**7. Regulation of specific pathways and its influence on cholesterol homeostasis**

Blood cholesterol concentration is a result of balance between cholesterol input and cholesterol output. When the input is surpass the output, blood cholesterol increases and by contrast, when cholesterol input is less than the output blood cholesterol levels decrease. Cholesterol input is attributed from the intestinal absorption of dietary and biliary cholesterol and cholesterol biosynthesis. On the other hand, the cholesterol output is mainly from LDLreceptor mediated LDL-cholesterol clearance, reverse transport by HDL, cholesterol catabo‐ lism by converting into bile acids, cholesterol and bile acids secretion in bile into the intestine lumen, and fecal excretion.

Metabolic nuclear receptors serve a central role in maintainingcellular and whole-body cholesterol homeostasis [40]. Two important transcriptional mechanisms to regulate choles‐ terol metabolism are the pathways mediated by sterol responsive element– binding protein (SREBP) and liver X receptor (LXR), which tightly regulate intracellular sterol concentrations. The SREBP pathway ensures that there is sufficientcholesterol to meet cellular requirements by directly activating expression of genesinvolved in the synthesis and uptake of cholesterol, and lipogenesis [41]. In the setting ofexcess free or unesterified cholesterol, SREBP-depend‐ entgene expression is suppressed. LXR and farnesoid X receptor (FXR),together with other members of the nuclear receptor superfamilypromote sterol storage, transport, and catabolism to prevent cholesterol accumulation [42]. LXRs respond to elevated cholesterol levels via transactivationof genes involved in sterol transport (ABCA1, ABCG1, ABCG5,and ABCG8), cholesterol efflux and high-density lipoprotein(HDL) metabolism (ABCA1, APOE, CETP, and PLTP), and sterol catabolism(CYP7A1) [42]. Other members of metabolicnuclear receptor family include receptors for bile acids (CAR and PXR), and fatty acids (peroxisome prolifer‐ aotr-activated receptors). Throughthe coordinated regulation of gene transcription, these nuclearreceptors regulate the key aspects of cellular and whole-body sterolhomeostasis, including cholesterol absorption and synthesis, lipoprotein synthesisand remodeling, lipo‐ protein uptake by peripheral tissues, reversecholesterol transport, and bile acid synthesis and absorption.

The amount of cholesterol that is synthesized in the liver is tightly regulated by dietary cholesterol levels. When dietary intake of cholesterol is high, synthesis is decreased and when dietary intake is low, synthesis is increased. However, cholesterol produced in other tissues is under no such feedback control. Cholesterol and similar oxysterols (the oxygenated deriva‐ tives of cholesterol, such as 22(R)-hydroxycholesterol, 2 4(S)-hydroxycholesterol, 27-hydrox‐ ycholesterol, and cholestenoic acid) act as regulatory molecules to maintain healthy levels of cholesterol. In tissues, many factor influence cholesterol balance through every cholesterol metabolic pathways.

#### **7.1. LDL receptor-mediated cholesterol clearance**

the hydrolysis of these stored cholesterol esters, yielding bioavailable cholesterol and fat‐

About 1 g of cholesterol is eliminated from the body per day, approximately equivalent to the amount that absorbed cholesterol and synthesize cholesterol. Approximately, half is excreted in the feces after conversion to bile acids in liver, and the remainder is excreted as cholesterol. Bile acids serve to remove unwanted cholesterol from the body and to aid in lipid digestion in the intestine. 7α-hydroxylase, the rate limiting enzyme of bile acid biosynthesis converts cholesterol into 7-hydroxycholesterol. 7-hydroxycholesterol is converted to one of the two primary bile acids, cholic acid and chenodeoxycholic acid. Bile acids are then delivered to the intestines where they aid in the absorption of lipids. Some of bile acids are modified to form secondary bile acids (lithocholic acid and deoxycholic acid) in the intestine by intestinal bacteria. However, the majority of bile acids delivered to intestine are recycled by re-absorp‐ tion in the ileum and returned to the liver by enterohepatic circulation. In liver, glyco- and tauroconjugate bile acids are formed and stored in gall bladder, from where they are released

into the intestinal lumen for aid fat/lipids digestion and absorption.

