Inhibition of Intestinal Bile Acid Transporter Slc10a2 Improves Triglyceride Metabolism and Normalizes Elevated Plasma Glucose Levels in Mice

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2−/− mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2 - Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes

Studies on the hormonal regulation of bile acid synthesis

The maintenance of a normal turnover of cholesterol is of vital importance, and disturbances of cholesterol metabolism may result in several important disease conditions. The major route for the elimination of cholesterol from the body is by hepatic secretion of cholesterol and bile acids (BAs) into the bile for subsequent fecal excretion. A better understanding of how hepatic cholesterol metabolism and BA synthesis are regulated is therefore of fundamental clinical importance, particularly for the prevention and treatment of cardiovascular disease. In the current thesis, the roles of known and newly recognized hormones in the regulation of BA synthesis were studied with the aim to broaden our understanding of how extrahepatic structures regulate BA synthesis in the liver. In normal humans, circulating levels of the intestinal fibroblast growth factor 19 (FGF19) were related to the amount of BAs absorbed from the intestine. The results support the concept that intestinal release of FGF19 signals to the liver suppressing BA synthesis. Thus, in addition to the liver - which harbors the full machinery for regulation of BA synthesis - the transintestinal flux of BAs is one important factor in this regulation. In mice, abrogation of the BA enterohepatic circulation by targeted deletion or pharmacological inhibition of the intestinal BA transporter ASBT reduces triglycerides in plasma and in the liver, concomitantly with a reduced hepatic triglyceride synthesis, Sucrose feeding results in an increased intestinal expression of the mouse orthologue of FGF19, FGF15, which can at least partly account for the reduced BA synthesis observed when this diet is fed. The powerful hepatic metabolic regulator FGF21 is induced in ASBT deficient mice, and is strongly upregulated by dietary sucrose. Specific inhibition of ASBT in leptin-deficient ob/ob mice reduces plasma triglyceride and glucose levels concomitantly with increased hepatic FGF21 expression. The ob/ob mice display reduced levels of hepatic LDL and HDL receptors, as well as of the rate-limiting enzyme of BA synthesis, Cyp7a1. These findings may in part explain the elevated plasma (particularly HDL) cholesterol levels in these animals. The Cyp7a1 response to dietary cholesterol is attenuated in ob/ob mice, and - in contrast to wt animals - the plasma cholesterol levels are increased. The HDL receptor SR-BI is positively regulated by leptin treatment of ob/ob mice, whereas Cyp7a1 is not. Selective stimulation of hepatic thyroid hormone receptor â with the drug GC-1 decreases plasma cholesterol and triglycerides dosedependently, and stimulates hepatic SR-BI and Cyp7a1 in normal mice. GC-1 also reduces elevated levels of plasma cholesterol in animals challenged with cholesterol and BAs. This indicates an important role for TRâ in reverse cholesterol transport, which might be useful in the treatment and prevention of atherosclerosis. It is concluded that hepatic BA synthesis in man is in part controlled from the intestine via FGF19, a novel pleiotropic metabolic regulator. Plasma glucose and triglycerides can be reduced by specific stimulation of BA synthesis. Thus, modulation of BA synthesis is a promising approach for the metabolic control of lipid and glucose metabolism which may be important in our attempts to treat and prevent cardiovascular disease

The PPAR pan-agonist tetradecylthioacetic acid promotes redistribution of plasma cholesterol towards large HDL

