Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.

Growth hormone (GH) has pleiotropic effects on cholesterol and lipoprotein metabolism. Pituitary GH is important for the normal regulation of hepatic LDL receptors (LDLR), for the enzymatic activity of bile acid regulatory cholesterol 7alpha-hydroxylase (C7alphaOH), and for the maintenance of resistance to dietary cholesterol. The present study aimed to determine whether GH has beneficial effects on plasma lipids and hepatic cholesterol metabolism in mice devoid of LDLR. Compared with wild-type controls, LDLR-deficient mice had approximately 250% elevated plasma total cholesterol and approximately 50% increased hepatic cholesterol levels; hepatic HMG CoA reductase activity was reduced by 70%, whereas C7alphaOH activity was increased by 40%. In LDLR mice, GH infusion reduced plasma cholesterol and triglycerides up to 40%, whereas HMG CoA reductase and C7alphaOH activities were stimulated by approximately 50% and 110% respectively. GH also stimulated HMG CoA reductase and C7alphaOH activities in control mice, whereas hepatic LDLR and plasma lipoproteins were unchanged. The effects of cholestyramine and atorvastatin on C7alphaOH in LDLR-deficient mice were potentiated by GH, and this was associated with a further reduction in plasma cholesterol. GH treatment reduces plasma cholesterol and triglycerides and stimulates C7alphaOH activity in mice devoid of LDLR, particularly in combination with resin or statin treatment. The potential of GH therapy in patients with homozygous familial hypercholesterolemia should be evaluated.     

Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPARalpha in rat liver

The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products.     

Loss of small heterodimer partner expression in the liver protects against dyslipidemia

Multiple studies suggest increased conversion of cholesterol to bile acids by cholesterol 7alpha-hydroxylase (CYP7A1) protects against dyslipidemia and atherosclerosis. CYP7A1 expression is repressed by the sequential activity of two nuclear hormone receptors, farnesoid X receptor (FXR) and small heterodimer partner (SHP). Here we demonstrate 129 strain SHP(-/-) mice are protected against hypercholesterolemia resulting from either a cholesterol/cholic acid (chol/CA) diet or from hypothyroidism. In a mixed 129-C57Bl/6 background, LDLR(-/-) and LDLR(-/-)SHP(-/-) mice had nearly identical elevations in hepatic cholesterol content and repression of cholesterol regulated genes when fed a Western diet. However, the LDLR(-/-)SHP(-/-) mice had greatly reduced elevations in serum VLDL and LDL cholesterol levels and triglyceride (TG) levels as compared with LDLR(-/-) mice. Additionally, the hepatic inflammation produced by the Western diet in the LDLR(-/-) mice was abolished in the LDLR(-/-)SHP(-/-) mice. CYP7A1 expression was induced 10-fold by the Western diet in the LDLR(-/-)SHP(-/-) mice but not in the LDLR(-/-) mice. Finally, hepatocyte-specific deletion of SHP expression was also protective against dyslipidemia induced by either a chol/CA diet or by hypothyroidism. While no antagonist ligands have yet been identified for SHP, these results suggest selective inhibition of hepatic SHP expression may provide protection against dyslipidemia.     

Pituitary control of lipoprotein and bile acid metabolism in male rats: growth hormone effects are not mediated by prolactin

Previous studies have established that growth hormone (GH) has many important effects on the regulation of cholesterol and lipoprotein metabolism. However, human GH (hGH) can also bind to prolactin receptors, eliciting prolactin receptor-mediated effects. In this study, we evaluated whether hGH can exert such responses in currently used animal models and whether prolactin affects lipoprotein and/or hepatic cholesterol metabolism. Normal and hypophysectomized (Hx) male rats were given either hGH or bovine GH, the latter unable to bind to the prolactin receptor. The hormones were continuously infused by use of subcutaneous osmotic mini-pumps for 7 days; blood and livers were collected after euthanasia. Both hormones stimulated hepatic LDL receptor expression and bile acid synthesis to a similar extent and normalized the altered plasma lipoprotein pattern in Hx rats. Prolactin, injected twice daily to Hx male rats, did not exert any effects on the plasma lipoprotein pattern or on cholesterol metabolism. We conclude that previously established effects of hGH on cholesterol metabolism are not mediated by prolactin in male rats.     

Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1-/-) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1-/- mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1-/- mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.     

Glucose and insulin induction of bile acid synthesis: mechanisms and implication in diabetes and obesity

Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.     

Leptin induces the hepatic high density lipoprotein receptor scavenger receptor B type I (SR-BI) but not cholesterol 7alpha-hydroxylase (Cyp7a1) in leptin-deficient (ob/ob) mice

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis.     

