**4. The role of cholesterol in the development of hypertensionassociated NASH**

As previously stated, dietary cholesterol intake is considered a risk factor for NAFLD/NASH.The liver is a crucial organ implicated in the regulation of cholesterol metabolism, including the synthesis and secretion of cholesterol, as well as the synthesis of BAs from cholesterol (a major pathway for hepatic cholesterol catabolism) and BA detoxification [35]. Disturbed cholesterol homeostasis in the liver is thought to be associated with the pathogenesis of NAFLD/NASH [35]. Our study showed that the HFC diet increased serum and hepatic levels of TC in the hypertensive SHR and SHRSP5/Dmcr strains, as well as the normotensive WKY strain [28]. It is worth noting that the increase in hepatic TC levels in the hypertensive rats was significantly lower than those in the normotensive WKY strain. Therefore, we postulated that more cholesterol was consumed for the synthesis of BAs in the livers of the hypertensive rats. In addition, serum TC levels in the hypertensive strains fed the control diet were markedly lower compared with those of the normotensive WKY strain, suggesting that the dysregulation of cholesterol metabolism may play an important role in the progression of hypertension-associated NASH.

In order to investigate the kinetics of cholesterol during the development of HFC-induced NASH in our hypertensive SHRSP5/Dmcr rat model, we evaluated the expression of proteins involved in de novo cholesterol synthesis, cholesterol uptake from bloodstream in the form of LDL, cholesterol secretion into blood in the form of very-low-density lipoprotein, and BA synthesis and detoxification [36].

Increased levels of hepatic BA were observed in NASH patients and were correlated with inflammation and fibrosis in the liver [47]. In our SHRSP5/Dmcr model, the HFC diet increased hepatic levels of CYP7A1 but decreased levels of CYP8B1, while CYP27A1 was downregulated and CYP7B1 was upregulated [36]. We used ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to further determine the hepatic levels of 21 types of BA in rats fed the HFC or control diet [48]. The HFC diet significantly increased total BA levels in the liver at 2 weeks, but decreased it at 8 weeks. We also investigated the composition of the total BA in the rats' livers. In the total BA pool, the relative proportions of CDCA species, which are hydrophobic and show high cytotoxicity [49], were markedly elevated at 8 and 14 weeks, whereas hydrophilic CA species, with lower toxicity, were significantly decreased at 14 weeks. The ratio of total CA to CDCA was prominently reduced by HFC feeding at 8 and 14 weeks. Most BAs (about 90% of the total) in the livers of rats fed the control diet were taurine-conjugated. In contrast, glycine-conjugated BAs were predominant in HFC-fed rats. In addition, canalicular transporters, BSEP and MRP2, were reduced in the livers of the rats during HFC feeding (2, 8, and 14 weeks), whereas MRP3, the basolateral transporter, was significantly increased at 8 and 14 weeks [36]. Therefore, the accumulation of total BAs in the rats' liver at 2 weeks of HFC feeding may have resulted from suppressed BA excretion to the bile duct, mediated by BSEP and MRP2 transporter proteins. Meanwhile, the decrease in total BA levels in the liver at 8 weeks may have been triggered by an increase in MRP3-mediated BA excretion to the blood. Furthermore, we demonstrated that the ratio of CA to CDCA was negatively correlated with liver injury (macrovesicular steatosis, serum ALT levels, and fibrotic area), whereas total glycol-BA/total tauro-BA was positively correlated. Therefore, the accumulation of BAs at 2 weeks of HFC-feeding, led by dysregulated BA synthesis and excretion, may trigger liver damage during the initial stages of NAFLD/NASH. Furthermore, a decrease in nuclear FXR, PXR, and CAR was observed in the livers of rats following HFC feeding. The downregulation of these nuclear receptors may be responsible for the increase in CYP7A1, as well as the decrease in BSEP and MRP2.

