Inhibition of PCSK9 prevents and alleviates cholesterol gallstones through PPARα-mediated CYP7A1 activation.

BACKGROUND & AIMS: Dysregulated cholesterol metabolism is the major factor responsible for cholesterol gallstones (CGS). Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol homeostasis and its inhibitors secure approval for treating various cholesterol metabolic disorders such as hypercholesterolemia and cardiovascular diseases, but its role in CGS remains unclear. Our study aims to clarify mechanisms by which PCSK9 promotes CGS formation and explore the application of the PCSK9 inhibitor, alirocumab, in preventing and treating CGS. APPROACH & RESULTS: The expressions of PCSK9 were notably increased in CGS patients' serum, bile, and liver tissues compared to those without gallstones. Moreover, among CGS patients, hepatic PCSK9 was positively correlated with hepatic cholesterol and negatively correlated with hepatic bile acids (BAs), suggesting PCSK9 was involved in disrupted hepatic cholesterol metabolism related to CGS. Mechanistically, in vitro experiments demonstrated that inhibition of PCSK9 enhanced nuclear expression of PPARα by diminishing its lysosomal degradation and subsequently activated CYP7A1 transcription. Finally, inhibition of PCSK9 prevented CGS formation and dissolved the existing stones in CGS mice by elevating the conversion of cholesterol into BAs through PPARα-mediated CYP7A1 activation. Additionally, serum PCSK9 level may function as a prognostic signature to evaluate the therapeutic efficacy of PCSK9 inhibitors. CONCLUSIONS: Inhibition of PCSK9 exerts preventive and therapeutic effects on CGS by activating PPARα-mediated CYP7A1 expression and facilitating the conversion of cholesterol into BAs, which highlights the potential of PCSK9 inhibition as a promising candidate for preventing and treating CGS in clinical applications. IMPACT AND IMPLICATIONS: PCSK9 plays a pivotal role in cholesterol metabolism and its inhibitors are approved for clinical use in cardiovascular diseases. Our study observes inhibition of PCSK9 prevents and dissolves CGS by activating PPARα-mediated CYP7A1 expression and facilitating the conversion of cholesterol into BAs. Mechanistically, PCSK9 inhibition enhanced the nuclear expression of PPARα by diminishing its lysosomal degradation and subsequently activated CYP7A1 transcription. Our study sheds light on the new function and mechanism of PCSK9 in CGS, providing a novel preventive and therapeutic target with potential clinical applications.

Resistance to Cholesterol Gallstone Disease: Hepatic Cholesterol Metabolism.

Cholesterol gallstone disease (CGD) is one of the most common digestive diseases, and it is closely associated with hepatic cholesterol metabolism. Cholesterol gallstones may be caused by abnormal hepatic cholesterol metabolism, such as excessive cholesterol biosynthesis within the liver, interfering with the uptake or export of cholesterol in the liver, and abnormal hepatic cholesterol esterification. In this review, we begin with a brief overview of the clinical diagnosis and treatment of gallstone disease (GSD). Then, we briefly describe the major processes of hepatic cholesterol metabolism and summarize the key molecular expression changes of hepatic cholesterol metabolism in patients with gallstones. We review and analyze the recent advances in elucidating the relationships between these key molecules and CGD, and some targets significantly impacting on CGD via hepatic cholesterol metabolism are also listed. We also provide a significant discussion on the relationship between CGD and nonalcoholic fatty liver disease (NAFLD). Finally, the new discoveries of some therapeutic strategies associated with hepatic cholesterol metabolism to prevent and treat CGD are summarized.

Maternal high-calorie diet feeding programs hepatic cholesterol metabolism and Abca1 promoter methylation in the early life of offspring.

