Lactoferrin increases 172ThrAMPK phosphorylation and ... - Nature

International Journal of Obesity (2009) 33, 991–1000 & 2009 Macmillan Publishers Limited All rights reserved 0307-0565/09 $32.00 www.nature.com/ijo

ORIGINAL ARTICLE Lactoferrin increases 172ThrAMPK phosphorylation and insulin-induced p473SerAKT while impairing adipocyte differentiation JM Moreno-Navarrete, FJ Ortega, W Ricart and JM Fernandez-Real Biomedical Research Institute of Girona, Hospital de Girona ‘Dr Josep Trueta’, and CIBER Fisiopatologia Obesidad y Nutricion (CB06/03/010) Instituto de Salud Carlos III, Girona, Spain Objective: Lactoferrin is a pleiotropic glycoprotein of the innate immune system with known effects on immunomodulation and cell differentiation. To gain an insight into the interaction among obesity, inflammation and insulin action, we aimed to examine the effects of lactoferrin on adipogenesis and the response to insulin in human hepatocarcinoma (HepG2) and 3T3-L1 cell lines. Design: The cells were cultured with increasing lactoferrin concentration under non-inflammatory, inflammatory and standard conditions. The response to insulin was evaluated through 473SerAKT phosphorylation. The effects of lactoferrin on adipogenesis were studied through the expression of different lipogenic markers, AMP-activated protein kinase (AMPK) activation, retinoblastoma (Rb) activity and Oil Red O staining in 3T3-L1 cells. Results: Lactoferrin increased dose-dependent insulin-induced 473SerAKT phosphorylation in both cell lines. Inflammationinduced decreased 473SerAKT phosphorylation was also rescued by lactoferrin. In addition, lactoferrin led to increased p172Thr AMPK during 3T3-L1 differentiation and to decreased adipogenesis (as shown by decreased expression of fatty acid synthase, acetyl-coenzyme A carboxylase-a and peroxisome proliferator-activated receptor-g in parallel with decreased formation of lipid droplets). Lactoferrin also increased dose-dependent Rb activity (expression and hypophosphorylation) during 3T3-L1 differentiation. Conclusion: Lactoferrin administration increased insulin-induced 473SerAKT phosphorylation, even in those conditions wherein the response to insulin was downregulated, and led to blunted adipogenesis in the context of increased p172ThrAMPK and Rb activity. International Journal of Obesity (2009) 33, 991–1000; doi:10.1038/ijo.2009.143; published online 4 August 2009 Keywords: lactoferrin; proinflammatory conditions; insulin action; adipogenesis; AMPK

Introduction Obesity-associated metabolic dysregulation is increasingly envisioned as a chronic inflammatory disease. Macrophage infiltration into the adipose tissue may have a role in insulin resistance-associated inflammatory activity. Macrophagesecreted factors are known to block adipogenesis and insulin action in adipocytes and hepatocytes by the downregulation of insulin receptor sustrate-1 (IRS-1), leading to decreased AKT phosphorylation.1–4 The mechanisms through which pro-inflammatory cytokines, like tumor necrosis factor-a, Correspondence: JM Moreno-Navarrete, Unit of Diabetes, Endocrinology and Nutrition Unit, Institut Investigacio´ Biome`dica de Girona, Hospital de Girona ‘Dr Josep Trueta’, Ctra. Franc- a s/n, Girona 17007, Spain. E-mail: [email protected] Received 17 February 2009; revised 8 June 2009; accepted 12 June 2009; published online 4 August 2009

interleukin-6 (IL-6) and IL-1b interact with cellular insulin signal transduction cascades have been described in the last years.5–9 Lactoferrin is a pleiotropic glycoprotein (80 kDa) and a prominent component of the first line of mammalian host defense, acting on specific lactoferrin receptors that exist in a variety of cells, like monocytes, lymphocytes, adipocytes, hepatocytes and endothelial cells.10 Lactoferrin expression is upregulated in response to inflammatory stimuli.10 Lactoferrin is able to bind and buffer several pathogen associated molecular patterns, such as lipopolysaccharide (LPS), viral components and soluble components of the extracellular matrix.11 This ability is associated with the putative lactoferrin anti-inflammatory activity, as shown in several studies.12 Lactoferrin administration led to decreased release of tumor necrosis factor-a and IL-6 in mice,13–15 to downregulated pro-inflammatory cytokine production in different

Lactoferrin and insulin response JM Moreno-Navarrete et al

992 cell lines acting via nuclear factor-kB,16 and to decreased LPSinduced binding of nuclear factor-kB to the tumor necrosis factor-a promoter.16 Lactoferrin also participates in the regulation of cellular growth and differentiation.17,18 In primary osteoblasts, lactoferrin stimulated proliferation and differentiation and even acted as a survival factor, thereby, inhibiting apoptosis induced by serum withdrawal and also inhibiting osteoclastogenesis in a murine bone marrow culture.19 To the best of our knowledge, effects of lactoferrin on the response to insulin (through p473SerAKT) have never been explored. We aimed to investigate the effects of different lactoferrin concentrations on insulin-induced 473SerAKT phosphorylation in human hepatocarcinoma (HepG2) and 3T3-L1 fibroblast mouse cell lines. We also evaluated whether lactoferrin rescues the decreased 473SerAKT phosphorylation induced by dexamethasone and macrophage conditioned media in the 3T3-L1 cell line. Finally, we studied the effects of lactoferrin on 3T3-L1 adipocyte differentiation.