The Role of Cholesterol in the Pathogenesis of Hypertension-Associated Nonalcoholic…

http://dx.doi.org/10.5772/intechopen.76199

115

Toxic BA accumulation in the liver induces hepatocyte injury, and BA hydrophobicity is correlated with cytotoxicity [12]. The order of BA hydrophobicity was reported to be CA < CDCA < DCA < LCA [12]. Hydrophobic BAs are potent inflammatory agents, whereas the hydrophilic BAs are anti-inflammatory [38]. Hydrophobic BAs stimulate ROS generation in hepatic mitochondria and lead to oxidative stress, hepatocyte apoptosis, and subsequent liver damage [50, 51]. BAs with detergent properties may also induce damage in hepatocyte membranes by binding to membrane components and disrupting the integrity of the plasma membrane [12, 52].

BA metabolism is tightly regulated to prevent the retention of excessive BAs in the liver [12]. Sulfation and glucuronidation of BAs, catalyzed by SULT2A1 and UGT, respectively, are major detoxification pathways of Bas [53, 54]. These reactions increase the solubility of BAs, enhance their fecal and urinary excretion, and reduce their toxicity. In addition, the nuclear receptors, PXR and CAR, protect hepatocytes from BA toxicity by regulating the transcription of genes involved in BA detoxification, including SULT and UGT [55, 56]. Our study showed that the HFC diet impaired BA detoxification by inducing the downregulation of PXR and CAR and further suppressing SULT2A1-catalyzed sulfation and UGT-catalyzed glucuronida-

**4.3. BA detoxification**

tion in the hypertensive SHRSP5/Dmcr rats [36].

### **4.1. De novo cholesterol synthesis and its uptake from blood**

Excessive intake of cholesterol may suppress de novo cholesterol synthesis via a feedback mechanism dependent on the transcriptional factor sterol regulator element-binding protein 2 (SREBP-2) [35]. SREBP-2 resides in the endoplasmic reticulum and remains there when cholesterol is abundant in hepatocytes; however, SREBP-2 is activated in response to low levels of cholesterol and translocated to the nucleus, where it triggers the expression of various genes, including low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). HMGCR is the rate-limiting enzyme for cholesterol biosynthesis. Our study showed that HMGCR was downregulated in the livers of SHRSP5/Dmcr rats during consumption of the HFC diet (2, 8, and 14 weeks), although SREBP-2 expression remained unchanged [36]. It was proposed that additional signaling, except SREBP-2, may be required for cholesterol synthesis in our rat model. The HFC diet decreased the expression of LDLR and LDLR-related protein 1, which are required for clearing cholesterol-contained lipoproteins from the blood by the liver [37]. Therefore, excessive intake of dietary cholesterol led to accumulation in the liver and consequently resulted in suppression of cholesterol synthesis and uptake.

#### **4.2. BA synthesis and excretion**

There are two major pathways of BA synthesis. The classic pathway is initiated by cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme, followed by the catalytic action of sterol 12 alpha-hydroxylase (CYP8B1) [38]. On the other hand, the initial step in the alternative (acidic) pathway is catalyzed by sterol 27-hydroxylase (CYP27A1), followed by oxysterol 7alpha-hydroxylase (CYP7B1). The major primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), are produced from cholesterol in the liver, while the secondary BAs, lithocholic acid (LCA) and deoxycholic acid (DCA), are generated from CDCA and CA in the intestines, respectively. After synthesis, conjugation of Bas is required for effective transport and detoxification [39]. BAs are conjugated with amino acids (taurine or glycine) or sulfate, mediated by BA coenzyme A synthase and BA amino acid transferase, and sulfotransferase (SULT2A1), respectively. Some BAs are glucuronidated by UDP-glucuronosyl *N*-transferases (UGT1A1, 2B4, and 2B7). Amino acid-conjugated BAs are excreted from the liver into the bile canaliculi via the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter protein located in the canalicular membrane of hepatocytes [40]. Multidrug-resistant protein 2 (MRP2) is another ABC transporter implicated in the transport of sulfated or glucuronidated BAs to bile, while MRP3, located in the basolateral membrane of hepatocyte, is responsible for the transport of BAs from the liver to the blood. In addition, bile acid-activated nuclear receptors (a group of transcriptional factors), such as farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), are implicated in the regulation of BA metabolism, including synthesis, transport, and detoxification [39]. Several studies have reported that activation of FXR, PXR, and CAR inhibits transcription of the CYP7A1 gene in hepatocytes, and therefore suppress BA synthesis [41–43]. Activation of FXR and PXR also induces expression of the BA transporter proteins, BSEP and MRP2 [44–46].