Maternal high-calorie diet feeding can dramatically increase the susceptibility of metabolic diseases in offspring. However, whether maternal high-calorie diet feeding can program hepatic cholesterol metabolism in the early life of offspring is less understood, and the epigenetic mechanisms underlying this intergenerational effect, especially during the early life of offspring, are unknown. Female C57BL/6J mice were randomly assigned to a high-calorie diet or control diet before and during gestation, and lactation. Lipid metabolism was evaluated in male offspring at weaning. Gene expressions and quantitative methylation levels of key genes associated with hepatic cholesterol metabolism were further evaluated in offspring at weaning age. We found that maternal high-calorie diet feeding resulted in higher body weight, hypercholesterolemia, elevated total cholesterol in liver homogenates, and fat deposits in the liver in offspring at weaning. For key genes that regulate cholesterol metabolism in liver, we showed lower Hmgcr and Ldlr, and higher Abca1 mRNA and protein expressions in offspring from dams fed with high-calorie diet at weaning age. We further found that maternal high-calorie diet feeding significantly decreased Abca1 methylation level in offspring, with lower methylation levels of both CpG 11 and CpG 22 sites. Interestingly, we found that Abca1 methylation level was negatively associated with hepatic Abca1 mRNA expression in offspring from dams fed with high-calorie diet and controls. However, the expressions of key genes associated with hepatic cholesterol metabolism were not significant between fetuses of dams fed with high-calorie diet and control diet. In conclusion, our results indicate that maternal high-calorie diet feeding results in aberrant lipid metabolism, including hypercholesterolemia and fat deposits in the liver of offspring as early as weaning age. Furthermore, maternal high-calorie feeding can program hepatic cholesterol metabolism and Abca1 methylation in the early life of offspring.

Glucocorticoid programming mechanism for hypercholesterolemia in prenatal ethanol-exposed adult offspring rats.

Our previous studies showed that prenatal ethanol exposure (PEE) elevated blood total cholesterol (TCH) level in adult offspring rats. This study was aimed at elucidating the intrauterine programming mechanism of hypercholesterolemia in adult rats induced by PEE. Pregnant Wistar rats were intragastrically administered ethanol (4 mg/kg∙d) from gestational day (GD) 9 to 20. The offspring rats were euthanized at GD20 and postnatal week 24. Results showed that PEE decreased serum TCH and HDL-C levels (female and male) as well as LDL-C level (female only) in fetal rats but increased serum TCH level and the TCH/HDL-C and LDL-C/HDL-C ratios in adult rats. Furthermore, PEE elevated serum corticosterone levels but inhibited hepatic insulin-like growth factor 1 (IGF1) signaling pathway, cholesterol synthesis and output in fetal rats. The conversed changes were observed in adult rats. Moreover, histone acetylation (H3K9ac and H3K14ac) and expression of hepatic reverse cholesterol transport (RCT) related genes, scavenger receptor BI and low-density lipoprotein receptor were decreased before and after birth by PEE. In HepG2 cells, cortisol negatively regulated the IGF1 signaling pathway and cholesterol metabolic genes, but this inhibition of the cholesterol metabolic genes could be reversed by glucocorticoid receptor antagonist RU486, whereas exogenous IGF1 treatment only reversed the downregulation of RCT genes by cortisol. We confirmed a "two programming" mechanism for PEE-induced hypercholesterolemia in adult rats. The "first programming" was a glucocorticoid (GC)-induced persistent reduction of RCT genes by epigenetic modifications, and the "second programming" was the negative regulation of cholesterol synthesis and output by the GC-IGF1 axis.

Two intrauterine programming mechanisms of adult hypercholesterolemia induced by prenatal nicotine exposure in male offspring rats.

Epidemiologic studies showed that low birth weight is associated with high cholesterol and an increased risk of cardiovascular diseases in adulthood. This study aimed to elucidate the intrauterine programming mechanisms of adult hypercholesterolemia. The results showed that prenatal nicotine exposure (PNE) caused intrauterine growth retardation and hypercholesterolemia in male adult offspring rats. Hepatic cholesterol synthesis and output were deceased in utero but increased in adults; hepatic reverse cholesterol transport (RCT) persistently deceased before and after birth. Meanwhile, PNE elevated serum corticosterone level and decreased hepatic IGF1 pathway activity in male fetuses, whereas converse changes were observed in male adults. The chronic stress model and cortisol-treated HepG2 cells verified that excessive glucocorticoid (GC)-induced GC-IGF1 axis programming enhanced hepatic cholesterol synthesis and output. In addition, PNE decreased the expression of specific protein 1 and P300 enrichment and H3K27 acetylation at the promoter region of genes responsible for RCT both in fetal and adult, male livers and reduced expression of those genes, similar alterations were also confirmed in cortisol-treated HepG2 cells, suggesting that excessive GC-related programming induced continuous RCT reduction by epigenetic modification. Taken together, the "2-programming" approach discussed above may ultimately contribute to the development of hypercholesterolemia in male adult offspring.-Zhou, J., Zhu, C., Luo, H., Shen, L., Gong, J., Wu, Y., Magdalou, J., Chen, L., Guo, Y., Wang, H. Two intrauterine programming mechanisms of adult hypercholesterolemia induced by prenatal nicotine exposure in male offspring rats.