Materials and methods THP-1 cell culture, differentiation and stimulation The human monocyte cell line THP-1 (American Type Culture Collection, Barcelona, Spain) was cultured in Rosswell Park Memmorial Institute (RPMI media 1640; Cat. No. 21870-076) 1640 medium containing 10% fetal bovine serum, 5 mM glucose, 2 mM L-glutamine, 50 mg ml1 gentamicine and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at 37 1C in a humidified 5%CO2 per 95 1C air atmosphere. The mature macrophage-like state was induced by treating THP-1 cells (1,2 106 cells) with 0,162 mM phorbol 12-myristate 13-acetate (PMA) (Sigma Chemical, Madrid, Spain) in 6-well culture dishes for 24 h. Differentiated, plastic-adherent cells were washed with cold Dulbecco’s phosphate buffered saline (D-PBS, Sigma Chemical) and then incubated with fresh medium without phorbol 12-myristate 13-acetate. Differentiated macrophages were treated with fresh medium or stimulated with 10 ng ml1 LPS (Sigma Chemical) for an additional 24 h. The supernatants (macrophage-conditioned media (MCM)) were collected, centrifuged at 900 g for 5 min, aliquoted and stored at –80 1C until testing.

Lactoferrin effects on insulin-induced p473SerAKT phosphorylation in HepG2 and 3T3-L1 cell lines HepG2 cells, undifferentiated 3T3-L1 and pre-differentiated 3T3-L1 cells were incubated in serum-free DMEM containing 25% non-stimulated THP-1 conditioned media (MCM) or 25% LPS-stimulated THP-1 conditioned media (LPS-MCM). Co-treatment with different lactoferrin (Sigma-Aldrich, Madrid, Spain) concentrations (0.01, 0.1, 1 and 10 mM) for 24 h was used to assay the effects on: (1) insulin-induced 473Ser AKT phosphorylation; (2) insulin-induced 473SerAKT phosphorylation after MCM and LPS-MCM stimulus; and (3) insulin-induced 473SerAKT phosphorylation after dexaInternational Journal of Obesity

methasone treatment. In this latter experiment, undifferentiated 3T3-L1 cells were incubated in media containing 20 mM glucose, 10 % fetal bovine serum, 100 U ml1 penicillin and 100 `ıg ml1 streptomycin, and insulin, dexamethasone and isobutylmethylxanthine (INS–DEX–IBMX) mixture, containing 10 mg ml1 bovine insulin (SigmaAldrich), 0.5 mM dexamethasone (Sigma-Aldrich) and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) for 2 days. After 24 h, all samples were stimulated with insulin (100 nM for 10 min) and the experiments were carried out in triplicate. The relative quantity of p473SerAKT, AKT and insulin receptor was measured by western blot.

Lactoferrin effects on 3T3-L1 differentiation The embryonic fibroblast mouse cell line 3T3-L1 (American Type Culture Collection) was maintained in DMEM containing 20 mM glucose, 10 % fetal bovine serum, 100 U ml1 penicillin and 100 mg ml1 streptomycin. Two days after confluence, INS–DEX–IBMX mixture was added for 2 days, followed by 5 days with insulin alone. Lactoferrin (0.1, 1 and 10 mM) was added in two steps of the differentiation process, and the experiment was carried out in triplicate. Differentiation was monitored by morphological assessment and Oil red O staining. For Oil Red O staining, cells were washed twice with phosphate buffer saline (PBS), fixed in 4 % formaldehyde for 1 h and stained for 30 min with 0.2 % Oil Red O solution in 60 % isopropanol. Cells were then washed several times with water, and excess water was evaporated by placing the stained cultures at approximately 32 1C. To determine the extent of adipose conversion, 0.2 ml of isopropanol was added to the stained culture dish. The extracted dye was immediately removed by gentle pipetting and its optical density was monitored spectrophotometrically at 500 nm using a multi-well plate reader (Model Anthos Labtec 2010 1.7 reader, Salzburg, Austria). The relative quantity of p172ThrAMP-activated protein kinase (p172ThrAMPK), AMPK, p79seracetyl-coenzyme A carboxylase alpha (p79SerACC), ACC, fatty acid synthase (FASN), peroxisome proliferator-activated receptor-g (PPAR-g), pSer807/811retipSer807/811 Rb), Rb and glucose transporter-4 (GLUT-4) noblastoma ( was measured by western blotting in triplicate. Density p172ThrAMPK p79SerACC and pSer807/811Rb values were normalized by total AMPK, total ACC and Rb, respectively. FASN, PPAR-g and GLUT-4 were normalized by b-actin levels. We also studied the insulin-induced effects on p473SerAKT/ AKT phosphorylation during the differentiation process (day 0, 1, 2, 4 and 7) under lactoferrin (1 mM) administration vs vehicle. Western blot analysis Cell lysates were washed in ice-cold PBS followed by homogenization assay using radio immunoprecipitation assay (RIPA) lysis buffer (10X: 0.5 M Tris-HCl, pH 7.4, 1. 5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10mM EDTA; Upstate,


