Cholesterol accumulation in the livers of <i>Sik3</i>^−/− mice after feeding with a high-cholesterol diet.

<p>(A) Male mice were fed a 2% cholesterol diet for 4 months (12–30 weeks) and then sacrificed (n = 5). (B) HE staining of the liver (sets at the <i>upper</i> and <i>lower left</i>). The arrows indicate eosin-negative foci which with autofluorescence (<i>lower right</i>: red, and nuclei are blue (DAPI)). The magnification is the same in each set. (C) Cholesterol and TG levels in the liver and serum were measured (n = 5). *** indicates <i>p</i><0.001. Means and SEM are shown. (D) FPLC analysis of serum lipids. (E) Serum levels of alanine aminotransferase (ALT) were monitored at the indicated time points. * and ** indicate <i>p</i><0.05 and <i>p</i><0.01, respectively. (F) Quantitative polymerase chain reaction analysis of inflammatory molecules in the liver.</p

<i>Sik3</i>^−/− mice are less tolerant to a cholic acid (CA)-containing diet.

<p>(A) Mice (n = 6, but <i>Sik3</i>^−/− mouse died before 1 month) were fed a diet supplemented with 0.25% cholic acid for 1 month (12–16 weeks) and then sacrificed. (B) HE staining of the liver (<i>left</i>), BSEP staining (<i>right</i>: BSEP is green and nuclei are blue (DAPI)). The magnification is the same in each set. (C) Photographs of gallbladders (scale, 1 mm). (D) The color of bile juice and bile sand in the gallbladder. (E) HE staining of the gallbladder. The magnification is the same in both panels. (F) The levels of bile acid (BA), cholesterol (Chol), and phospholipids (PL) in bile juice from the gallbladder were measured. * and ** indicate <i>p</i><0.05 and <0.01, respectively. Means and SEM are shown. (G) Serum BA and alanine aminotransferase (ALT) levels were monitored at the indicated periods. All ALT data points are <i>p</i><0.001, except day 0. (H) Serum alkaline phosphatase (ALP) and total bilirubin (T-Bil) levels were measured. (I) Cholesterol and TG levels in the liver and serum were measured. *** indicates <i>p</i><0.001. (J) FPLC analysis of serum lipids.</p

Gene expression profile in the liver.

<p>(A) One-year-old male mice (n = 5) were fasted for 4 h, and the liver mRNA levels were measured using quantitative polymerase chain reaction (qPCR). Red and blue indicate the up- and down-regulated genes in <i>Sik3</i>^−/− mice, respectively. +, fold increase; -, fold decrease. The threshold is set at <i>p = </i>0.1. The values marked with an asterisk (*) were obtained using PCR-array kits (n = 3). The abbreviations for the genes and the PCR primers used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037803#pone.0037803.s007" target="_blank">Table S2</a>. Mit, mitochondria; TCA, tricarboxylic acid cycle. (B) Intracellular signaling molecules and their activation status in the liver were examined by western blot analysis. (C) Immunohistochemical analysis of SIK3 substrates (CRTC2 and HDAC5) in the liver.</p

<i>Sik3</i>^−/− mice are lean, hypolipidemic, and hypoglycemic.

<p>(A) C57BL/6 mice (male: n = 4) were fed various diets (HF, high fat; HS, high sucrose; HChol, high cholesterol) for 2 weeks, and liver mRNA was examined by quantitative PCR. *, **, and *** indicate <i>p</i><0.05, <0.01, and <0.001, respectively. Means and SEM are shown. (B) The body weight of male mice (n = 6) was monitored. All data points show <i>p</i><0.001. (C) One-year-old male mice (n = 5) were sacrificed (scale: 1 mm), and the indicated tissues were weighed (D). (E) Histology of liver and mesenteric fatty tissue is shown. Each magnification is the same. (F) Cholesterol (Chol) and triglycerides (TG) in the liver and serum were measured (n = 5). (G) Serum cholesterol and TG were separated using FPLC. (H) The food consumption of each group (n = 12). (I) Rectal temperature (n = 12). (J) Oxygen consumption (VO₂, voluntarily O₂ consumption) and (K) average respiratory quotient (RQ) during the day and night (n = 5). (L) Mice (n = 5) were fasted and their blood glucose levels were monitored at the indicated time points. All data points show <i>p</i><0.001. (M) After 4-h fasting, the serum levels of insulin, leptin, free fatty acid (FFA), and ß-hydroxybutyrate were measured. (N) After 4-h fasting, glucose (1.5 g/kg) was intraperitoneally injected (GTT, glucose tolerance test) and blood glucose levels were monitored (n = 5). (O) After 24-h fasting, lactate (1.5 g/kg) was injected intraperitoneally (LTT, lactate tolerance test; n = 5).</p

<i>Sik3</i>^−/− mice are less tolerant to a cholesterol-containing high-fat diet.

<p>(A) Male mice were fed a high-fat and high-sucrose diet supplemented with 2% cholesterol (HF/HS/HChol) for 4 months (12–30 weeks) and then sacrificed (n = 6). (B) HE staining of the liver (sets at the <i>upper</i> and <i>lower left</i>), BSEP-staining (<i>lower right</i>: BSEP is green and nuclei are blue (DAPI)). The magnification is the same in each set. (C) Cholesterol and TG levels in the liver and serum were measured (n = 6). *** indicates <i>p</i><0.001. Means and SEM are shown. (D) FPLC analysis of serum lipids. (E) Serum levels of alanine aminotransferase (ALT) were monitored at the indicated time points. ** indicates <i>p</i><0.01. (F) Quantitative polymerase chain reaction analysis of inflammatory molecules (tumor necrosis factor-α and STAT3) in the liver.</p

Excess bile acids in the serum and liver of <i>Sik3</i>^−/− mice.

<p>(A) The serum levels of ALT, alkaline phosphatase, and bile acids are shown. The age at serum collection of the mice fed a chow diet (n = 4–6) are shown, while those of mice fed an HF, HChol, or HF/HS/HChol diet (n = 5–6) was 30 weeks (feeding 12–30 weeks). @ in the ALT panel indicates the maximum values for the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037803#pone-0037803-g004" target="_blank">Figure 4E</a>. (B) Bile acids were extracted from the liver (n = 3) and normalized by liver weigh. (C) Gain of body weight after feeding mice with a high cholic acid (CA)-containing diet. Wild type (n = 6) and <i>Sik3</i>^−/− mice (n = 5) were fed a diet supplemented with 0.25% CA for 1 month. For the control group, wild-type mice (n = 9) were fed with a chow diet.</p