190 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**7. Regulation of specific pathways and its influence on cholesterol**

Blood cholesterol concentration is a result of balance between cholesterol input and cholesterol output. When the input is surpass the output, blood cholesterol increases and by contrast, when cholesterol input is less than the output blood cholesterol levels decrease. Cholesterol input is attributed from the intestinal absorption of dietary and biliary cholesterol and cholesterol biosynthesis. On the other hand, the cholesterol output is mainly from LDLreceptor mediated LDL-cholesterol clearance, reverse transport by HDL, cholesterol catabo‐ lism by converting into bile acids, cholesterol and bile acids secretion in bile into the intestine

Metabolic nuclear receptors serve a central role in maintainingcellular and whole-body cholesterol homeostasis [40]. Two important transcriptional mechanisms to regulate choles‐ terol metabolism are the pathways mediated by sterol responsive element– binding protein (SREBP) and liver X receptor (LXR), which tightly regulate intracellular sterol concentrations. The SREBP pathway ensures that there is sufficientcholesterol to meet cellular requirements by directly activating expression of genesinvolved in the synthesis and uptake of cholesterol, and lipogenesis [41]. In the setting ofexcess free or unesterified cholesterol, SREBP-depend‐ entgene expression is suppressed. LXR and farnesoid X receptor (FXR),together with other members of the nuclear receptor superfamilypromote sterol storage, transport, and catabolism to prevent cholesterol accumulation [42]. LXRs respond to elevated cholesterol levels via transactivationof genes involved in sterol transport (ABCA1, ABCG1, ABCG5,and ABCG8), cholesterol efflux and high-density lipoprotein(HDL) metabolism (ABCA1, APOE, CETP, and

ty acids.

**homeostasis**

lumen, and fecal excretion.

**6.4. Excretion of cholesterol**

Cellular cholesterol increase is due to the uptake of cholesterol containing lipoproteins by receptors. LDL receptor regulates the cellular transport of LDL particles. One mechanism for regulating LDL receptor expression and controlling the expression of all the enzymes in the cholesterol biosynthetic pathway involves sterol-sensitive response elements (SREs). SREs are found in the promoters of the genes coding for the enzymes of cholesterol biosynthesis pathway and LDL receptors. Transcription factors of SRE activation are SREBPs. Three major SREBP isoforms, SREBP-1a, -1c, and -2, have been identified and differ in relative abundance in the liver and other various tissues. SREBP-1a is a potent activator of all SREBP-responsive genes and functions to maintain basal levels of cholesterol and fatty acid synthesis. SREBP-1c selectively activates genes involved in fatty acid synthesis, while SREBP-2 preferentially regulates genes important for cholesterol homeostasis by activating the transcription of HMG-CoA synthase, HMG-CoA reductase, LDL receptor [41].

Due to their ability to bind SREs, SREBP-2 plays an instrumental role in cholesterol homeo‐ stasis. These transcription-regulatory proteins are bound by another protein called SREBP cleavage activating proteins (SCAPs). SCAP, in turn, can bind reversibly with another endoplasmic reticulum-resident membrane protein, INSIG. SCAPs bind to SREBP-2 in the endoplasmic reticulum where a regulatory domain within SCAP responds to the level of oxysterols present in the cell. When the intracellular cholesterol and oxysterols concentrations decrease, the SREBP/SCAP complex moves to Golgi apparatus, leaving INSIG. Two proteases localized in Golgi, site-1 and -2 proteases (S1P and S2P) cleave SREBP-2 to release the tran‐ scription activation domain of SREBP-2. SREBP-2 preferentially activates transcription of target genes of LDL receptor [41]. When oxysterol levels are high, the SCAP/SREBP complex remains in the endoplasmic reticulum, preventing cleaved SREBP-2 from promoting gene expression. In addition to the up-regulation of LDLR transcription, nuclear SREBP-2 increases the transcription of PCSK9, a sterol-responsive protein that accelerates LDLR turnover in the liver, thereby limiting lipoprotein uptake. As high concentrations of cellular cholesterol suppress SREBP-2 cleavage and release from endoplasmic reticulum, PCSK9 transcription is reduced, which subsequently increases LDLR levels, helping to maintain cholesterol homeo‐ stasis [43].

