**Cholesterol: Biosynthesis, Functional Diversity, Homeostasis and Regulation by Natural Products**

J. Thomas1, T.P. Shentu1 and Dev K. Singh2\*

*1Department of Medicine, University of Illinois, Chicago 2Division of Developmental Biology, Department of Pediatrics Children's Hospital of University of Illinois, University of Illinois at Chicago USA* 

#### **1. Introduction**

418 Biochemistry

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Spannhake, E. W., Brindley, D. N. and Natarajan, V. (2005). Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NFkappaB activation and interleukin-8 secretion in human bronchial epithelial cells. Most of the discussions on cardiovascular diseases include the relative level of total plasma cholesterol whereas complete lipid profile is very important as clinical diagnotics for cardiovascular risk. For example, the status of triglycerides, low density lipoprotein cholesterol (LDL cholesterol), high density lipoprotein cholesterol (HDL cholesterol), thyroid functions, insulin and lipid peroxides etc as these lipids are related to cardiovascular disease either as markers of another underlying disturbance or along the same pathways of cholesterol metabolism. According to latest World Health Organization (WHO) report about 50% of the heart attacks occur in individual with high level of cholesterol. The most abundant sterol in animal system is in the form of cholesterol whereas plants lack cholesterol but they contain structurally similar other sterol and similar biosynthetic pathway exist both in plants and animals as well as some prokaryotes also synthesize some specific sterols. In 1948, the Framingham Heart Study - under the direction of the National Heart Institute (now known as the National Heart, Lung, and Blood Institute or NHLBI) - embarked on an ambitious project in health research. At the time, little was known about the general causes of heart disease and stroke, but the death rates for CVD had been increasing steadily since the beginning of the century and had become an American epidemic. The Framingham Heart Study became a joint project of the National Heart, Lung and Blood Institute and Boston University. The concern about cholesterol was largely fueled by this study and others that provided strong evidence that when large populations are observed, persons with higher than average serum total cholesterol have a higher incidence of coronary artery disease (CAD). Laboratory reports often mention two main types of cholesterol, the HDL cholesterol (often termed the "good" cholesterol) and LDL cholesterol (often mis-named the "bad" one. Even if the total LDL is lowered, the important fraction is actually the small LDL, which is more easily oxidized into a potentially atherogenic particle than its larger, more buoyant counterpart. Bacteria or other infectious agents are being looked at as part of the culprits as causative factors in initiating injury to the arterial wall. Cholesterol is then attracted to this 'rough' site on the blood vessel

<sup>\*</sup> Corresponding Author

wall in an attempt to heal the wall so that blood will flow smoothly over the injured area. Cholesterol itself is not the cause of CAD. The blood cholesterol is rather only a reflection of other metabolic imbalances in the vast majority of cases. If we assume blood lipid (fats) status are associated with some risk of cardiovascular disease. The question is which lipids are the important markers, and even more important what should we do about them.

Lowering cholesterol too aggressively or in artificial circumstances or having too low total cholesterol is also undesirable. Plasma cholesterol level below 200mg% is desirable whereas as 200-239mg% and above 240mg% is considered as borderline and high level of cholesterol, respectively. Ingested cholesterol comes from animal sources (plants and prokaryotes do not contain cholesterol (Gylling and Miettinen, 1995) such as eggs, meat, dairy products, fish, and shellfish or biosynthesized from the breakdown of carbohydrates, lipids, or proteins available in the food. One study has estimated that the complete abolition of dietary cholesterol absorption would reduce plasma cholesterol by up to 62% (Gylling and Miettinen, 1995). About 50% of dietary cholesterol is absorbed through intestinal enterocytes, while the rest is excreted through feces (Ostlund et al., 1999). It is estimated that half of ingested cholesterol enters the body while the other half excreated in the feces. Normally, the more cholesterol we absorb, the less our bodies make. There is slight increase in plasma cholesterol with increase in the amount of cholesterol ingested each day, usually is not changed more than 15 percent by altering the amount of cholesterol in the diet. Although the response of individuals differs markedly. Cholesterol is integeral part of membranes and perform a number of vital functions in the cell and due to this property of cholesterol, each cell has the capability to biosynthesize cholesterol if it is required. Cholesterol is a component of steroid hormones, including pregnenolone, estrogens, progesterone, testosterone, vitamin D and bile acids. Bile acids are involved in lipid digestion, absorption, and excretion.