993 Billerica, MA, USA) supplemented with a protease inhibitor cocktail (Sigma, Madrid, Spain) at 4 1C for 30 min. Cellular debris were eliminated by centrifugation of the diluted samples at 10 000 g for 10 min (4 1C). Protein concentration was determined using Lowry assay. RIPA protein extracts (50 ug) were separated by SDS-PAGE and transferred to nitrocellulose membranes by conventional procedures. Membranes were immunoblotted with AKT, p473SerAKT, p79SerACC, ACC, pSer807/811Rb (Cell Signaling Technology, Isaza S.A., Barcelona, Spain), p172ThrAMPK, AMPK, FASN, PPAR-g, Rb and anti-b-actin antibodies (Santa Cruz Biotechnology, Quimigen S.L., Madrid, Spain), insulin receptor (UpstateMillipore, Billerica, MA, USA) and GLUT-4 (Chemicon International, Billerica, MA, USA). Anti-rabbit immunoglobulin G and anti-mouse immunoglobulin G coupled to horseradish peroxidase was used as secondary antibody. Horseradish peroxidase activity was detected by chemiluminescence and quantification of protein expression was carried out using Scion image software (Scion Corp., Frederick, MD, USA).

Cytokine antibody array A commercially available Custom Human Cytokines Antibody Array for Bionova (Ray Biotech, Norcross, GA, USA) was used to determine the level of the 20 cytokines cited in Figure 1. The assay was carried out according to the manufacturer’s instructions. In brief, membranes were blocked with a blocking buffer, and then incubated overnight with 1 ml of the culture supernatants at 4 1C. The membranes were washed and incubated with 1 ml of primary biotin-conjugated antibody at room temperature for 2 h. After washing, 2 ml of horseradish peroxidase-conjugated streptavidin was added and incubated

MCM

for 30 min at room temperature. The membranes were developed by using detection buffer, exposed to X-ray film and processed by autoradiography. Detectable spots were scanned and analyzed for densitometry with Scion Image software.

MTT-based cell viability assays and cell counting The ability of lactoferrin to affect 3T3-L1 and HepG2 cell viability was determined using a standard colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) reduction assay. Cells in exponential growth were harvested by trypsinization and seeded at a concentration of approximately 2.5 103 cells per 200 ml per well into 96-well plates, and left during an overnight period for attachment. The medium was then removed and fresh medium with various concentrations of human lactoferrin (hLf) (1, 10, 20 mM) were added to the cultures in parallel. Control cells without agents were cultured using the same conditions with comparable media changes. Compounds were not renewed during the entire period of cell exposure. After treatment (5 days), the medium was removed and replaced by fresh drug-free medium (100 ml per well), and MTT (5 mg ml1 in PBS) was added to each well at a volume of 1/10. After incubation for 2–3 h at 37 1C, the supernatants were carefully aspirated, 100 ml of dimethyl sulfoxide were added to each well, and the plates agitated to dissolve the crystal product. Absorbances were measured at 570 nm. The cell viability effects from exposure of cells to each compound were analyzed as percentages of the control cell absorbances, which were obtained from control wells treated with appropriate

LPS-MCM

Figure 1 Cytokine antibody array of 20 cytokines to assay the lipopolysaccharide (LPS) effects on THP-1 cells, showing a high proinflammatory cytokine production in these cells.

International Journal of Obesity


994 Human lactoferrin effects on insulin-induced 473SerAKT phosphorylation under non-inflammatory and inflammatory conditions Lactoferrin led to increased dose-dependent (1.16, 1.65, 1.9 and 2.2-fold, respectively, Po0.005 for the three highest concentrations) insulin-induced p473SerAKT/AKT ratio (after hLf 0.01, 0.1 1 and 10 mM) (Figure 2a). Conditioned media from LPS-stimulated THP-1 cells led to a three-fold decrease in insulin-induced 473SerAKT phosphorylation in HepG2 cells (Figure 2b). Lactoferrin recovered 473Ser AKT phosphorylation dose dependently by 1.8, 4 and 5.15-fold, respectively (Figure 2b). Conditioned media from unstimulated THP-1 cells did not change 473SerAKT phosphorylation significantly in these HepG2 cells. Again, lactoferrin increased the response to insulin dose dependently (Figure 2b). In the absence of insulin, we did not observe any increase in p473SerAKT after hLf administration. However, using 150 mg of total protein in the western blot (instead of 50 mg of total protein), we found a significant increase in p473SerAKT with the two highest lactoferrin concentration (1 and 10 mM, respectively) (Figure 3a). Lactoferrin administration did not increase total AKT or insulin receptor protein levels (Figure 3b).

concentrations of the compounds vehicles that were processed simultaneously. For each treatment, cell viability was evaluated as a percentage using the following equation: (A570 of treated sample/A570 of untreated sample) 100. Cell counting was assessed by trypan blue dye exclusion using a Neubauer hemocytometer, after 7 days differentiation of 3T3-L1 cells, in triplicate. Sensitivity to hLf was then expressed as the percentage of cell viability for each concentration of the drug. Percent cell viability for 1, 10 and 20 mM of hLf was 97.9, 86.3 and 87.4, respectively, in 3T3-L1 cells; and 94.9, 85.4 and 80.9, respectively, in HepG2 cells.