Tetradecylthioacetic acid (TTA) is a synthetic fatty acid with a sulfur substitution in the β-position. This modification renders TTA unable to undergo complete β-oxidation and increases its biological activity, including activation of peroxisome proliferator activated receptors (PPARs) with preference for PPARα. This study investigated the effects of TTA on lipid and lipoprotein metabolism in the intestine and liver of mice fed a high fat diet (HFD). Mice receiving HFD supplemented with 0.75% (w/w) TTA had significantly lower body weights compared to mice fed the diet without TTA. Plasma triacylglycerol (TAG) was reduced 3-fold with TTA treatment, concurrent with increase in liver TAG. Total cholesterol was unchanged in plasma and liver. However, TTA promoted a shift in the plasma lipoprotein fractions with an increase in larger HDL particles. Histological analysis of the small intestine revealed a reduced size of lipid droplets in enterocytes of TTA treated mice, accompanied by increased mRNA expression of fatty acid transporter genes. Expression of the cholesterol efflux pump Abca1 was induced in the small intestine, but not in the liver. Scd1 displayed markedly increased mRNA and protein expression in the intestine of the TTA group. It is concluded that TTA treatment of HFD fed mice leads to increased expression of genes involved in uptake and transport of fatty acids and HDL cholesterol in the small intestine with concomitant changes in the plasma profile of smaller lipoproteins

Disruption of the ileal BA transporter gene Slc10a2

<p><b>activates enzymes in BA synthesis.</b> (A) mRNA levels of cholesterol 7α-hydroxylase (Cyp7a1) in livers of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> animals. (B) Cyp7a1 enzymatic activity in pooled microsomal samples from livers of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> animals. Results shown are mean of two analyses. (C) Protein levels of Cyp7a1 in livers of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice determined in pooled microsomal samples by immunoblot using a Cyp7a1-specific antibody. 20 µg and 40 µg of protein were used per sample, respectively. The results are representative of three separate immunoblots. Note the loading order of the samples. (<b>D</b>) Serum levels of the Cyp7a1 activity marker 7α-hydroxy-4-cholesten-3-one (C4) were assayed as an indirect measure of Cyp7a1 enzymatic activity in pooled serum samples of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> animals. Data show mean of two separate measurements. (<b>E</b>) hepatic mRNA levels of 12α-hydroxylase (Cyp8b1), and (<b>F</b>) hepatic small heterodimer partner (Shp) mRNA levels of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice. mRNA levels for the wt mice were normalized to 1. Data are expressed as mean ± standard error (SEM) for the mRNA analysis. wt littermates (n = 10), <i>Slc10a2+/−</i> (n = 9) and <i>Slc10a2−/−</i> (n = 10) for A-F. Significances of differences between groups was tested by Dunnetts test; a p-value < <0.01 is denoted **.</p

Inhibition of Slc10a2 reduces mRNA levels of enzymes in fatty acid and glucose metabolism.

<p>Ob/ob mice were treated with the specific Slc10a2 protein inhibitor, AZD7806 or control vehicle (Controls) (see experimental procedures). (<b>A</b>) Hepatic mRNA levels of Srebp1c, and its target genes acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd1). (<b>B</b>) Hepatic mRNA levels of glucokinase (Gk), pyruvate kinase (Lpk), glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pepck) mRNA levels of the control vehicle treated animals are normalized to 1. Data are represented as mean ± standard error (SEM). Significances of differences between groups was tested by Student’s t test, a p-value <0.05 is denoted *.</p

Ablation of the Slc10a2 gene lowers the levels of plasma s TGs but not cholesterol.

<p>(A) Total plasma cholesterol and TGs from wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice. (B) Plasma profiles of cholesterol and TGs (C) were analysed from wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> animals by fast protein liquid chromatography (FPLC). Lines represent means of all individuals from each group, respectively. Lipoprotein fractions are indicated, n = 9–10. (D) Hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase mRNA levels and enzymatic activity of wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice. HMG-CoA reductase enzymatic activity was analyzed from pooled hepatic microsomal samples and represents mean of two measurements. (n = 9–10). (E) Hepatic mRNA levels of the sterol transporters ATP-binding cassette sub-family G member 5 (Abcg5) and Abcg8 in wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice. (F) Hepatic mRNA levels of sterol regulatory element-binding protein 1c (Srebp1c) and Srebp2, in wt, <i>Slc10a2+/−</i> and <i>Slc10a2−/−</i> mice. mRNA levels in wt mice were normalized to 1. Data are expressed as mean ± standard error (SEM) for the mRNA and total plasma cholesterol and TGs analyzes. wt littermates (n = 10), <i>Slc10a2</i>+/− (n = 9) and <i>Slc10a2</i>−/− (n = 10) for A-F. Significances of differences between groups was tested by Dunnetts test, a p-value <0.05 is denoted *.</p

Inhibition of the Slc10a2 protein improves plasma glucose and TGs in ob/ob mice.

