Dietary procyanidins selectively modulate intestinal farnesoid X receptor-regulated gene expression to alter enterohepatic bile acid recirculation: elucidation of a novel mechanism to reduce triglyceridemia

Scope: Understanding the molecular basis by which dietary procyanidins modulate triglyceride and cholesterol homeostasis has important implications for the use of natural products in the treatment and prevention of cardiovascular disease. Methods: To determine whether modulation of bile acid (BA) homeostasis contributes to the hypotriglyceridemic action of grape seed procyanidin extract (GSPE) we examined the effect on genes regulating BA absorption, transport and synthesis in vitro, in Caco-2 cells, and in vivo, in wild type (C57BL/6) and farnesoid x receptor knockout (Fxr −/− ) mice. Results: We provide novel evidence demonstrating that GSPE is a naturally occurring geneselective bile acid receptor modulator (BARM). Mechanistically, GSPE down-regulates genes involved in intestinal BA absorption and transport in an Fxr-dependent manner, resulting in decreased enterohepatic BA recirculation. This correlates with increased fecal BA output, decreased serum triglyceride and cholesterol levels, increased hepatic cholesterol 7␣-hydroxylase (Cyp7a1), and decreased intestinal fibroblast growth factor 15 (Fgf15) expression. GSPE also increased hepatic HmgCoA reductase (Hmgcr) and synthase (Hmgcs1) expression, while concomitantly decreasing sterol regulatory element-binding protein 1c (Srebp1c). Conclusion: GSPE selectively regulates intestinal Fxr-target gene expression in vivo, and modulation of BA absorption and transport is a critical regulatory point for the consequential hypotriglyceridemic effects of GSPE

Understanding the role of the intestine in the molecular hypotriglyceridemic actions of a grape seed procyanidin extract

Hypertriglyceridemia is a prevalent condition that is associated with cardiovascular disease. Grape seed procyanidin extract (GSPE), a natural compound rich in procyanidins, has recently been shown to reduce serum triglyceride (TG) levels in vivo in normolipidemic animals. This effect was shown to be mediated via farnesoid X receptor (FXR), a member of the nuclear hormone receptor (NHR) superfamily. NHRs are ligand-inducible, and in some cases ligand-independent, transcription factors that interact with DNA to regulate gene expression. Activation of FXR by its endogenous ligand, bile acids (BA), modulates TG and BA homeostasis via regulation of hepatic and intestinal gene expression. Activation of FXR in the liver by BAs increases the expression of small heterodimer partner (SHP), which then acts as a repressor to decrease hepatic expression of sterol response element binding protein 1c (SREBP1c), a key transcription factor regulating lipogenic gene expression, thereby lowering serum TG levels. Studies have shown that in the liver, GSPE acts as a co-agonist ligand for FXR resulting in enhancement of BA-bound FXR activation in vitro, and that the TG-lowering ability of GSPE is lost in vivo in both, FXR and SHP knockout mouse models. Recently, utilizing a FXRE-luciferase reporter mouse model, it was shown that the intestine has the highest bile acid-induced FXR signaling under physiological conditions. FXR activation in the intestine induces the expression of several FXR target-genes including intestinal bile acid-binding protein (IBABP), organic solute transporters (OST) α/β, and fibroblast growth factor (FGF) 15/19, while repressing the expression of the apical sodium dependent bile acid transporter (ASBT), which contributes to bile acid enterohepatic recirculation and bile acid homeostasis. The overall aim of this research was to further investigate the effects of GSPE to aid in the understanding of its molecular hypotriglyceridemic mode of action. Based on the fact that FXR is a bridge between the liver and the intestine to control bile acid levels and to regulate bile acid synthesis and enterohepatic flow, the first aim was to discern whether or not GSPE exerts any effects on intestinal FXR which could then contribute to the TG-lowering effect of GSPE. Therefore, the effects of GSPE on the regulation of known FXR target-genes in the intestine was investigated, to provide further insight into the inter-relationship between the intestine and the liver in the regulation of lipid homeostasis by GSPE. Studies have previously established the ability of GSPE to lower serum TG levels in a normolipidemic state, therefore, the second aim was to determine the potential of GSPE to lower serum TG levels in a hypertriglyceridemic state, and to identify the underlying molecular events. Our results indicate that in the intestine, GSPE may act as a gene-selective bile acid receptor modulator (BARM), rather than a bile acid-dependent co-agonist of FXR, as previously reported to occur in the liver. In the course of the studies conducted herein, GSPE treatment resulted in alterations in the expression of ileal FXR-target genes in different ways, i.e., GSPE acted as a FXR co-agonist thereby suppressing ASBT gene expression, while in contrast it acted a FXR modulator by decreasing CDCAinduced IBABP and OSTα/β mRNA expression. Therefore, these results indicate that GSPE may impair bile acid enterohepatic recirculation, which is achieved by decreasing the intestinal up-take of bile acids, as well as by decreasing the amount of BA that return to the liver via the portal circulation. Furthermore, GSPE also induced a rapid and transit increase in FGF19 expression in vitro in Caco2 cells. We hypothesize that the above-mentioned effects induced by GSPE on intestinal FXR-target gene expression may therefore be contributing to changes in overall body BA homeostasis and also its serum TG lowering ability. Additionally, our results show that GSPE treatment effectively reduces serum TG levels by 27% when assessed in a fructose-fed rat model representing a hypertriglyceridemic state. Consequently, GSPE may represent a promising natural compound for the treatment of hypertriglyceridemia and its’ related conditions

