorticoids, or sex steroids. In the Leydig cell, 17α-hydroxylase/17,20-desmolase prevails to produce DHEA, which 17β-HSD1 then converts to androstenediol. DHEA can also undergo conversion, via 3β-HSD, to androstenedione, which 17β-HSD1 then converts to testosterone.
In addition, the testis can also use 5α-reductase, which is located in the SER, to convert testosterone to dihydrotestosterone (DHT). However, extratesticular tissue is responsible for most of the production of DHT. The conversion of testosterone to DHT is especially important in certain testosterone target cells (see pp. 1097–1099).
The Leydig cells of the testes make ~95% of the circulating testosterone. Although testosterone is the major secretory product, the testes also secretes pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, androsterone, and DHT. The conversion of testosterone to DHT by Leydig cells is minor compared with its production in certain testosterone target cells (see p. 1085). Androstenedione is of major importance because it serves as a precursor for extraglandular estrogen formation. In men who are between the ages of 25 and 70 years, the rate of testosterone production remains relatively constant (Table 54-1). Figure 54-5 summarizes the changes in plasma testosterone levels as a function of age in human males. N54-6
Androgen Production and Turnover
BLOOD PRODUCTION RATE—HORMONE DELIVERED TO THE BLOOD (µg/day)
PLASMA CONCENTRATION (µg/L)
Testosterone Secretion and Production Rates
Contributed by Emile Boulpaep, Walter Boron
In the text, we noted that plasma testosterone levels are relatively constant in males between the ages of 25 and 70 years. As for any substance in the blood, the stability of plasma levels of testosterone indicates that the rate of testosterone production is equal to the rate of testosterone removal. However, the stability of plasma testosterone levels says nothing about the individual rates of production and removal.
It is important to distinguish between the secretion rate of a hormone and the production rate. Secretion refers to the release of the hormone from a specific organ or gland, and may be determined by selectively catheterizing the artery and vein supplying that tissue and ascertaining the arterial-venous difference in the concentration of that substance. For example, the concentration of testosterone is 400 to 500 µg/L in effluent of the spermatic vein; this level is ~75 times higher than the concentration found in the arterial blood. Thus, if we knew the blood flow out of the spermatic vein, then we could compute the rate of secretion of testosterone by the testis.
Production rate refers to the total appearance of the hormone in the circulation as the result of the secretion by all tissues in the body. Thus, the secretion rate for the testes equals the whole-body production rate only when other tissues make no contribution.
In the steady state, the amount of testosterone cleared from the circulation equals the amount produced. Thus,
Here, PR is the whole-body production rate, MCR is the metabolic clearance rate, and [S] is the concentration of the substance in the plasma. MCR is defined in the same way as renal clearances (see Table 33-2). That is, MCR is the virtual number of liters per day that are fully cleared of testosterone. This clearance is due to the metabolism of testosterone, which is discussed on pages 1099–1100. Because the mean metabolic clearance rate for testosterone is ~1000 L/day, and the testosterone concentration is about 6.5 µg/L (range, 3 to 10 µg/L), the production rate must be about 6500 µg/day. Evidence for this high clearance rate is the fact that the plasma half-life of testosterone is only 10 to 20 minutes.
Adipose tissue, skin, and the adrenal cortex also produce testosterone and other androgens
Several tissues besides the testes—including adipose tissue, skin, adrenal cortex, brain, and muscle—produce testosterone and several other androgens. These substances may be synthesized de novo from cholesterol or produced by peripheral conversion of precursors. Moreover, the peripheral organs and tissues may convert sex steroids to less active forms (see Fig. 54-6). Notable sites of extragonadal conversion include adipose tissue and the skin. Androstenedione is converted to testosterone in peripheral tissues. In this case, androstenedione is the precursor for the hormone testosterone. Testosterone can be converted to estradiol or DHT or go “backward” by reversible interconversion to androstenedione. Thus, a potent hormone such as testosterone may also serve as a precursor for a weaker hormone (androstenedione), a hormone with different activities (estradiol), or a more potent hormone having similar activities (DHT). This last example may be illustrated by the effects of DHT on hair follicles, sebaceous glands, and the sex accessory organs. In these tissues, the androgenic effects of circulating testosterone are amplified by its conversion by 5α-reductase to DHT, which has a much higher affinity for the androgen receptor (AR; see p. 1085). Some tissues, including the brain, aromatize testosterone to estradiol, and thus the action of this metabolite occurs via the estrogen receptor.
