PMC

Liver microarray analysis. Male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. RNA from individual rats was pooled within experimental groups, and the pooled samples were analyzed by Affymetrix arrays. A, data were analyzed, and significantly altered KEGG pathways (p < 0.05) are highlighted in darker blue, and pathways in lighter blue tended to be altered (p < 0.1). B, heat map of selected genes involved in liver fatty acid import and oxidation regulated by SPH treatment.

Induction of skeletal muscle Ucp3 gene expression is abolished by pharmacological removal of bile acids. A–C, male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A, in skeletal muscle, SPH-fed rats exhibited reduced expression of Pparα and its target genes Acox1 and Mcad. B, muscle gene expression of Pparβ/δ and its target gene Adrp and Angptl4 was enhanced by SPH treatment. C, expression of genes related to fatty acid oxidation Cpt-1b and Ucp2 and -3 was enhanced in the SPH-treated rats. D, three groups of rats (n = 6) were pair-fed the same diets, plus the SPH diet with 0.5 weight % c'am for 46 days. Pharmacological removal of bile acids by 0.5 weight % cholestyramine completely abolished Ucp3 induction. mRNA levels are normalized to Gtf2b and relative to the expression in the casein-fed rats. Data are presented as mean ± S.E. Significant differences from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01.

Reduction in fed-state plasma glucose is attenuated by pharmacological removal of bile acids. A–D, male Wistar rats (n = 6) were fed high fat diets (45 kcal% fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A, fed-state plasma insulin level was decreased, and glucagon levels tended to be lower (p = 0.06) in the SPH-treated rats. B and C, liver gene expression of the rate-limiting gluconeogenic enzyme Pck1 was decreased, and liver glycogen concentration was increased by SPH feeding. D, fed state but not fasted plasma glucose level was lower in the SPH-fed rats. E, three groups of rats (n = 6) were pair-fed the same diets, plus the SPH diet with 0.5 weight % c'am for 46 days. Pharmacological removal of bile acids by 0.5 weight % cholestyramine attenuated the glucose lowering effect of SPH. Pck1 mRNA levels are normalized to Gtf2b. Data are presented as means ± S.E. Significant difference from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01.

Elevated plasma bile acid concentration is an underlying factor for the increased energy expenditure and decreased adiposity elicited by SPH feeding. A–F, male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A and B, SPH-fed rats had elevated plasma BAs, whereas total liver BAs were unchanged. C–E, SPH-fed rats showed reduced body weight gain. Energy efficiency, calculated as body weight gain per energy intake, and white adipose tissue masses were reduced by SPH treatment. F, energy digestibility was equal in SPH- and casein-fed rats. G–M, three groups of rats (n = 6) were pair-fed the SPH diet, the casein diet, and the SPH diet with 0.5 weight % cholestyramine. G–I, inclusion of cholestyramine to the SPH diet attenuated the increase in plasma BA concentrations, without modulating liver total BAs or growth. J, inclusion of cholestyramine attenuated the reduction in body fat mass determined by dual x-ray absorptiometry. K and L, three groups of rats (n = 6) were pair-fed the SPH diet, the casein diet, and the SPH diet with 0.5 weight % c'am, and energy expenditure was calculated by indirect calorimetry. Cholestyramine treatment attenuated the increase in O₂ consumption, CO₂ elimination, and heat production. Data are presented as mean ± S.E. Significant differences from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01.

Bile acids induce fat oxidation in brown and white adipose tissues. A–G, male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A, SPH treatment increased ex vivo palmitate-CoA oxidation capacity in iBAT. B–D, mRNA levels of Ucp1 and type 2 iodothyronine deiodinase (Dio2) were increased by SPH treatment, whereas the level of Pgc-1α mRNA was not significantly increased. E–G, mRNA levels of Ucp1, Dio2, and Pgc-1α in white adipose tissue depots of subcutaneous (inguinal, iWAT) and abdominal (epididymal, eWAT, and mesenteric, MeWAT, respectively) fat. H–K, three groups of rats were pair-fed the same diets, plus the SPH diet with 0.5 weight % c'am for 46 days. The reduction in abdominal (mesenteric + epididymal + perirenal and retroperitoneal) fat mass was attenuated by inclusion of cholestyramine in the SPH diet. I and J, epididymal WAT ex vivo palmitate-CoA oxidation capacity and mRNA levels. K, adipocyte cell size in eWAT was increased by inclusion of cholestyramine to the SPH diet. L, increased in vitro palmitate-CoA oxidation capacity in adipocytes of eWAT origin after 24 h of preincubation with taurocholic acid. mRNA levels are normalized to general transcription factor IIB (Gtf2b). Data are presented as mean ± S.E. (A–J, n = 4–6; L, n = 3). Significant differences from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01.