In this chapter, mode of intracellular and extracellular cholesterol transport through acceptors-donors and thereafter cholesterol trafficking pathways will be described in detail. Furthermore, we will discuss the regulation of cholesterol at enzymatic/transcriptional level and diverse functions of cholesterol in our body. Taken together, this book chapter will address recent advances in cholesterol metabolism both *in vitro* and *in vivo* models related to absorptions, biosynthesis, transport, excretion and therapeutic targets for new drugs and natural compounds.

#### **1.1 Cholesterol biosynthetic pathway**

As early as 1926, studies by Heilbron, Kamm and Owens suggested that squalene is precursor of cholesterol biosynthesis (Garrett & Grisham, 2007). In the same year, H.J. Channon, demonstrated first time that animals fed on shark oil produced more cholesterol in the tissues. In 1940, Bloch and Rittenberg, first time demonstrated that mice fed on radiolabled acetate showed significant radiolabeled cholesterol (Bloch et al., 1945; Kresge et al., 2005). In 1952, Konard Bloch and Robert Langdon showed conclusively that squalene as well as cholesterol are synthesized from acetate for which Fyodor Lynen and Bloch were awarded the Noble Prize in Medicine/Physiology in 1964. Cholesterol is biosynthesized from 2-carbon metabolic intermediate, acetyl-CoA hooked end to end involving a number of enzymatic reactions and finally get converted into the 27-carbon molecule of cholesterol. Metabolism (catabolism) of lipids, carbohydrates and proteins lead to the formation Acetyl-CoA. Proteins are generally are not catabolized for the purpose of energy and usually broken down into amino acids for denovo protein biosynthesis, under excessive protein

wall in an attempt to heal the wall so that blood will flow smoothly over the injured area. Cholesterol itself is not the cause of CAD. The blood cholesterol is rather only a reflection of other metabolic imbalances in the vast majority of cases. If we assume blood lipid (fats) status are associated with some risk of cardiovascular disease. The question is which lipids are the

Lowering cholesterol too aggressively or in artificial circumstances or having too low total cholesterol is also undesirable. Plasma cholesterol level below 200mg% is desirable whereas as 200-239mg% and above 240mg% is considered as borderline and high level of cholesterol, respectively. Ingested cholesterol comes from animal sources (plants and prokaryotes do not contain cholesterol (Gylling and Miettinen, 1995) such as eggs, meat, dairy products, fish, and shellfish or biosynthesized from the breakdown of carbohydrates, lipids, or proteins available in the food. One study has estimated that the complete abolition of dietary cholesterol absorption would reduce plasma cholesterol by up to 62% (Gylling and Miettinen, 1995). About 50% of dietary cholesterol is absorbed through intestinal enterocytes, while the rest is excreted through feces (Ostlund et al., 1999). It is estimated that half of ingested cholesterol enters the body while the other half excreated in the feces. Normally, the more cholesterol we absorb, the less our bodies make. There is slight increase in plasma cholesterol with increase in the amount of cholesterol ingested each day, usually is not changed more than 15 percent by altering the amount of cholesterol in the diet. Although the response of individuals differs markedly. Cholesterol is integeral part of membranes and perform a number of vital functions in the cell and due to this property of cholesterol, each cell has the capability to biosynthesize cholesterol if it is required. Cholesterol is a component of steroid hormones, including pregnenolone, estrogens, progesterone, testosterone, vitamin D and bile acids. Bile acids are

In this chapter, mode of intracellular and extracellular cholesterol transport through acceptors-donors and thereafter cholesterol trafficking pathways will be described in detail. Furthermore, we will discuss the regulation of cholesterol at enzymatic/transcriptional level and diverse functions of cholesterol in our body. Taken together, this book chapter will address recent advances in cholesterol metabolism both *in vitro* and *in vivo* models related to absorptions, biosynthesis, transport, excretion and therapeutic targets for new drugs and

