Table 1.
Proteins detected that are involved in signaling or metabolic pathways.
Fig 1.
Analysis of the effects of LF on the phosphorylation of HSL and PLIN by PKA and determination of PKA activity.
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. (A) Phosphorylation of HSL Ser660 and (B) PLIN Ser497 by PKA. (C) 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. *p < 0.05, ***p < 0.001 HSL, hormone-sensitive lipase; LF, lactoferrin; PLIN, perilipin; PKA, protein kinase A; SD, standard deviation.
Fig 2.
Analysis of the effects of LF on the activation of ERK1/2 and Ras.
(A) 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) 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. *p < 0.05, **p < 0.01, ***p < 0.001. ERK, extracellular signal-regulated kinase; LF, lactoferrin; SD, standard deviation.
Fig 3.
Analysis of the effect of LF on CREB activation.
(A) 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) HSL and (C) 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. *p < 0.05, **p < 0.01, ***p < 0.001. AC, adenylyl cyclase; CREB, cAMP response element binding protein; HSL, hormone-sensitive lipase; LF, lactoferrin; SD, standard deviation.
Fig 4.
The influence of PKA activity inhibition on the downstream factor CREB.
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 t test. **p < 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.
Fig 5.
The influence of PKA activity inhibition on the HSL expression level and lipolysis by LF.
(A) 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 t test, and the data represent the mean ± SD values of triplicate determinations of one of the three identical experiments. *p < 0.05, **p < 0.01. (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. *p < 0.05, **p < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. LF, lactoferrin; SD, standard deviation.
Fig 6.
LF-induced lipolysis in LRP1-silenced adipocytes.
(A) 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 t test. **p < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. (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 t test vs LF untreated control. **p < 0.01; n.s., no significant difference. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. (C) LRP1 silencing by siRNA. Adipocytes were transiently transfected with negative control siRNA (siNC) or LRP1 siRNA (siLRP1) (see Materials and methods). 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.
Fig 7.
The influence of a βAR-blocker on the activities of lipolysis inducers.
(A) Activation of lipolysis in adipocytes by ISO with or without a βAR inhibitor. The amount of glycerol in the medium was analyzed 24 h after adding 0.1 mM of ISO with or without atenolol (β1AR blocker) to quantitate lipolysis. The statistical significance of the differences in the data for LF treated vs. untreated cells was evaluated using the Student’s t test. ***p < 0.001, n.s.; no significant difference. The data represent the mean ± SD values of triplicate determinations of one of the three identical experiments. (B) Activation of lipolysis by LF with or without βAR inhibitor. The amount of glycerol in the medium was analyzed 24 h after adding 1 mg/ml of LF with or without atenolol (β1AR blocker) to quantify lipolysis. The statistical significance of the data of LF treated and untreated cells were evaluated using Student’s t test. *p < 0.05, †p < 0.1, n.s.; no significant difference. The data represent the mean ± SD values of triplicate determinations of one of the three identical experiments. LF, lactoferrin; SD, standard deviation.
Fig 8.
Hypothetical model depicting the induction of lipolysis by LF in mature adipocytes.
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; Gαs, Gs alpha subunit; HSL, hormone-sensitive lipase; LF, lactoferrin; LRP1, low-density lipoprotein receptor-related protein 1; PKA, protein kinase A; PLIN, perilipin.