Hepatic <i>Inhbe</i> mRNA expression in mice.

<p>(A) Hepatic <i>Inhbe</i> mRNA levels were assessed by qRT-PCR in control animals (C57BL/6J mice; n = 6) and an animal model of type 2 diabetes at 8–12 weeks of age (db/db mice; n = 6). Animals in a freely fed state were sacrificed in the daytime. (B) INHBE protein levels in plasma were assessed by ELISA in control animals (C57BL/6J mice; n = 5) and an animal model of type 2 diabetes at 8 weeks of age (db/db mice; n = 5) in a freely fed state. (C) <i>Inhbe</i> mRNA expression in the liver, skeletal muscles and epididymal adipose tissue of an animal model of type 2 diabetes at 8 weeks of age (db/db mice; n = 5) were assessed by qRT-PCR; ND, not determined. (D) Insulin increased <i>Inhbe</i> mRNA expression in mouse primary hepatocytes. Insulin-induced expression of <i>Inhbe</i> mRNA was observed in cells treated with culture medium containing 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and glucocorticoid. Data are expressed as mean ± SEM of three wells. (E) Hepatic <i>Inhbe</i> mRNA levels in the siNON- and siINHBE-treated groups on days 2 (n = 6), 7 (n = 6), and 14 (n = 10) after the first dosing. (F) Hepatic Inhbe protein levels measured by ELISA in the siNON- (n = 6) and siINHBE-treated groups (n = 6) on day 7 after the first dosing. Data are expressed as mean ± SEM. Differences between the two groups were assessed by unpaired <i>t</i>-test (A, E) or Mann-Whitney U test (D). *: <i>P</i> < 0.05 (A), ^##: <i>P</i> < 0.01 (D).</p

Knock down of hepatic <i>Inhbe</i> mRNA in db/db mice suppressed body weight gain.

<p>(A) Cumulative food intake in the siNON- and siINHBE-treated groups on days 2 (n = 6 for each group), 7 (n = 6 for each group), and 14 (n = 10 for each group) after the first dosing. Cumulative food intake of same mouse as ones in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194798#pone.0194798.g003" target="_blank">Fig 3E</a> is indicated at each time point which was nearest in sacrifice of animals. (B) Body weight in the siNON- and siINHBE-treated groups on days 2 (n = 6 for each group), 7 (n = 6 for each group), and 14 (n = 10 for each group) after the first dosing. Body weight of same mouse as ones in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194798#pone.0194798.g003" target="_blank">Fig 3E</a> is obtained at point in time nearest to the time when the animals were sacrificed and at time point before first dosing (Day0). Data are expressed as mean ± SEM. Differences between the two groups were assessed by unpaired <i>t</i>-test. **: <i>P</i> < 0.01.</p

Inhibin βE (<i>INHBE</i>) is a possible insulin resistance-associated hepatokine identified by comprehensive gene expression analysis in human liver biopsy samples

<div><p>The liver plays a major role in whole-body energy homeostasis by releasing secretory factors, termed hepatokines. To identify novel target genes associated with insulin resistance, we performed a comprehensive analysis of gene expression profiles using a DNA chip method in liver biopsy samples from humans with varying degrees of insulin resistance. Inhibin βE (<i>INHBE</i>) was identified as a novel putative hepatokine with hepatic gene expression that positively correlated with insulin resistance and body mass index in humans. Quantitative real time-PCR analysis also showed an increase in <i>INHBE</i> gene expression in independent liver samples from insulin-resistant human subjects. Additionally, <i>Inhbe</i> gene expression increased in the livers of db/db mice, a rodent model of type 2 diabetes. To preliminarily screen the role of <i>Inhbe in vivo</i> in whole-body energy metabolic status, hepatic mRNA was knocked down with siRNA for <i>Inhbe</i> (siINHBE) in db/db mice. Treatment with siINHBE suppressed body weight gain during the two-week experimental period, which was attributable to diminished fat rather than lean mass. Additionally, treatment with siINHBE decreased the respiratory quotient and increased plasma total ketone bodies compared with treatment with non-targeting siRNA, both of which suggest enhanced whole-body fat utilization. Our study suggests that <i>INHBE</i> functions as a possible hepatokine to alter the whole-body metabolic status under obese insulin-resistant conditions.</p></div

Hepatic <i>INHBE</i> mRNA expression was increased in humans with high insulin resistance and body mass index.

<p>(A) Correlation between the homeostasis model assessment of insulin resistance (HOMA-IR) level and <i>INHBE</i> mRNA expression in the livers of human subjects. <i>INHBE</i> mRNA level was quantified by DNA chip. (B) Hepatic <i>INHBE</i> mRNA levels (DNA chip) in human subjects in the low (n = 6) and high HOMA-IR groups (n = 9). (C) Hepatic <i>INHBE</i> mRNA level by quantitative real-time reverse-transcription PCR (qRT-PCR) in human subjects in the low (n = 5) and high HOMA-IR groups (n = 5). (D) Correlation between body mass index (BMI) and mRNA expression of <i>INHBE</i> (DNA chip) in the livers of human subjects. Data in (B) and (C) are expressed as mean ± SEM. Differences between the two groups were assessed by Mann-Whitney U test (B) or unpaired <i>t</i>-test (C). ^#: <i>P</i> < 0.05 (B), *: <i>P</i> < 0.05 (C).</p

Clinical characteristics of subjects for DNA chip and qRT-PCR analyses.