Perinatal maternal high-fat diet promotes alterations in hepatic lipid metabolism and resistance to the hypolipidemic effect of fish oil in adolescent rat offspring.

SCOPE: Maternal high-fat diet (HFD) promotes obesity and metabolic disturbances in offspring at weaning and adult life. We investigated metabolic consequences of maternal HFD in adolescent rat offspring and the potential benefic effects of fish oil (FO) (n-3 polyunsaturated fatty acid source). METHODS AND RESULTS: Female rats received isocaloric, standard diet (STD: 9% fat) or HFD (28.6%) before mating, and throughout pregnancy and lactation. After weaning, male offspring received standard diet and, from 25th to 45th day, received oral administration of soybean oil (SO) or FO. HFD offspring showed higher body weight and adiposity, which was not attenuated by FO. In STD offspring, FO reduced serum triglyceride and cholesterol, as expected, but not in HFD offspring. Liver of HFD offspring groups showed increased free cholesterol and FO-treated HFD group showed lower expression of Abcg8, suggesting decreased cholesterol biliary excretion. HFD offspring presented higher hepatic expression of lipogenic markers, Srebf1 mRNA and acetyl CoA carboxylase (ACC). Serum n-3 PUFA were decreased in FO-treated HFD compared to FO-treated STD offspring, which may explain the reduced hypolipidemic FO effect. CONCLUSION: Maternal HFD impaired the ability of FO to reduce adiposity and serum lipids in adolescent offspring, suggesting a potential predisposition to future development of metabolic disorders.

Modulation of Hepatic Protein Kinase Cß Expression in Metabolic Adaptation to a Lithogenic Diet.

BACKGROUND & AIMS: Dietary factors are likely an important determinant of gallstone development, and difficulty in adapting to lithogenic diets may predispose individuals to gallstone formation. Identification of the critical early diet-dependent metabolic markers of adaptability is urgently needed to prevent gallstone development. We focus on the interaction between diet and genes, and the resulting potential to influence gallstone risk by dietary modification. METHODS: Expression levels of hepatic protein kinase C (PKC) isoforms were determined in lithogenic diet-fed mice, and the relationship of hepatic cholesterol content and PKCß expression and the effect of hepatic PKCß overexpression on intracellular signaling pathways were analyzed. RESULTS: Lithogenic diet feeding resulted in a striking induction of hepatic PKCß and PKCδ mRNA and protein levels, which preceded the appearance of biliary cholesterol crystals. Unlike PKCß deficiency, global PKCδ deficiency did not influence lithogenic diet-induced gallstone formation. Interestingly, a deficiency of apolipoprotein E abrogated the diet-induced hepatic PKCß expression, whereas a deficiency of liver X receptor-α further potentiated the induction, suggesting a potential link between the degree of hepatic PKCß induction and the intracellular cholesterol content. Furthermore, our results suggest that PKCß is a physiologic repressor of ileum basal fibroblast growth factor 15 (FGF15) expression and activity of hepatic proto-oncogene serine/threonine-protein kinase Raf-1/mitogen-activated protein (MAP) kinase kinase/extracellular signal-regulated kinases 1/2 (Raf-1/MEK/ERK1/2) cascade proteins, and the complex interactions between these pathways may determine the degree of hepatic ERK1/2 activation, a potent suppressor of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase expression. We found that PKCß regulated Raf-1 activity by modulating the inhibitory Raf-1Ser259 phosphorylation. CONCLUSIONS: Our results demonstrate a novel interaction between the hepatic PKCß/Raf-1 regulatory axis and ileum PKCß/FGF15/ERK axis, which could modulate the bile lithogenecity of dietary lipids. The data presented are consistent with a two-pronged mechanism by which intestine and liver PKCß signaling converges on the liver ERK1/2 pathway to control the hepatic adaptive response to a lithogenic diet. Elucidating the impact and the underlying mechanism(s) of PKCß action could help us understand how different types of dietary fat modify the risk of gallstone formation, information that could help to identify novel targets for therapeutic approaches to combat this disease.

Hepatic Mttp deletion reverses gallstone susceptibility in L-Fabp knockout mice.

Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.