Statistical analysis Data are expressed as the mean±s.e.m. The experiments were carried out in triplicate. The effects of lactoferrin treatment were evaluated using unpaired t-test and Wilcoxon’s test.

Results

pAKT/AKT density

LPS effects on THP-1 cells THP-1 spontaneously released IL-8, GRO and monocyte chemotactic protein-1 (cytokines present in MCM media) (Figure 1). THP-1 stimulated cells released more IL-8, GRO, IL-1b, monocyte chemotactic protein-1, MIP-1a, MIP-1b, intercellular adhesion molecule IL-6 and tumor necrosis factor-a (cytokines present in LPS-MCM media) than non stimulated THP-1 cells (Figure 1). 2.5

Lactoferrin effects on insulin-induced p473SerAKT phosphorylation in non-differentiated and differentiated 3T3-L1 cells under normal and inflammatory conditions Lactoferrin (1 and 10 mM) led to increased p473SerAKT in predifferentiated (after INS–DEX–IBMX) 3T3-L1 cells in the absence of insulin (Figure 3a). The highest lactoferrin

*

2

*

*

1.5 1 0.5 0

hLf (μM) Insulin (100 nM)

hLf (μM)

0

0 0.01 0.1 1

10

Insulin (100 nM)

-

+

+

+

+

+

p473SerAKT

* 0 -

0 +

0.01 0.1 + +

1 +

AKT

10 +

pAKT/AKT density

hLf 0 uM hLf 0.01 uM hLf 0.1 uM

hLf1 uM

2.5 *

2 *

1.5

++

+

1

p473SerAKT

AKT

0.5 0 0

0

Insulin (100 nM) +

+

hLf (μM)

0.01 0.01 0.10 +

+

+

0.1

1.0

1.0

+

+

+

LPS-non stimulated conditionated medium LPS- stimulated conditioned medium

Figure 2 (a) Effects of increasing lactoferrin concentration in the response to insulin in human hepatocarcinoma (HepG2) cells. *Po0.05 vs. insulin-stimulated control. (b) Effects of increasing lactoferrin concentration in the response to insulin in HepG2 cells under inflammatory conditions during 24 h. *Po0.05 vs insulinstimulated control. þ Po 0.05 vs insulin-stimulated control with lipopolysaccharide (LPS)-stimulated macrophage-conditioned media (MCM). þ þ Po 0.005 vs insulin-stimulated control with LPS-stimulated MCM.



995 150 μg total protein Insulin (100nM) hLf (μM)

- -

-

-

+

-

10 1 0.1 0.01 0

0

50 μg total protein -

-

-

-

+

hLf (μM) 10 1 0.1 0.01 0

-

IR

10 1 0.1 0.01 0 0 Non-differentiated 3T3-L1

p473SerAKT

β-Actin AKT Pre-differentiated (after INS-ISODEX) 3T3-L1

p473SerAKT

IR

β-Actin AKT

IR

p473SerAKT HepG2

AKT

β-Actin

p473Ser AKT, AKT (a) and insulin receptor (IR) protein expression (b) in non-differentiated and predifferentiated (after INS–DEX–IBMX mixture) 3T3-L1 and human hepatocarcinoma (HepG2) cells in absence of insulin.

Figure 3 Effects of increasing lactoferrin concentration on

concentration (10 mM) also led to increased p473SerAKT in non-differentiated 3T3-L1 cells. All these effects were observed only with 150 mg of total protein in the western blot. Lactoferrin administration did not increase AKT or insulin receptor protein levels (Figure 3b). After insulin administration, lactoferrin led to a dosedependent increase in p473SerAKT/AKT ratio (0.01, 0.1, 1 and 10 mM) in both pre-differentiated (after INS–DEX–IBMX mixture) and non-differentiated 3T3-L1 cells. p473SerAKT/ AKT ratio optical density was significantly higher in nondifferentiated 3T3-L1 cells compared with pre-differentiated (after INS–DEX–IBMX mixture) 3T3-L1 cells (0.66±0.11 vs 0.083±0.01 arbitrary units, P ¼ 0.01). Lactoferrin administration recovered the relatively low p473SerAKT/AKT ratio in pre-differentiated (after INSFDEX–IBMX mixture) 3T3-L1, which was increased 1.5, 6, 13.6 and 24.2-fold after 0.01, 0.1, 1 and 10 mM lactoferrin, respectively (Figure 4a). In non-differentiated 3T3-L1 cells, this increase was less marked 1.2, 1.36, 1.42 and 2.1-fold, respectively, being only significant at the highest lactoferrin dose (Figure 4b). Conditioned media from LPS-stimulated THP-1 cells led to decreased 473SerAKT phosphorylation by 73% and 67% (in differentiated and non-differentiated 3T3-L1 cells, respectively) (P ¼ 0.01, P ¼ 0.005). In pre-differentiated (INS–DEX–IBMX) 3T3-L1 cells, insulin-induced 473SerAKT phosphorylation was recovered dose dependently after co-treatment with lactoferrin (0.01, 0.1 and 1 mM), increasing p473SerAKT/AKT 3.2, 17.8 and 33-fold (P ¼ 0.006, o0.001 and 0.007, respectively) (Figure 4a).