<p>Clinical characteristics of subjects for DNA chip and qRT-PCR analyses.</p

Knock down of hepatic <i>Inhbe</i> mRNA in db/db mice altered body composition.

<p>Body fat, lean mass, and bone volumes were measured by CT analysis in the siNON- (n = 10) and siINHBE-treated groups (n = 10) on day 10 or 11 after the first dosing. Body fat volume (A), body lean mass volume (B), and bone volume (C) values for each animal were plotted versus body weight values. Body fat volume (A) and body lean mass volume (B) in the siINHBE- versus siNON-treated group were significantly decreased and increased, respectively, by analysis of covariance (<i>P</i> < 0.01). (D) Body fat and lean mass percentages were calculated by dividing body fat volume and body lean mass volume by total volume, respectively. Data are expressed as mean ± SEM of 10 animals. Difference between the two groups in body fat percentages in (D) was assessed by unpaired <i>t</i>-test. Difference between the two groups in body lean mass percentages in (D) was assessed by Mann-Whitney U test (D). **: <i>P</i> < 0.01 (for body fat percentages), ^##: <i>P</i> < 0.01 (for body lean mass percentages).</p

Knock down of hepatic <i>Inhbe</i> mRNA in db/db mice caused a switch from carbohydrates to fats as the respiratory substrate and increased fat metabolism.

<p>(A) Average energy daytime and nighttime respiratory quotients (RQ) on day 8 after the first dosing in the siNON- (n = 4) and siINHBE-treated groups (n = 4). (B) RQ values over a 24-h period on day 8 after the first dosing in the siNON- (n = 4) and siINHBE-treated groups (n = 4). (C) Average energy expenditure (EE) in the siNON- (n = 4) and siINHBE-treated groups (n = 4) during the daytime and nighttime periods of day 8 after the first dosing. (D) Average locomotor activity in the siNON- (n = 4) and siINHBE-treated groups (n = 4) during the daytime and nighttime periods of day 8 after the first dosing. (E) Plasma total ketone bodies (TKB) on days 2 (n = 6 for each group), 7 (n = 6 for each group), and 14 (n = 10 for each group) after the first dosing. Data are expressed as mean ± SEM. Differences between the two groups were assessed by unpaired <i>t</i>-test. **: <i>P</i> < 0.01, *: <i>P</i> < 0.05.</p

Generation of SGLT5-deficient mice and their fructose and mannose uptake by renal BBMV s.

<p>(A) Schematic representation of the strategy for targeting the <i>Slc5a10</i> gene. A targeting vector was constructed by inserting a neomycin resistant (<i>neo</i>) gene cassette to disrupt exons 3–6 of the <i>Slc5a10</i> genomic locus on a BAC genomic clone. Arrows indicate PCR primers for genotyping. (B) A representative result of genotyping the offspring obtained by intercrossing heterozygous-deficient mice. Wild type and null alleles are detected as signals of 900 bp and 350 bp, respectively. <i>Wt</i>: Wild type mice, <i>He</i>: Heterozygous null mutant, <i>Ho</i>: Homozygous null mutant. (C) Sodium-dependent uptake of fructose and (D) mannose in BBMVs of WT mice (+/+) and SGLT5-deficient mice (−/−). (E) Sodium-independent uptake of fructose and (F) mannose in BBMVs of WT mice (+/+) and SGLT5-deficient mice (−/−). Data are presented as means ± S.D. Data are derived from 3 independent experiments.</p

Food and water intake in WT (+/+) mice and SGLT5-deficient mice (−/−). Daily intake of

<p>(<b>A</b>) <b>food and</b> (<b>B</b>) <b>water of mice at 17 weeks of age.</b> (C) Calculated daily energy intake. Data are presented as means ± S.E.M (n = 8–10). ### P<0.001 versus respective plain water control.</p

Influence of the long-term consumption of fructose on tissue weight and lipid metabolism.

<p>(A) Plasma triglyceride levels of WT mice (+/+) and SGLT5-deficient mice (−/−). (B) Plasma total cholesterol levels. (C) Weight of epididymal fat. (D) Weight of the liver. (E) Hepatic triglyceride levels. (F) Histopathological analysis of the liver sections. Two sections per mouse were stained with Sudan III. Representative images are shown (scale bar, 50 µm). Data are presented as means ± S.E.M (<i>n</i> = 8–10). * <i>P</i><0.05, *** <i>P</i><0.001 versus WT mice given 30% fructose water. # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001 versus respective plain water controls.</p