10 research outputs found
Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes
<div><p>Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and <i>in vitro</i> studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1.</p></div
Analysis of the effect of LF on CREB activation.
<p><b>(A)</b> Phosphorylation of CREB-Ser133 in adipocytes treated with LF. Phosphorylated CREB was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to the protein expression level of CREB. Changes in protein expression levels of <b>(B)</b> HSL and <b>(C)</b> AC isomers (AC1, 2, and 6) in the presence or absence (0 min) of 1 mg/ml LF normalized to the protein expression level of β-actin. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. AC, adenylyl cyclase; CREB, cAMP response element binding protein; HSL, hormone-sensitive lipase; LF, lactoferrin; SD, standard deviation.</p
Analysis of the effects of LF on the activation of ERK1/2 and Ras.
<p><b>(A)</b> Activation of ERK1/2 (Thr202/Tyr204) after treatment of adipocytes with LF. Phosphorylated ERK1/2 was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to protein expression levels of ERK1/2 are shown. <b>(B)</b> Ras activation through c-Raf in adipocytes treated with LF. Activated Ras captured from cell lysates using a pull-down assay kit (see Experimental Procedures) before (0 min) and after treatment with 1mg/ml of LF. Activated Ras eluted from the beads was detected using western blot analysis. Intensity levels normalized to the total protein determined by BCA. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. ERK, extracellular signal-regulated kinase; LF, lactoferrin; SD, standard deviation.</p
Proteins detected that are involved in signaling or metabolic pathways.
<p>Peptide information used for quantitation of these proteins is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141378#pone.0141378.s001" target="_blank">S1 Table</a>. Details include function, accession number, peptide count, and peptides used for quantitation, confidence score, <i>p</i>-value (ANOVA), description, gene name, trivial name, fold-change (LF-treated group/untreated control group), and <i>p</i>-value (Student <i>t</i> test).</p><p>*<i>p</i> < 0.05,</p><p>**<i>p</i> < 0.01,</p><p>***<i>p</i> < 0.001</p><p>ERK, extracellular signal-regulated kinase; HSL, hormone-sensitive lipase; LF, lactoferrin; PKA, protein kinase A; PLIN, perilipin.</p><p>Proteins detected that are involved in signaling or metabolic pathways.</p
LF-induced lipolysis in LRP1-silenced adipocytes.
<p><b>(A)</b> Activation of lipolysis by LF. To quantitate lipolysis, the amount of glycerol in the medium was analyzed 24 h after adding 1 mg/ml of LF. The statistical significance of the differences between LF treated and untreated cells was evaluated using the Student <i>t</i> test. **<i>p</i> < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(B)</b> Activation of HSL by LF treatment. Phosphorylation of HSL was detected in the presence or absence of 1 mg/ml LF 15 min after the addition of LF. Phosphorylation levels normalized to protein expression levels are shown. The statistical significance was evaluated using the Student <i>t</i> test vs LF untreated control. **<i>p</i> < 0.01; n.s., no significant difference. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(C)</b> LRP1 silencing by siRNA. Adipocytes were transiently transfected with negative control siRNA (siNC) or LRP1 siRNA (siLRP1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141378#sec002" target="_blank">Materials and methods</a>). LRP1 protein expression was monitored by immunoblotting during each assay. Distinctive data is shown. β-actin was used as a loading control. HSL, hormone-sensitive lipase; LF, lactoferrin; LRP1, lipoprotein receptor-related protein 1; SD, standard deviation.</p
Analysis of the effects of LF on the phosphorylation of HSL and PLIN by PKA and determination of PKA activity.
<p>Phosphorylation of HSL and PLIN by PKA was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to protein expression levels of HSL and PLIN are shown. <b>(A)</b> Phosphorylation of HSL Ser660 and <b>(B)</b> PLIN Ser497 by PKA. <b>(C)</b> Analysis of PKA activity in adipocytes treated with LF. PKA activity in adipocytes was detected using an ELISA before (0 min) and after treatment with LF. Kinase activity normalized to the total protein determined by BCA is shown. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *<i>p</i> < 0.05, ***<i>p</i> < 0.001 HSL, hormone-sensitive lipase; LF, lactoferrin; PLIN, perilipin; PKA, protein kinase A; SD, standard deviation.</p
The influence of PKA activity inhibition on the HSL expression level and lipolysis by LF.
<p><b>(A)</b> Change in expression levels of HSL by LF. The protein expression levels of HSL were detected 3 h after the treatment with 1 mg/ml of LF with or without H-89, a selective PKA inhibitor. Changes are normalized to the β-actin protein expression level. The statistical significance of the data compared with the LF untreated sample was evaluated using Student’s <i>t</i> test, and the data represent the mean ± SD values of triplicate determinations of one of the three identical experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(B)</b> Activation of lipolysis by LF. The amount of glycerol in the medium was analyzed after the treatment with 1 mg/ml of LF with or without H-89, a selective PKA inhibitor, to quantify lipolysis. The statistical significance of the data compared with LF untreated cells was evaluated using Dunnett’s multiple comparison test. *<i>p</i> < 0.05, **<i>p</i> < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. LF, lactoferrin; SD, standard deviation.</p
The influence of PKA activity inhibition on the downstream factor CREB.
<p>Phosphorylation level of CREB-Ser133 was detected 15 min after the treatment with 1 mg/ml of LF with or without H-89, a selective PKA inhibitor. Adipocytes were pre-incubated in H-89 starting at 30 min before the addition of LF. Phosphorylation level was normalized to the CREB protein expression level. The statistical significance of the differences in the data for LF treated vs. untreated samples was evaluated using the Student <i>t</i> test. **<i>p</i> < 0.01, n.s.; no significant difference. The data represent the mean ± SD of triplicate determinations of one of the three identical experiments. LF, lactoferrin; PKA, protein kinase A: CREB, cAMP response element binding protein: HSL, hormone sensitive lipase: SD, standard deviation.</p
Hypothetical model depicting the induction of lipolysis by LF in mature adipocytes.
<p>T, Transcription; P, Phosphorylation; solid arrow, direct interaction; dashed arrow, indirect interaction. AC, adenylyl cyclase; CREB, cAMP response element binding protein; ERK, extracellular signal-regulated kinase; Gα<sub>s</sub>, G<sub>s</sub> alpha subunit; HSL, hormone-sensitive lipase; LF, lactoferrin; LRP1, low-density lipoprotein receptor-related protein 1; PKA, protein kinase A; PLIN, perilipin.</p