In

non-differentiated 3T3-L1 cells, insulin-induced AKT phosphorylation was only recovered at the highest lactoferrin dose (1.77, 2.13 and 2.82-fold (P ¼ 0.3, 0.4 and 0.02, respectively) (Figure 4b). INS–DEX–IBMX mixture decreased insulin-induced p473Ser AKT action that was recovered with insulin in the last 5 days of differentiation process. Lactoferrin (1 mM) only recovered insulin-induced p473SerAKT during the INS–DEX– IBMX mixture treatment (Figure 5). 473Ser

Human lactoferrin effects on 3T3-L1 differentiation The increase in lipid droplets, the synthesis of lipogenic proteins and AMPK activity (p172ThrAMPK) were evaluated during 3T3-L1 differentiation. Administration of lactoferrin led to dose-dependent reduction in lipid droplets (Figure 5). In fact, at the highest lactoferrin dose (10 mM), lipid accumulation was similar to that present in non-differentiated 3T3-L1 fibroblasts (Figure 6). In agreement with these observations, the lipogenic proteins, FASN, ACC and PPAR-g were downregulated after lactoferrin treatment (1 and 10 mM). FASN decreased by 44 and 66%, respectively (Po0.001 and o0.001), ACC by 22% and 96% (P ¼ 0.03 and Po0.001, respectively) and PPAR-g by 73% and 84% (Po0.001 and o0.001, respectively) compared with untreated, differentiated 3T3-L1 fibroblasts (Figure 7). Furthermore, hLf led to 11.4 and 15.3-fold (P ¼ 0.03 and 0.009, after 1 and 10 mM, respectively) increased phosphoInternational Journal of Obesity


996 Non-differentiated 3T3-L1

Pre-differentiated (after INS-ISO-DEX) 3T3-L1

2.5 pAKT/AKT density

pA K T /A K T density

2.5 2 **

1.5

** ++

1 *

0.5

+

+

2 *

1.5 1

+

0.5

0

0

hLf (μM)

0

0

0

Insulin (100nM) -

+

+

+

+

+

-

-

+

-

+

-

LPS-stimulated MCM

1

1

10

0

0

0

0.1

1

1

10

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

-

+

-

-

-

+

-

+

-

+

-

+

-

0.01 0.01 0.1 0.1

0.01 0.01 0.1

p473SerAKT

AKT

Differentiation cocktail*

Without Differentiation cocktail*

Figure 4 - Effects of increasing lactoferrin concentration in the response to insulin in pre-differentiated (after INSFDEX–IBMX mixture) (a) and non-differentiated (b) 3T3-L1 under normal and inflammatory conditions during 24 h. *Po 0.05 vs insulin-stimulated control. **Po 0.005 vs insulin-stimulated control. þ Po 0.05 vs insulin-stimulated control with lipopolysaccharide (LPS)-stimulated macrophage-conditioned media (MCM). þ þ Po 0.005 vs insulin-stimulated control with LPSstimulated MCM. There was a significant decrease of p472SerAKT/AKT ratio in 3T3-L1 pre-differentiated (after INS–DEX–IBMX mixture) cells in control and hLf 0.01 mM.

hLf (1μM) 7th

4th 2ond 1st

0

Differentiation day

7th

4th 2ond 1st

p473SerAKT

0

Insulin (100 nM)

AKT

pAKT/AKT density

2.5 2 1.5 ++

1 0.5

++ **

**

0 0 day 0 day- 1st hLf day

1st 2ond 2ond day- day dayhLf hLf

4th day

4th dayhLf

7th day

7th dayhLf

Figure 5 Insulin action during 3T3-L1 differentiation after lactoferrin (hLf, 1 mM) or vehicle administration. **Po 0.005 vs insulin-stimulated p473SerAKT at baseline. þþ Po 0.005 vs insulin-stimulated p473SerAKT after vehicle administration. Blunted insulin action was reversible during the differentiation process. hLf (1 mM) treatment attenuated the blunted insulin action.

rylation of AMPK (p172ThrAMPK). In parallel to p172ThrAMPK GLUT-4 levels (5.1 fold, P ¼ 0.03) and p79SerACC (4.7 fold, P ¼ 0.02) increased accordingly at the highest lactoferrin dose (10 mM). International Journal of Obesity