#### **7.2. Regulation of cholesterol biosynthesis**

SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triacylglycerols, and phospholipids, as well as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor required to synthesize these molecules. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways [41]. High cholesterol/oxysterol levels acting on SCAP ultimately stop the maturation of SREBPs, resulting in the down regulation of key enzymes such as HMG-CoA reductase, thus, reducing the amount of cholesterol produced by the liver. To compensate the decreased cholesterol synthesis a homeostatic response in which cells increase the density of LDL receptors on their surfaces. This increases the clearance rate of LDL particles from the plasma and reduces plasma LDL cholesterol and its related health risks. The decrease in cholesterol synthesis also promotes an increase of HDL, thus, clearing even more cholesterol from the plasma.

Elevated levels of cellular cholesterol are accompanied by the increased production of oxysterols, which are specificligands of LXRs, allowing LXRs to function as cholesterol sensors [44]. LXRs respond to elevated cholesterol levels via transactivationof genes involved in sterol transport (ABCA1, ABCG1, ABCG5,and ABCG8), cholesterol efflux and HDL metabolism (ABCA1, APOE, CETP, and PLTP), and sterol catabolism(CYP7A1). Additionally, LXRs also play a central role in regulatingcellular lipid content through activation of SREBP-1c, whichis the master regulator of de novo lipogenesis [40]. In response to activation, LXRs act in a coordinated fashion to maintain cholesterol homeostasis by directing the tissue-specific expression of genes involved in sterol transport and metabolism [45]. A principal function of LXR in macrophages is to promote cholesterol removal from the cell through the induction of ABCA1, ABCG1, and apolipoprotein E. LXR also induces genes involvedin lipoprotein metabolism, including LPL, CETP, and PLTP [45].

#### **7.3. Regulation of cholesterol absorption and secretion**

At the intestine, cholesterol is absorbed into enterocytes by a mechanism involving Niemann Pick C1-like protein 1 (NPC1L1) [46]. The NPC1L1 protein is abundant on intestinal brush border membranes. It functions as a sterol transporter to mediate intestinal cholesterol absorption and counterbalances hepatobiliary cholesterol excretion [46]. NPC1L1, is not under control of a nuclear receptor LXR [47]. In the enterocyte, cholesterol is readily esterified by the action of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) and released into lymph in associa‐ tion with chylomicrons. The ATP-binding cassette (ABC) transporter protein ABCA1 andthe ABC half-transporters, ABCG5 and ABCG8, are LXR target genesin the intestine and partici‐ pate in cholesterol absorption. ABCA1 and ABCG5/8 counteract cholesterol absorption via effluxof cholesterol from the enterocyte into the gut lumen. LXR agonists exerttheir effect on cholesterol absorption through upregulationof ABCG5 and ABCG8, which is necessary for the majority of sterols secreted into bile [40, 48].