Repressed de novo bile acid synthesis and indications of increased biliary BA secretion. A–H, male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A, plasma alanine aminotransferase was increased in the SPH-fed rats but the elevation did not indicate hepatocellular damage. B, liver gene expression of Ucp2, a marker of oxidative stress and linked to bile obstruction, was borderline (p = 0.053) reduced by SPH treatment. C, hepatic concentrations of cholesterol and sterol esters were markedly decreased in the SPH-treated rats. D, elevated plasma bile acid concentrations in SPH-treated rats were accompanied with lower liver expression of genes involved in de novo bile acid synthesis, Cyp7a1, and in bile acid conjugation, Baat. E, liver expression of genes encoding for apical transporters involved in bile generation Abcb4 (also called Mdr2) was strongly induced, whereas expression of the Abcb11 (also called Bsep) tended (p = 0.09) to be higher. Expressions of Abcc2 (also called Mrp2) and Abcb1b (also called Mdr1) were not altered by SPH treatment. F, liver gene expressions of genes involved in GSH synthesis, glutamate-cysteine ligase, catalytic unit (Gclc), and modulator unit (Gclm) were unaltered, and glutathione synthase (Gss) was induced by SPH treatment. G, liver cytosolic GSH was increased in the SPH-treated rats. H, no significant difference was observed in 5 days fecal bile acid excretion. mRNA levels are normalized to Gtf2b. Data are presented as mean ± S.E. Significant difference from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01.

Reduced fed-state plasma TAG concentrations by elevated plasma bile acid levels. A–L, male Wistar rats (n = 6) were fed high fat diets (45 kcal % fat) ad libitum for 46 days with either SPH or casein as the sole protein source. A–D, in the liver, SPH-fed rats exhibited reduced expression of Pparα and its target genes Cpt-1a and Acox1 but increased expression of Pparβ/δ and its target gene Adrp. E–G, ex vivo liver mitochondrial CPT-1 capacity was enhanced, and liver mRNA level of the CPT-1 regulator Acc2 was reduced without significantly altering liver mitochondrial CPT-1 capacity in the presence of the CPT-1 inhibitor malonyl-CoA (5 μm). H, SPH-fed rats had elevated fasting plasma hydroxybutyrate. I–L, liver TAG and fed-state plasma TAG concentrations were reduced, whereas liver Vldlr gene expression was increased in the SPH-treated rats. M–O, two groups of rats (n = 5) were pair-fed the SPH and the casein diets for 25 days. M, plasma metabolomic analysis by NMR revealed that VLDL concentration was significantly reduced by SPH treatment. Peaks on the positive y axis scale are significantly elevated by SPH feeding, and peaks on the negative y axis are significantly higher in casein-fed rats. R2 is the full cross-validated regression coefficient. N and O, no significant alterations were found in plasma cholesterol levels measured by biochemical assay. P–S, three groups of rats (n = 6) were pair-fed the SPH diet, the casein diet, and the SPH-diet with 0.5 weight % c'am for 46 days. Pharmacological removal of bile acids by 0.5 weight % cholestyramine attenuated the lipid-lowering effects of SPH in liver, and fed-state plasma TAG. mRNA levels are normalized to Gtf2b. Data are presented as mean ± S.E. Significant differences from casein-fed rats are denoted as follows: *, p < 0.05; **, p < 0.01. Significant difference between SPH-fed and SPH + cholestyramine-fed rats is denoted by ##, p < 0.01.