<p>Ob/ob mice were treated with a specific Slc10a2 protein inhibitor, (AZD7806), or control vehicle (vehicle) (see experimental procedures). (<b>A</b>) fasting levels of blood glucose and plasma insulin. (<b>B</b>) plasma total TGs and cholesterol. (<b>C</b>) Hepatic mRNA levels of fibroblast growth factor 21 (FGF21) and cholesterol 7α-hydroxylase (Cyp7a1). (<b>D</b>) Liver cholesterol and TGs. (<b>E</b>) Distal ileum mRNA levels of FGF15, small heterodimer partner (Shp), Ileal BA binding protein (Ibabp) and farnesoid X receptor (Fxr) from ob/ob animals treated with a Slc10a2 protein inhibitor. mRNA levels of the control vehicle treated animals are normalized to 1. Data are represented as mean ± standard error (SEM). Significances of differences between groups was tested by Student’s t test, a p-value <0.05 is denoted *. P<0.01 is denoted **.</p

<i>Slc10a2</i>−/− mice display lower hepatic TGs and cholesterol than wild type mice.

<p>(A) Liver TGs and cholesterol content were analyzed from a total of four groups; wt and <i>Slc10a2−/−</i> mice fed either standard mouse chow or a sucrose-rich (SR) diet (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037787#s2" target="_blank">Materials and Methods</a>) for two weeks. (B) Hepatic mRNA levels of ATP-citrate lyase (Acl), acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd1), enzymes involved in fatty acid synthesis from wt or <i>Slc10a2−/−</i> animals, as in (A). (C) Sterol regulatory element-binding protein 1 (Srebp1) immunoblots were performed on pooled liver cytoplasmic and nuclear protein preparations, respectively, from wt or <i>Slc10a2−/−</i> mice fed a regular chow or a SR diet using an antibody specific for the N’-terminus of Srebp1. An antibody against lamin was used as nuclear loading control. The blot is representative of three separate immunoblots. Hepatic mRNA expression levels of (D), glucokinase (Gk); pyruvate kinase (Lpk); (E) glucose-6-phosphate dehydrogenase (G6pdh); malic enzyme (Me). mRNA values in the wt group fed regular chow were normalized to 1. (F) Fibroblast growth factor 21, FGF(21). mRNA values in the wt group on regular chow were normalized to 1. Data are expressed as mean ± standard error (SEM) for the mRNA, hepatic cholesterol and TG analysis. wt (n = 6), <i>Slc10a2−/−</i> (n = 5), wt + SR (n = 6) and <i>Slc10a2+/−</i> +SR (n = 6) for A-D. Significances of differences between groups were tested by Student’s t test, a p-value <0.05 is denoted *. p<0.01 is denoted **.</p

A sucrose-rich (SR) diet normalizes elevated CYP7A1 levels and induces the expression of the metabolic regulators FGF15 and FGF21 in <i>Slc10a2</i>−/− mice.

<p>Livers from wt and <i>Slc10a2</i>−/− mice fed regular chow or a SR diet were analysed for A), mRNA levels of Cyp7a1; B), Serum C4 levels, a marker of Cyp7a1 enzymatic activity; C), hepatic mRNA levels of SHP; and for D), HMGCoA synthase, HMGCoA reductase and SREBP2 mRNA. In panel E, FGF15, SHP and IBABP mRNA levels were determined in samples from distal ileum of wt and <i>Slc10a2</i>−/− mice fed regular diet or the SR diet. Data are expressed as mean ± standard error (SEM), n = 5–6. mRNA expression for the wt group on regular chow is normalized to 1. Significances of differences between groups were tested by Student’s t test, a p-value <0.05 is denoted *. p<0.01 is denoted **.</p