As early as 1926, studies by Heilbron, Kamm and Owens suggested that squalene is precursor of cholesterol biosynthesis (Garrett & Grisham, 2007). In the same year, H.J. Channon, demonstrated first time that animals fed on shark oil produced more cholesterol in the tissues. In 1940, Bloch and Rittenberg, first time demonstrated that mice fed on radiolabled acetate showed significant radiolabeled cholesterol (Bloch et al., 1945; Kresge et al., 2005). In 1952, Konard Bloch and Robert Langdon showed conclusively that squalene as well as cholesterol are synthesized from acetate for which Fyodor Lynen and Bloch were awarded the Noble Prize in Medicine/Physiology in 1964. Cholesterol is biosynthesized from 2-carbon metabolic intermediate, acetyl-CoA hooked end to end involving a number of enzymatic reactions and finally get converted into the 27-carbon molecule of cholesterol. Metabolism (catabolism) of lipids, carbohydrates and proteins lead to the formation Acetyl-CoA. Proteins are generally are not catabolized for the purpose of energy and usually broken down into amino acids for denovo protein biosynthesis, under excessive protein

important markers, and even more important what should we do about them.

involved in lipid digestion, absorption, and excretion.

natural compounds.

**1.1 Cholesterol biosynthetic pathway** 

consumption or during certain disease states, certain proteins can be catabolized to acetyl-CoA. Non-essential fatty acids, trans-fatty acids, and saturated fats, and refined carbohydrates are general source of excessive acetyl-CoAwhich pressurize our body to biosynthesize cholesterol. In other words, cholesterol is formed from excess calories which usually are generated most often from carbohydrates and fats.

Fig. 1. Cholesterol Biosynthetic Pathway

The process of cholesterol synthesis has five major steps:


**A**cetyl-CoA units are converted to mevalonate by a series of reactions that begins with the formation of **HMG-CoA (Figure 1).** Unlike the HMG-CoA formed during ketone body synthesis in the mitochondria, this form is synthesized in the cytoplasm. However, the pathway and the necessary enzymes are the same as those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. Acetoacetyl-CoA and a third mole of acetyl-CoA are converted to HMG-CoA by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase, HMGR (this enzyme is bound in the endoplasmic reticulum, ER). HMGR absolutely requires NADPH as a cofactor and two moles of NADPH are consumed during the conversion of HMG-CoA to mevalonate. The reaction catalyzed by HMGR is the rate limiting step of cholesterol biosynthesis, and this enzyme is subject to complex regulatory controls which will be discussed in separate section of this book chapter.

Fig. 2. Post squalene pathway of cholesterol and other sterol Biosynthesis

Mevalonate is then activated by three successive phosphorylations, yielding 5 pyrophosphomevalonate. Phosphorylation mevalonate and successive reactions maintain its solubility, since otherwise these are insoluble in water. After phosphorylation, an ATPdependent decarboxylation yields isopentenyl pyrophosphate, IPP, an activated isoprenoid molecule. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate, DMPP. One molecule of IPP condenses with one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Finally, the NADPH-requiring enzyme, squalene synthase catalyzes the head-to-tail condensation of two molecules of FPP, yielding squalene (squalene synthase also is tightly associated with the endoplasmic reticulum). Squalene undergoes a two step cyclization to yield lanosterol catalyzed by sequalene mono-

pathway and the necessary enzymes are the same as those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. Acetoacetyl-CoA and a third mole of acetyl-CoA are converted to HMG-CoA by the action of HMG-CoA synthase. HMG-CoA is converted to mevalonate by HMG-CoA reductase, HMGR (this enzyme is bound in the endoplasmic reticulum, ER). HMGR absolutely requires NADPH as a cofactor and two moles of NADPH are consumed during the conversion of HMG-CoA to mevalonate. The reaction catalyzed by HMGR is the rate limiting step of cholesterol biosynthesis, and this enzyme is subject to complex regulatory

**Cholesteryloleate**

Mevalonate is then activated by three successive phosphorylations, yielding 5 pyrophosphomevalonate. Phosphorylation mevalonate and successive reactions maintain its solubility, since otherwise these are insoluble in water. After phosphorylation, an ATPdependent decarboxylation yields isopentenyl pyrophosphate, IPP, an activated isoprenoid molecule. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate, DMPP. One molecule of IPP condenses with one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Finally, the NADPH-requiring enzyme, squalene synthase catalyzes the head-to-tail condensation of two molecules of FPP, yielding squalene (squalene synthase also is tightly associated with the endoplasmic reticulum). Squalene undergoes a two step cyclization to yield lanosterol catalyzed by sequalene mono-

3β-hydroxysteroid-∆24-reductase.