Human lactoferrin effects on Rb activity during 3T3-L1 differentiation Retinoblastoma activity (as showed by increased Ser807/811Rb phosphorylation and decreased total Rb levels) decreased


997

Non-differentiated 3T3-L1 control

Differentiated 3T3-L1 hLf (0.1 μM)

Differentiated 3T3-L1 control

0.4 0.35 O.D. (500 nM)

0.3

*

0.25 0.2 0.15

**

**

0.1 0.05 0 Differentiated 3T3-L1 hLf (1 μM)

N-Dif

Differentiated 3T3-L1 hLf (10 μM)

Dif

0.1 μM

1 μM

10 μM

Dif - hLf

Figure 6 Oil red O staining after 3T3-L1 differentiation with increasing lactoferrin concentrations (0.1, 1, 10 mM).

1.2

Density

1 *

0.8

**

0.4 0.2

Density

Differentiatited

Differentiatited

AMPK

** **

**

**

**

0 hLf (μM)

hLf (μM)

ACC FASN PPAR-γ

*

0.6

20 18 16 14 12 10 8 6 4 2 0

p172ThrAMPK

*

p79SerACC

** 0 -

0 +

0.1 +

1 +

10 +

ACC FASN

**

pACC/ACC pAMPK/AMPK

PPAR-γ

GLUT-4

** GLUT-4

*

** *

**

0 -

0 +

0.1 +

**

1 +

*

10 +

β-Actin

N-Dif Dif 0.1 1

10

Dif+hLf (μM)

Figure 7 Effects of lactoferrin on lipogenic proteins and AMP-activated protein kinase (AMPK) phosphorylation during 3T3-L1 differentiation (7 days). *Po 0.05 vs differentiated control. **Po 0.005 vs differentiated control.

during differentiation of 3T3-L1 cells. Lactoferrin increased Rb activity (hypophosphorylation and Rb expression) dose-dependently in parallel to the inhibition of adipogenesis (Figure 8).

Human lactoferrin effects on cell viability during 3T3-L1 differentiation When differentiating 3T3-L1 cells, the cell number was significantly increased when compared with non-differentiated International Journal of Obesity


998 hLf (μM) Non-D D

0.1

1

10 Phospho-Rb (Ser807/811)

Rb

β-Actinn

25.00

1.2

*

0.8 0.6

*

*

*

0.4

*

15.00 10.00 5.00

0.2

0.00

0 N-Dif

Figure 8 Effects of lactoferrin on

Dif

pSer807/811

hLf (0.1uM) hLf (1uM) hLf (10uM) pSer807/811

retinoblastoma (

N-Dif

Dif

hLf (0.1uM) hLf (1uM) hLf (10uM)

Rb) and Rb protein levels during 3T3-L1 differentiation. *Po 0.05 vs differentiated control.

3T3-L1 control cells (1 200 000 216 000 vs 680 000 154 000 cells ml1, P ¼ 0.001). 3T3-L1 cells treated with 0.1, 1 and 10 mM of lactoferrin during differentiation led to significantly decreased cell count (575 000 142 000, 650 000±163 000 and 390 000±118±000±cell ml1, respectively, P ¼ 0.001). Lactoferrin did not affect percentage cell viability of 3T3-L1 cells in the MTT assay (97.9, 86.3 and 87.4% after 1, 10 and 20 mM of hLf were, respectively).

Discussion Lactoferrin is an important modulator of inflammation. Its antioxidant20 and anti-inflammatory activities (blocking nuclear factor-kB activation in response to LPS16) could explain the positive effects of lactoferrin in intracellular insulin action. LPS-stimulated THP-1 conditioned media induced a significant decrease in response to insulin in HepG2, 3T3-L1 fibroblasts and in pre-differentiated (after INS–DEX–IBM mixture) 3T3-L1 cells, as demonstrated by decreased insulin-induced 473SerAKT phosphorylation. Lactoferrin upregulated insulin-induced 473SerAKT phosphorylation in non-differentiated and pre-differentiated (after INS–DEX–IBM mixture) 3T3-L1 cells and in HepG2 cells. At the dose of 1 mM and higher, lactoferrin increased the response to insulin in all cell lines tested. This effect was especially observed in those conditions where insulin action was blunted, such as pre-differentiated (after INS–DEX–IBMX mixture) 3T3-L1 cells and in cells under treatment with pro-inflammatory media. The differentiation International Journal of Obesity