Increased levels of hepatic BA were observed in NASH patients and were correlated with inflammation and fibrosis in the liver [47]. In our SHRSP5/Dmcr model, the HFC diet increased hepatic levels of CYP7A1 but decreased levels of CYP8B1, while CYP27A1 was downregulated and CYP7B1 was upregulated [36]. We used ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to further determine the hepatic levels of 21 types of BA in rats fed the HFC or control diet [48]. The HFC diet significantly increased total BA levels in the liver at 2 weeks, but decreased it at 8 weeks. We also investigated the composition of the total BA in the rats' livers. In the total BA pool, the relative proportions of CDCA species, which are hydrophobic and show high cytotoxicity [49], were markedly elevated at 8 and 14 weeks, whereas hydrophilic CA species, with lower toxicity, were significantly decreased at 14 weeks. The ratio of total CA to CDCA was prominently reduced by HFC feeding at 8 and 14 weeks. Most BAs (about 90% of the total) in the livers of rats fed the control diet were taurine-conjugated. In contrast, glycine-conjugated BAs were predominant in HFC-fed rats. In addition, canalicular transporters, BSEP and MRP2, were reduced in the livers of the rats during HFC feeding (2, 8, and 14 weeks), whereas MRP3, the basolateral transporter, was significantly increased at 8 and 14 weeks [36]. Therefore, the accumulation of total BAs in the rats' liver at 2 weeks of HFC feeding may have resulted from suppressed BA excretion to the bile duct, mediated by BSEP and MRP2 transporter proteins. Meanwhile, the decrease in total BA levels in the liver at 8 weeks may have been triggered by an increase in MRP3-mediated BA excretion to the blood. Furthermore, we demonstrated that the ratio of CA to CDCA was negatively correlated with liver injury (macrovesicular steatosis, serum ALT levels, and fibrotic area), whereas total glycol-BA/total tauro-BA was positively correlated. Therefore, the accumulation of BAs at 2 weeks of HFC-feeding, led by dysregulated BA synthesis and excretion, may trigger liver damage during the initial stages of NAFLD/NASH. Furthermore, a decrease in nuclear FXR, PXR, and CAR was observed in the livers of rats following HFC feeding. The downregulation of these nuclear receptors may be responsible for the increase in CYP7A1, as well as the decrease in BSEP and MRP2.

#### **4.3. BA detoxification**

involved in de novo cholesterol synthesis, cholesterol uptake from bloodstream in the form of LDL, cholesterol secretion into blood in the form of very-low-density lipoprotein, and BA

Excessive intake of cholesterol may suppress de novo cholesterol synthesis via a feedback mechanism dependent on the transcriptional factor sterol regulator element-binding protein 2 (SREBP-2) [35]. SREBP-2 resides in the endoplasmic reticulum and remains there when cholesterol is abundant in hepatocytes; however, SREBP-2 is activated in response to low levels of cholesterol and translocated to the nucleus, where it triggers the expression of various genes, including low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). HMGCR is the rate-limiting enzyme for cholesterol biosynthesis. Our study showed that HMGCR was downregulated in the livers of SHRSP5/Dmcr rats during consumption of the HFC diet (2, 8, and 14 weeks), although SREBP-2 expression remained unchanged [36]. It was proposed that additional signaling, except SREBP-2, may be required for cholesterol synthesis in our rat model. The HFC diet decreased the expression of LDLR and LDLR-related protein 1, which are required for clearing cholesterol-contained lipoproteins from the blood by the liver [37]. Therefore, excessive intake of dietary cholesterol led to accumulation in the liver and consequently resulted in suppression of cholesterol synthesis and uptake.

