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

Metabolic Effects of a Grape Seed Procyanidin Extract on Risk Factors of Cardiovascular Disease

Bile acid (BA) recirculation and synthesis are tightly regulated via communication along the gut-liver axis and assist in the regulation of triglyceride (TG) and cholesterol homeostasis. Serum TGs and cholesterol are considered to be treatable risk factors for cardiovascular disease, which is the leading cause of death both globally and in the United States. While pharmaceuticals are common treatment strategies, nearly one-third of the population use complementary and alternative (CAM) therapy alone or in conjunction with medications, consequently it is important that we understand the mechanisms by which CAM treatments function at the molecular level. It was previously demonstrated that one such CAM therapy, namely a grape seed procyanidin extract (GSPE), reduces serum TGs via the farnesoid X receptor (Fxr). GSPE treatment also induces the expression of hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate limiting enzyme for de novo BA synthesis. Herein, we demonstrate that both gene and protein expression of Cyp7a1 is increased due to the fact that GSPE selectively regulates intestinal Fxr target genes involved in BA uptake and transport. Apical sodium dependent bile acid transporter (Asbt) expression is decreased with a concomitant reduction in fibroblast growth factor 15 (Fgf15), leading to a lack of repression on hepatic Cyp7a1. The subsequent 47% decrease in serum BAs and 69% increase in fecal BA excretion results in a significant reduction in serum TG and cholesterol. These Fxr dependent effects are lost in Fxr-/- mice, clearly demonstrating the critical role of this nuclear receptor. In a subsequent study we confirm that GSPE represses Asbt expression, while the BA sequestrant cholestyramine (CHY) induces expression. Treatment with either GSPE or CHY increases expression of Cyp7a1, with co-administration augmenting the increase. In the liver, GSPE and CHY independently induce expression of genes regulating cholesterol and lipid synthesis; however, when combined the expression of cholesterogenic and lipogenic genes induced by CHY is attenuated. Taken together these data indicate that GSPE has the potential for use either alone or as a complementary therapy in the treatment of hypertriglyceridemia and hypercholesterolemia. These findings, combined with the ability of low molecular weight procyanidins (LMW-PCNs) to modify intracellular proteins and signaling pathways led us to optimize a protocol for isolating LMW-PCNs from the seeds of grapes grown at the University of Nevada, Reno (UNR) vineyard. An ethyl acetate based extraction process utilizing whole seeds was found to be both time and cost effective, while preserving the anti-oxidant properties of the procyanidin-rich extract. This protocol will provide the basis for further extractions in order to conduct in vitro and in vivo testing, potentially allowing for the development of a value added product from the UNR vineyards

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

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

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 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

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

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

Average weekly mouse weight (g) during dietary intervention.

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