**25-hydroxycholesterol Cholic acid** 

**Biles acids Steroid hormones**

**Oxysterol**

**ACAT 25-hydroxylase CYP7A**

controls which will be discussed in separate section of this book chapter.

lanosterol 14α-demethylase

3β-hydroxysteroid-∆14-reductase

3β-hydroxysteroid C-4 sterol demethylase complex

3β-hydroxysteroid-∆8–∆7-sterol isomerase (EBP)

3β-hydroxysteroid-∆5-desaturase (lathosterol dehydrogenase)

**Desmosterol**

Fig. 2. Post squalene pathway of cholesterol and other sterol Biosynthesis

lanosterol

**Choleta-8(9),24-dien-3-ol (Zymosterol)**

**Cholesta-7,24-dien-3-ol**

**7-dehydrodesmosterol**

3β-hydroxysteroid -∆7-reductase (DHCR7)

**SLOS**

**4,4-dimethylcholesta-8,(9),24-dien-3b-ol**

oxygenase and sequalene 2, 3 epoxidase enzymes. Sequalene mono oxygenase is the second committed step in cholesterol biosynthesis and lead to the formation squalene 2, 3 epoxide. This enzymatic reaction require supernatant protein factor (SPF) and NADPH as a cofactor to introduce molecular oxygen as an epoxide at the 2, 3 position of squalene. The activity of supernatant protein factor itself is regulated by phosphorylation/dephosphorylation (Singh et al., 2003). Through a series of 19 additional reactions, lanosterol is converted to cholesterol. The first sterol intermediate, lanosterol, is formed by the condensation of the 30 carbon isoprenoid squalene as explained above and figure 1., and subsequent enzymatic reactions define the 'post-squalene' half of the pathway figure 2.

The conversion of lanosterol to cholesterol involves the reduction of the C-24 double bond, removal of three methyl groups at the C-14 and C-4 positions, and 'migration' of the C-8(9) double bond (Figure 2) (for a review, see (Herman, 2003)). Some of the enzymatic reactions must occur in sequence; for example, Δ8–Δ7 isomerization cannot precede C-14α demethylation. The saturation of the C-24 double bond of lanosterol can occur at multiple points in the pathway, creating two immediate precursors for cholesterol, desmosterol [cholesta-5(6), 24-dien-3β-ol] and 7-dehydrocholesterol (7DHC), whose relative abundance may vary among different tissues. Desmosterol, in particular, appears to be abundant in the developing mammalian brain (Herman, 2003). Several post-squalene sterol intermediates serve additional cellular functions as well. The C-14 demethylated derivatives of lanosterol, 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol and 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol, have meiosis-stimulating activity and accumulate in the ovary and testis, respectively (Rozman et al., 2002). 7-Dehydrocholesterol is the immediate precursor for vitamin D synthesis. Selected human enzymes of post-squalene cholesterol biosynthesis have also been identified based on homology to sterol biosynthetic enzymes from *Arabidopsis thaliana* (Herman, 2003; Waterham et al., 2001). In addition, cholesterol and other sterol intermediates can be converted to oxysterols that can act as regulatory signaling molecules and bind orphan nuclear receptors such as LXRα (Fitzgerald et al., 2002).

Normal healthy adults synthesize cholesterol at a rate of approximately 1g/day and consume approximately 0.3g/day. A relatively constant level of cholesterol in the body (150 - 200 mg/dL) is maintained primarily by controlling the level of *de novo* synthesis which is partly regulated in part by the dietary intake of cholesterol. Cholesterol from both diet and synthesis is utilized in the formation of membranes and in the synthesis of the steroid hormones and bile acids. The greatest proportion of cholesterol is used in bile acid synthesis.