The adrenal cortex (see p. 1021) is another source of androgen production in both males and females. Normal human adrenal glands synthesize and secrete the weak androgens DHEA, conjugated DHEA sulfate, and androstenedione. Essentially, all the DHEA in male plasma is of adrenal origin. However, <1% of the total testosterone in plasma is derived from DHEA. As summarized in Table 54-1, the plasma concentration of androstenedione in males is only ~25% that of testosterone. About 20% of androstenedione is generated by peripheral metabolism of other steroids. Although the adrenal gland contributes significantly to the total androgen milieu in males, it does not appear to have significant effects on stimulation and growth of the male accessory organs (Box 54-1). This occurs primarily as a result of DHT production from circulating testosterone.
Testosterone and Aging Men
For a long time, the abrupt hormonal alterations that signal the dramatic changes of female menopause were believed to have no correlate in men. We now know that men do experience a gradual decline in their serum testosterone levels (see Fig. 54-5) N54-6 and that this decline is closely correlated with many of the changes that accompany aging: decreases in bone formation, muscle mass, growth of facial hair, appetite, and libido. The blood hematocrit also decreases. Testosterone replacement can reverse many of these changes by restoring muscle and bone mass and correcting the anemia.
Although the levels of both total and free testosterone decline with age, levels of LH are frequently not elevated. This finding is believed to indicate that some degree of hypothalamic-pituitary dysfunction accompanies aging.
Testosterone acts on target organs by binding to a nuclear receptor
Most testosterone in the circulation is bound to specific binding proteins. About 45% of plasma testosterone binds to sex hormone–binding globulin (SHBG) and ~55% binds to serum albumin and corticosteroid-binding globulin (CBG; see p. 1021). A small fraction (~2%) of the total circulating testosterone circulates free, or unbound, in plasma. The free form of testosterone enters the cell by passive diffusion and subsequently exerts biological actions or undergoes metabolism by other organs such as the prostate, liver, and intestines (see the next section). The quantity of testosterone entering a cell is determined by the plasma concentration and by the intracellular milieu of enzymes and binding proteins.
Once it diffuses into the cell, testosterone either binds to a high-affinity AR in the nucleus or undergoes conversion to DHT, which also binds to the AR. This receptor functions as a homodimer (AR/AR) and is a member of the family of nuclear receptors (see Table 3-6) that includes receptors for glucocorticoids, mineralocorticoids, progesterone, estrogens, vitamin D, thyroid hormone, and retinoic acid. The gene coding for the AR is located on the X chromosome. The androgen-AR complex is a transcription factor that binds to hormone response elements on DNA located in the promoter regions of the target genes. Interaction between the androgen-AR complex and nuclear chromatin causes marked increases in transcription, which ultimately lead to the synthesis of specific proteins. As a result of these synthetic processes, specific cell functions ensue, including growth and development. Thus, presence of the AR in a cell or tissue determines whether that tissue responds to androgens.
Whether the active compound in any tissue is DHT or testosterone depends on the presence or absence in that tissue of the microsomal enzyme 5α-reductase, which converts testosterone to DHT. The biological activity of DHT is 30 to 50 times higher than that of testosterone. Some tissues, including the brain, aromatize testosterone to estradiol, and thus the action of this metabolite occurs via the estrogen receptor.
Some of the effects of androgens may be nongenomic. For example, androgens may stimulate hepatic microsomal protein synthesis by a mechanism independent of binding to the AR. Other evidence indicates that the action of androgens on the prostate gland may occur via the adenylyl cyclase/PKA system (see pp. 56–57) and could result in gene activation under some circumstances.
Metabolism of testosterone occurs primarily in the liver and prostate
Only small amounts of testosterone enter the urine without metabolism; this urinary testosterone represents <2% of the daily testosterone production. The large remaining balance of testosterone and other androgens is converted in the liver to 17-ketosteroids and in the prostate to DHT. The degradation products of testosterone are primarily excreted in the urine and feces as water-soluble conjugates of either sulfuric acid or glucuronic acid.