*

20.00

1 Rb density

Phospho-Rb / Rb density

1.4

cocktail, with high insulin and dexamethasone concentration, decreased insulin-induced 473SerAKT phosphorylation in the first phase of differentiation process,21 thus explaining the relative ‘insulin resistance’ of pre-differentiated (after INS–DEX–IBMX treatment) 3T3-L1 cells. This effect was reversible, because during the second phase of the differentiation process (in which only insulin is present) insulin-induced 473SerAKT phosphorylation was recovered. Impaired insulin action in these cells was also rescued by increasing lactoferrin concentration. Under inflammatory conditions, lactoferrin dose dependently rescued the low response to insulin in both HepG2 and 3T3-L1 cells. Lactoferrin could have an insulinotropic activity, increasing AKT phosphorylation on serine 473 in the absence of insulin. Different antioxidant and anti-inflammatory actions of lactoferrin could also be involved in the improvement of insulin action under inflammatory conditions. Recently, Yagi et al.22,23 reported that lactoferrin suppressed the adipogenic differentiation of MC3T3-G2/PA6 cells. Lactoferrin co-treatment decreased lipid droplets and the mRNA expression of several adipogenesis markers (CEBP-a, PPAR-g, aP2 and adiponectin) while inducing osteogenesis (increasing the expression of Runx2, osteocalcin and Sox9). These findings fit with existing hypotheses that a reciprocal or inverse relationship exists between adipogenesis and osteogenesis in the marrow microenvironment.24,25 However, these authors did not study the effects of insulin. According to our data, lactoferrin also downregulated 3T3-L1 differentiation, decreasing the formation of lipid droplets. Red oil density was negatively associated with lactoferrin concentrations. Lacto-


999 ferrin also decreased dose dependently different lipogenic proteins (FASN, ACC and PPAR-g) in parallel with increased AMPK activation (p172ThrAMPK) and GLUT-4 levels. This latter observation could be related with the lactoferrin interference on adipogenic differentiation. In fact, AMPK activation leads to increased fat oxidation by the upregulation of the transcription factor NRF-1, which stimulates mitochondrial biogenesis and inhibits both lipolysis and lipogenesis, affecting directly the enzymes engaged in lipid metabolism and downregulating PPAR-g expression.26 Hyperphosphorylation of Rb (inhibition) is necessary to promote clonal expansion and adipocyte differentiation of 3T3-L1 cells.27,28 We found decreased Rb activity and protein levels during 3T3-L1 cells differentiation. Lactoferrin increased dose dependently Rb activity (hypophosphorylation and Rb expression) in parallel to its inhibitory effects on adipogenesis. Thus, lactoferrin could inhibit adipogenic differentiation through the increase of hypophosphorylated Rb (Rb activity) levels, inducing cell cycle arrest.29 In MTTbased cell viability assays to evaluate lactoferrin cytotoxicity, we found that lactoferrin (10 mM) decreased cell viability of 3T3-L1 by only 15%. Cell counting corroborated the negative effects of lactoferrin on 3T3-L1 expansion during the differentiation process. We did not find changes in GLUT-4 expression between pre-adipocytes and adipocytes. We used dexamethasone as a component of the differentiation cocktail that could have influenced the results. In fact, dexamethasone did not change GLUT-4 expression levels while decreasing GLUT-4 translocation and glucose uptake in 3T3-L1 cells.30 Dexamethasone also led to decreased GLUT-4 expression in adipocytes in one study,31

Study limitations We studied the lactoferrin effects during a 24 h co-incubation. The observed effects suggest a mechanism whereby changes in gene/protein expression are required. Shorter exposure times need to be further evaluated to test if the same enhancement of insulin-stimulated AKT activation can be achieved. The 24 h conditioned media may still contain LPS, and some the actions of hLf may be explained (a least in part) by its ability bind to and buffer LPS. The effects of hLf on increased insulin sensitivity and GLUT-4 expression should be evaluated further in vivo. To the best of our knowledge, this is the first study showing an insulin sensitizing effect of lactoferrin, even under conditions associated with insulin resistance (proinflammatory medium and dexamethasone administration). Further investigation is necessary to evaluate the possible benefits of this protein in metabolic disturbances associated with insulin resistance and obesity.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This work was partially supported by research grants from the Ministerio de Educacio´n y Ciencia (SAF2008-0273).