#### **7.4. Regulation of cholesterol transport**

liver, thereby limiting lipoprotein uptake. As high concentrations of cellular cholesterol suppress SREBP-2 cleavage and release from endoplasmic reticulum, PCSK9 transcription is reduced, which subsequently increases LDLR levels, helping to maintain cholesterol homeo‐

SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triacylglycerols, and phospholipids, as well as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor required to synthesize these molecules. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways [41]. High cholesterol/oxysterol levels acting on SCAP ultimately stop the maturation of SREBPs, resulting in the down regulation of key enzymes such as HMG-CoA reductase, thus, reducing the amount of cholesterol produced by the liver. To compensate the decreased cholesterol synthesis a homeostatic response in which cells increase the density of LDL receptors on their surfaces. This increases the clearance rate of LDL particles from the plasma and reduces plasma LDL cholesterol and its related health risks. The decrease in cholesterol synthesis also promotes

Elevated levels of cellular cholesterol are accompanied by the increased production of oxysterols, which are specificligands of LXRs, allowing LXRs to function as cholesterol sensors [44]. LXRs respond to elevated cholesterol levels via transactivationof genes involved in sterol transport (ABCA1, ABCG1, ABCG5,and ABCG8), cholesterol efflux and HDL metabolism (ABCA1, APOE, CETP, and PLTP), and sterol catabolism(CYP7A1). Additionally, LXRs also play a central role in regulatingcellular lipid content through activation of SREBP-1c, whichis the master regulator of de novo lipogenesis [40]. In response to activation, LXRs act in a coordinated fashion to maintain cholesterol homeostasis by directing the tissue-specific expression of genes involved in sterol transport and metabolism [45]. A principal function of LXR in macrophages is to promote cholesterol removal from the cell through the induction of ABCA1, ABCG1, and apolipoprotein E. LXR also induces genes involvedin lipoprotein

At the intestine, cholesterol is absorbed into enterocytes by a mechanism involving Niemann Pick C1-like protein 1 (NPC1L1) [46]. The NPC1L1 protein is abundant on intestinal brush border membranes. It functions as a sterol transporter to mediate intestinal cholesterol absorption and counterbalances hepatobiliary cholesterol excretion [46]. NPC1L1, is not under control of a nuclear receptor LXR [47]. In the enterocyte, cholesterol is readily esterified by the action of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) and released into lymph in associa‐ tion with chylomicrons. The ATP-binding cassette (ABC) transporter protein ABCA1 andthe ABC half-transporters, ABCG5 and ABCG8, are LXR target genesin the intestine and partici‐

an increase of HDL, thus, clearing even more cholesterol from the plasma.

metabolism, including LPL, CETP, and PLTP [45].

**7.3. Regulation of cholesterol absorption and secretion**

stasis [43].

**7.2. Regulation of cholesterol biosynthesis**

192 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

The hepatic nuclear receptor, PPARα, exert control over many aspects of reversecholesterol transport. Hepatic synthesisof apoA-I and apoA-II, the two major apolipoproteins in discoi‐ dalHDL ( HDL2), is regulated via PPARα activation and transcriptional regulation ofRORα (NR1F1), a widely expressed nuclear receptor that is activatedby cholesterol or cholesterol sulfate ligands [49]. ABCA1, which helps in reverse transport from peripheral tissues, is PPARγ/LXR-regulated cholesterol/phospholipid transporter [50]. PLTP is activatedby both LXR and FXR whereas CETP is transactivated by LXR [40,51].

#### **7.5. Excretion of cholesterol**

Bile acids are synthesized in hepatocytes and this production is tightly controlled by the nuclear receptor transcription factors, LXR-*α* and LXR-*β*. Activation of LXRs by specific oxysterol derivatives leads to the regulation of bile acid synthesis by stimulating cholesterol 7α-hydroxylase (*CYP7A1)* transcription to convertcholesterol to bile acids. FXR, a bile acid receptor, plays a central role of lipid metabolism in liver cells. FXR may play the major roles in bileacid metabolism, reverse cholesterol transport, and protecthepatocytes against choles‐ tasis by feedback inhibition ofbile acid synthesis by CYP7A1; stimulation of bile acid efflu‐ xfrom hepatocytes by bile salt export pump; inhibition of bile acid uptakeinto hepatocytes by Na+ -taurocholate co-transporting polypeptide; and regulation of reverse cholesteroltransport by inducing ApoCII and PLTP [40, 52].