Grape Seed Procyanidins and Cholestyramine Differentially Alter Bile Acid and Cholesterol Homeostatic Gene Expression in Mouse Intestine and Liver

Bile acid (BA) sequestrants, lipid-lowering agents, may be prescribed as a monotherapy or combination therapy to reduce the risk of coronary artery disease. Over 33% of adults in the United States use complementary and alternative medicine strategies, and we recently reported that grape seed procyanidin extract (GSPE) reduces enterohepatic BA recirculation as a means to reduce serum triglyceride (TG) levels. The current study was therefore designed to assess the effects on BA, cholesterol and TG homeostatic gene expression following co-administration with GSPE and the BA sequestrant, cholestyramine (CHY). Eight-week old male C57BL/6 mice were treated for 4 weeks with either a control or 2% CHY-supplemented diet, after which, they were administered vehicle or GSPE for 14 hours. Liver and intestines were harvested and gene expression was analyzed. BA, cholesterol, non-esterified fatty acid and TG levels were also analyzed in serum and feces. Results reveal that GSPE treatment alone, and co-administration with CHY, regulates BA, cholesterol and TG metabolism differently than CHY administration alone. Notably, GSPE decreased intestinal apical sodium-dependent bile acid transporter (Asbt) gene expression, while CHY significantly induced expression. Administration with GSPE or CHY robustly induced hepatic BA biosynthetic gene expression, especially cholesterol 7 alpha-hydroxylase (Cyp7a1), compared to control, while co-administration further enhanced expression. Treatment with CHY induced both intestinal and hepatic cholesterologenic gene expression, while co-administration with GSPE attenuated the CHY-induced increase in the liver but not intestine. CHY also induced hepatic lipogenic gene expression, which was attenuated by co-administration with GSPE. Consequently, a 25% decrease in serum TG levels was observed in the CHY+GSPE group, compared to the CHY group. Collectively, this study presents novel evidence demonstrating that GSPE provides additive and complementary efficacy as a lipid-lowering combination therapy in conjunction with CHY by attenuating hepatic cholesterol synthesis, enhancing BA biosynthesis and decreasing lipogenesis, which warrants further investigation

Average weekly mouse weight (g) during dietary intervention.

<p>Average weekly mouse weight (g) during dietary intervention.</p

GSPE decreases the expression of intestinal apical cholesterol transporters, but not in combination with cholestyramine.

<p>Gene expression was analyzed (A) <i>Abcg5</i>, (B) <i>Abcg8</i>, and (C) <i>Npc1l1</i>. Statistical differences are shown as: *p≤0.05, ** p≤0.01.</p

Effects on intestinal cholesterol synthesis and transporter gene expression following treatments.

<p>Gene expression was analyzed for (A) <i>Srebf2</i>, (B) <i>Hmgcs1</i>, (C) <i>Hmgcr</i>, (D) <i>Acat2</i>, (E) <i>Mttp</i>, and (F) <i>Scarb1</i>. Statistical differences are shown as: *p≤0.05, **** p≤0.0001.</p

Hepatic cholesterol and lipogenic homeostatic gene expression following treatments.

<p>Gene expression was analyzed for (A) <i>Srebf2</i>, (B) <i>Hmgcs1</i>, (C) <i>Hmgcr</i>, (D) <i>Ldlr</i>, (E) <i>Srebf1c</i>, (F) <i>Acc1</i>, (G) <i>Fasn</i>, (H) <i>Scd1</i>, and (I) <i>ApoA5</i>. Statistical differences are shown as: *p≤0.05, ** p≤0.01, ***p≤0.001, **** p≤0.0001.</p

Expression of genes involved in basolateral intestinal cholesterol transport following treatments.

<p>Gene expression was analyzed for (A) <i>Abca1</i>, (B) <i>ApoA1</i>, and (C) <i>Ldlr</i>. Statistical differences are shown as: *p≤0.05, ** p≤0.01.</p

GSPE and cholestyramine induce the hepatic expression of genes regulating bile acid synthesis.

<p>Gene expression was analyzed for (A) <i>Cyp7a1</i>, (B) <i>Cyp8b1</i>, (C) <i>Cyp27a1</i>, and (D) <i>Cyp7b1</i>. Statistical differences are shown as: *p≤0.05, ** p≤0.01, ***p≤0.001, **** p≤0.0001.</p

Serum Biochemical analysis following treatments.

<p>Serum analysis was performed for (A) bile acids (BA), (B) cholesterol (CHOL), (C) triglyceride (TG), (D) non-esterified fatty acids (NEFA), (E) alanine aminotransferase (ALT), and (F) aspartate aminotransferase (AST). Normal upper and lower limits for ALT and AST are represented by the dashed lines in (E) and (F). Statistical differences are shown as: *p≤0.05, **p≤0.01, ***p≤0.001, **** p≤0.0001.</p