Antiatherogenic effect of taurine in high fat diet fed rats

The role of taurine on atherogenesis induced by high fat diet in rats, a species which depends entirely on taurine for conjugation of bile acids has been investigated. Wistar male rats were fed on (p.o.) taurine in addition to high fat diet (11% coconut oil w/w) for 6 months. High fat diet caused significant increase of serum total cholesterol (2 fold), serum triglycerides (92.6%), LDL cholesterol (92.3%) and body weight gain (2.8 fold). Taurine administration significantly reduced serum cholesterol (37%), triglycerides (94.5%), LDL cholesterol (34%), body weight (46%). It also significantly reduced aortic cholesterol and thiobarbituric acid reactive substances and there was a significant increase of reduced glutathione. Taurine significantly increased fecal bile acids which may have resulted in significant decrease of serum cholesterol. Aortic lesion index was significantly decreased in the taurine administered group suggesting the antiatherogenic effect of taurine. It is concluded that taurine attenuated the atherogenesis possibly by its hypocholesterolemic and antioxidant property.     

Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice

Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity.     

Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley-Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1(-/-) mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.     

Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis

Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.     

Resistance of SHP-null mice to bile acid-induced liver damage

The orphan nuclear hormone receptor SHP (gene designation NROB2) is an important component of a negative regulatory cascade by which high levels of bile acids repress bile acid biosynthesis. Short term studies in SHP null animals confirm this function and also reveal the existence of additional pathways for bile acid negative feedback regulation. We have used long term dietary treatments to test the role of SHP in response to chronic elevation of bile acids, cholesterol, or both. In contrast to the increased sensitivity predicted from the loss of negative feedback regulation, the SHP null mice were relatively resistant to the hepatotoxicity associated with a diet containing 0.5% cholic acid and the much more severe effects of a diet containing both 0.5% cholic acid and 2% cholesterol. This was associated with decreased hepatic accumulation of cholesterol and triglycerides in the SHP null mice. There were also alterations in the expression of a number of genes involved in cholesterol and bile acid homeostasis, notably cholesterol 12alpha-hydroxylase (CYP8B1), which was strongly reexpressed in the SHP null mice, but not the wild type mice fed either bile acid containing diet. This contrasts with the strong repression of CYP8B1 observed with short term bile acid feeding, as well as the effects of long term feeding on other bile acid biosynthetic enzymes such as cholesterol 7alpha-hydroxylase (CYP7A1). CYP8B1 expression could contribute to the decreased toxicity of the chronic bile acid treatment by increasing the hydrophilicity of the bile acid pool. These results identify an unexpected role for SHP in hepatotoxicity and suggest new approaches to modulating effects of chronically elevated bile acids in cholestasis.     

Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a pivotal role in fatty acid β‑oxidation in heart, adipose tissue and skeletal muscle. However, key functions of ACSL1 in the liver remain largely unknown. We investigated acute effects of hepatic ACSL1 deficiency on lipid metabolism in adult mice under hyperlipidemic and normolipidemic conditions. We knocked down hepatic ACSL1 expression using adenovirus expressing a ACSL1 shRNA (Ad-shAcsl1) in mice fed a high-fat diet or a normal chow diet. Hepatic ACSL1 depletion generated a hypercholesterolemic phenotype in mice fed both diets with marked elevations of total cholesterol, LDL-cholesterol and free cholesterol in circulation and accumulations of cholesterol in the liver. Furthermore, SREBP2 pathway in ACSL1 depleted livers was severely repressed with a 50% reduction of LDL receptor protein levels. In contrast to the dysregulated cholesterol metabolism, serum triglycerides, free fatty acid and phospholipid levels were unaffected. Mechanistic investigations of genome-wide gene expression profiling and pathway analysis revealed that ACSL1 depletion repressed expressions of several key enzymes for bile acid biosynthesis, consequently leading to reduced liver bile acid levels and altered bile acid compositions. These results are the first demonstration of a requisite role of ACSL1 in bile acid biosynthetic pathway in liver tissue. Furthermore, we discovered that Acsl1 is a novel molecular target of the bile acid-activated farnesoid X receptor (FXR). Activation of FXR by agonist obeticholic acid repressed the expression of ACSL1 protein and mRNA in the liver of FXR wild-type mice but not in FXR knockout mice.     

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7alpha-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7alpha-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7alpha-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.     

Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding

Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7alpha-hydroxylase (Cyp7a1(-/-)) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1(-/-) males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1(+/+) animals. Although bile acid pool size contracted markedly in all the Cyp7a1(-/-) mice, the female Cyp7a1(-/-) mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1(-/-) males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1(+/+) males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1(-/-) mice by either of these manipulations. In the Cyp7a1(-/-) mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.     

Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7alpha-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12alpha-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.     

Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression

Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.