References 1 Lumeng CN, Deyoung SM, Saltiel AR. Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins. Am J Physiol Endocrinol Metab 2007; 292: E166–E174. 2 Constant VA, Gagnon A, Landry A, Sorisky A. Macrophageconditioned medium inhibits the differentiation of 3T3-L1 and human abdominal preadipocytes. Diabetologia 2006; 49: 1402–1411. 3 Zhou L, Sell H, Eckardt K, Yang Z, Eckel J. Conditioned medium obtained from in vitro differentiated adipocytes and resistin induce insulin resistance in human hepatocytes. FEBS Lett 2007; 581: 4303–4308. 4 Yarmo MN, Landry A, Molgat AS, Gagnon A, Sorisky A. Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes. Exp Cell Res 2009; 315: 411–418. 5 Jager J, Gre´meaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF. Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology 2007; 148: 241–251. 6 Krogh-Madsen R, Plomgaard P, Moller K, Mittendorfer B, Pedersen BK. Influence of TNF-alpha and IL-6 infusions on insulin sensitivity and expression of IL-18 in humans. Am J Physiol Endocrinol Metab 2006; 291: E108–E114. 7 Lagathu C, Yvan-Charvet L, Bastard JP, Maachi M, Quignard-Boulange´ A, Capeau J et al. Long-term treatment with interleukin-1beta induces insulin resistance in murine and human adipocytes. Diabetologia 2006; 49: 2162–2173. 8 Stephens JM, Lee J, Pilch PF. Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction. J Biol Chem 1997; 272: 971–976. 9 Senn JJ, Klover PJ, Nowak IA, Mooney RA. Interleukin-6 induces cellular insulin resistance in hepatocytes. Diabetes 2002; 51: 3391–3399. 10 Ward PP, Paz E, Conneely OM. Multifunctional roles of lactoferrin: a critical overview. Cell Mol Life Sci 2005; 62: 2540–2548. 11 Otsuki K, Yakuwa K, Sawada M, Hasegawa A, Sasaki Y, Mitsukawa K et al. Recombinant human lactoferrin has preventive effects on lipopolysaccharide-induced preterm delivery and production of inflammatory cytokines in mice. J Perinat Med 2005; 33: 320–323. 12 Conneely OM. Antiinflammatory activities of lactoferrin. J Am Coll Nutr 2001; 20: 389S–395S, discussion 396S-397S. 13 Hayashida K, Kaneko T, Takeuchi T, Shimizu H, Ando K, Harada E. Oral administration of lactoferrin inhibits inflammation and nociception in rat adjuvant-induced arthritis. J Vet Med Sci 2004; 66: 149–154. 14 Kruzel ML, Harari Y, Chen CY, Castro GA. The gut. A key metabolic organ protected by lactoferrin during experimental systemic inflammation in mice. Adv Exp Med Biol 1998; 443: 167–173. 15 Zimecki M, Artym J, Chodaczek G, Kocieba M, Kruzel ML. Protective effects of lactoferrin in Escherichia coli-induced bacteremia in mice: relationship to reduced serum TNF alpha level and increased turnover of neutrophils. Inflamm Res 2004; 53: 92–96. 16 Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, MattsbyBaltzer I. Lactoferrin down-regulates the LPS-induced cytokine



1000 17

18

19 20

21

22

23

production in monocytic cells via NF-kappa B. Cell Immunol 2002; 220: 83–95. Zhou Y, Zeng Z, Zhang W, Xiong W, Wu M, Tan Y et al. Lactotransferrin: a candidate tumor suppressor-deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway. Int J Cancer 2008; 123: 2065–2072. Letchoumy PV, Mohan KV, Stegeman JJ, Gelboin HV, Hara Y, Nagini S. In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion, and angiogenesis during experimental oral carcinogenesis. Oncol Res 2008; 17: 193–203. Naot D, Grey A, Reid IR, Cornish J. LactoferrinFa novel bone growth factor. Clin Med Res 2005; 3: 93–101. Maneva A, Taleva B, Maneva L. Lactoferrin-protector against oxidative stress and regulator of glycolysis in human erythrocytes. Z Naturforsch C 2003; 58: 256–262. Buren J, Lai YC, Lundgren M, Eriksson JW, Jensen J. Insulin action and signalling in fat and muscle from dexamethasone-treated rats. Arch Biochem Biophys 2008; 474: 91–101. Yagi M, Suzuki N, Takayama T, Arisue M, Kodama T, Yoda Y et al. Lactoferrin suppress the adipogenic differentiation of MC3T3G2/PA6 cells. J Oral Sci 2008; 50: 419–425. Yagi M, Suzuki N, Takayama T, Arisue M, Kodama T, Yoda Y et al. Effects of lactoferrin on the differentiation of pluripotent mesenchymal cells. Cell Biol Int 2009; 33: 283–289.


24 Rosen CJ, Bouxsein ML. Mechanisms of disease: is osteoporosis the obesity of bone? Nat Clin Pract Rheumatol 2006; 2: 35–43. 25 Gimble JM, Zvonic S, Floyd ZE, Kassem M, Nuttall ME. Playing with bone and fat. J Cell Biochem 2006; 98: 251–266. 26 Rossmeisl M, Flachs P, Brauner P, Sponarova J, Matejkova O, Prazak T et al. Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores. Int J Obes Relat Metab Disord 2004; 28 Suppl 4: S38–S44. 27 Usui I, Haruta T, Iwata M, Takano A, Uno T, Kawahara J et al. Retinoblastoma protein phosphorylation via PI 3-kinase and mTOR pathway regulates adipocyte differentiation. Biochem Biophys Res Commun 2000; 275: 115–120. 28 Abella A, Dubus P, Malumbres M, Rane SG, Kiyokawa H, Sicard A et al. Cdk4 promotes adipogenesis through PPARgamma activation. Cell Metab 2005; 2: 239–249. 29 Son HJ, Lee SH, Choi SY. Human lactoferrin controls the level of retinoblastoma protein and its activity. Biochem Cell Biol 2006; 84: 345–350. 30 Sakoda H, Ogihara T, Anai M, Funaki M, Inukai K, Katagiri H et al. Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction. Diabetes 2000; 49: 1700–1708. 31 Oda N, Nakai A, Mokuno T, Sawai Y, Nishida Y, Mano T et al. Dexamethasone-induced changes in glucose transporter 4 in rat heart muscle, skeletal muscle and adipocytes. Eur J Endocrinol 1995; 133: 121–126.