The influence of adiposity and acute exercise on circulating hepatokines in normal weight and overweight/obese men

Hepatokines are liver-secreted proteins with potential to influence glucose regulation and other metabolic parameters. This study investigated differences in adiposity status on five novel hepatokines and characterised their response to acute moderate-intensity exercise in groups of normal weight and overweight/obese men. Twenty-two men were recruited into normal weight and overweight/obese groups (BMI: 18.5 to 24.9 and 25.0 to 34.9 kg∙m-2). Each completed two experimental trials, exercise and control. During exercise trials, participants performed 60 min of moderate-intensity treadmill exercise (~60% V̇O2 peak) and then rested for 6 h. Participants rested throughout control trials. Circulating fibroblast growth factor-21 (FGF21), follistatin, leukocyte cell-derived chemotaxin 2 (LECT2), fetuin-A and selenoprotein-P (SeP) were measured throughout. Fasted (resting) FGF21 and LECT2 were higher in overweight/obese individuals (129% and 55%; P ≤ 0.01) and correlated with indices of adiposity and insulin resistance; whereas circulating follistatin was lower in overweight/obese individuals throughout trial days (17%, P < 0.05). In both groups, circulating concentrations of FGF21 and follistatin were transiently elevated after exercise for up to 6 h (P ≤ 0.02). Circulating fetuin-A and SeP were no different between groups (P ≥ 0.19) and, along with LECT2, were unaffected by exercise (P ≥ 0.06). These findings show that increased adiposity is associated with a modified hepatokine profile, which may represent a novel mechanism linking excess adiposity to metabolic health. Furthermore, acute perturbations in circulating FGF21 and follistatin after exercise may contribute to the health benefits of an active lifestyle

Effects of vanadate on insulin-stimulated Akt phosphorylation in H4IIEC hepatocytes.

<p>H4IIEC cells were serum-starved overnight. Then the cells were treated with the indicated concentrations of H₂O₂ for 3 h. Then, the cells were treated with 1mM of sodium orthovanadate for 30 min, and stimulated with insulin (1 ng/mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies.</p

Effects of H2O2 on PTP1B activity in H4IIEC hepatocytes.

<p>(A) H4IIEC cells were serum-starved overnight and then treated with the indicated concentrations of H2O2. After snap-freezing in liquid N2, cells were disrupted through scraping into ice-cold RIPA buffer containing a protease inhibitor cocktail, followed by brief sonication and clearing of the resulting lysates by centrifugation at 15,000 rpm for 15 min. PTP1B activity was determined using a CycLex® Protein Tyrosine Phosphatase 1B (PTP1B) Fluorometric Assay Kit. Fluorescence values from three independent experiments were normalized to total protein concentrations and are expressed as mean fold increases over control ± S.D. ** <i>p</i><0.01, versus untreated control. (B) Protein levels of PTP1B were measured by Western blotting.</p

Dual action of H2O2 on insulin signal transduction can be explained by different thresholds for the repression of PTP1B activity and enhancement of JNK activation.

<p>Dual action of H2O2 on insulin signal transduction can be explained by different thresholds for the repression of PTP1B activity and enhancement of JNK activation.</p

Time course of extracellular H2O2 concentration following its administration to H4IIEC hepatocytes.

<p>H4IIEC cells were incubated with the indicated concentrations of H2O2 for the indicated periods of time, and then the concentration of H2O2 in the culture medium was measured by ferrous oxidation of xylenol orange (FOX) assay.</p

Effects of antioxidant <i>N</i>-acetyl-L-cysteine on H₂O₂-induced alterations of insulin signal transduction in H4IIEC hepatocytes.

<p>(A–B) H4IIEC cells were serum-starved and treated with 10 mM of N-acetyl-L-cysteine overnight. Then the cells were treated with the indicated concentrations of H₂O₂ for 3 h, and stimulated with insulin (1 ng/mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies. (C) Quantitative data from densitometric analysis of p-Akt signals from three independent experiments were normalized to the values for total Akt and are expressed as mean fold increases over control ± S.E.M. . * <i>p</i><0.05, versus treatment with insulin alone; ** <i>p</i><0.01, versus treatment with insulin alone.</p

Effects of H2O2 on JNK activation in H4IIEC hepatocytes.

<p>(A) H4IIEC cells were serum-starved overnight and then treated with the indicated concentrations of H2O2. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated antibodies. Signals were detected chemiluminescence. Representative blots are shown. (B) Quantitative data from densitometric analysis of p-JNK signals from three independent experiments were normalized to the values for total JNK and are expressed as mean fold increases over control ± S.D. ** <i>p</i><0.01, versus untreated control.</p

Effects of H₂O₂ on the expression of mRNAs encoding antioxidative enzymes in H4IIEC hepatocytes.

<p>H4IIEC cells were serum-starved overnight and treated with the indicated concentrations of H₂O₂ for 3 h. Levels of the mRNAs encoding GPX1, GPX3, and catalase were measured by real-time-PCR. Values represent the mean ± S.D (<i>n</i> = 6).</p

Effects of JNK knockdown on H₂O₂-induced alterations of insulin signal transduction in H4IIEC hepatocytes.

<p>H4IIEC cells were transfected with siRNA specific to JNK1 or negative control. 24 h later, the cells were serum-starved overnight. Then the cells were treated with the indicated concentrations of H₂O₂ for 3 h, and stimulated with insulin (1 ng/mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies. (A) Protein levels of total JNK, phosphorylated INK, and phosphorylated serine residue of IRS-1 in the cells transfected with JNK. (B) Protein levels of phosphorylated Akt in the cells stimulated with insulin.</p

Linear regression analysis of total adiponectin concentrations with clinical parameters.

*<p><i>p</i><0.05.</p><p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034952#pone-0034952-t001" target="_blank">TABLE 1</a> for abbreviations.</p