There are two major pathways of BA synthesis. The classic pathway is initiated by cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme, followed by the catalytic action of sterol 12 alpha-hydroxylase (CYP8B1) [38]. On the other hand, the initial step in the alternative (acidic) pathway is catalyzed by sterol 27-hydroxylase (CYP27A1), followed by oxysterol 7alpha-hydroxylase (CYP7B1). The major primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), are produced from cholesterol in the liver, while the secondary BAs, lithocholic acid (LCA) and deoxycholic acid (DCA), are generated from CDCA and CA in the intestines, respectively. After synthesis, conjugation of Bas is required for effective transport and detoxification [39]. BAs are conjugated with amino acids (taurine or glycine) or sulfate, mediated by BA coenzyme A synthase and BA amino acid transferase, and sulfotransferase (SULT2A1), respectively. Some BAs are glucuronidated by UDP-glucuronosyl *N*-transferases (UGT1A1, 2B4, and 2B7). Amino acid-conjugated BAs are excreted from the liver into the bile canaliculi via the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter protein located in the canalicular membrane of hepatocytes [40]. Multidrug-resistant protein 2 (MRP2) is another ABC transporter implicated in the transport of sulfated or glucuronidated BAs to bile, while MRP3, located in the basolateral membrane of hepatocyte, is responsible for the transport of BAs from the liver to the blood. In addition, bile acid-activated nuclear receptors (a group of transcriptional factors), such as farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), are implicated in the regulation of BA metabolism, including synthesis, transport, and detoxification [39]. Several studies have reported that activation of FXR, PXR, and CAR inhibits transcription of the CYP7A1 gene in hepatocytes, and therefore suppress BA synthesis [41–43]. Activation of FXR and PXR

also induces expression of the BA transporter proteins, BSEP and MRP2 [44–46].

synthesis and detoxification [36].

114 Cholesterol - Good, Bad and the Heart

**4.2. BA synthesis and excretion**

**4.1. De novo cholesterol synthesis and its uptake from blood**

Toxic BA accumulation in the liver induces hepatocyte injury, and BA hydrophobicity is correlated with cytotoxicity [12]. The order of BA hydrophobicity was reported to be CA < CDCA < DCA < LCA [12]. Hydrophobic BAs are potent inflammatory agents, whereas the hydrophilic BAs are anti-inflammatory [38]. Hydrophobic BAs stimulate ROS generation in hepatic mitochondria and lead to oxidative stress, hepatocyte apoptosis, and subsequent liver damage [50, 51]. BAs with detergent properties may also induce damage in hepatocyte membranes by binding to membrane components and disrupting the integrity of the plasma membrane [12, 52].

BA metabolism is tightly regulated to prevent the retention of excessive BAs in the liver [12]. Sulfation and glucuronidation of BAs, catalyzed by SULT2A1 and UGT, respectively, are major detoxification pathways of Bas [53, 54]. These reactions increase the solubility of BAs, enhance their fecal and urinary excretion, and reduce their toxicity. In addition, the nuclear receptors, PXR and CAR, protect hepatocytes from BA toxicity by regulating the transcription of genes involved in BA detoxification, including SULT and UGT [55, 56]. Our study showed that the HFC diet impaired BA detoxification by inducing the downregulation of PXR and CAR and further suppressing SULT2A1-catalyzed sulfation and UGT-catalyzed glucuronidation in the hypertensive SHRSP5/Dmcr rats [36].
