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Endocrinology, doi:10.1210/en.2007-0179
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Endocrinology Vol. 148, No. 9 4267-4275
Copyright © 2007 by The Endocrine Society

Hyperglycemia Enhances Adipogenic Induction of Lipid Accumulation: Involvement of Extracellular Signal-Regulated Protein Kinase 1/2, Phosphoinositide 3-Kinase/Akt, and Peroxisome Proliferator-Activated Receptor {gamma} Signaling

Chia Chi Chuang1, Rong Sen Yang1, Keh Sung Tsai1, Feng Ming Ho and Shing Hwa Liu

Institute of Toxicology (C.C.C., S.H.L.), and Departments of Orthopaedics (R.S.Y.) and Laboratory Medicine (K.S.T.), College of Medicine, National Taiwan University, and Departments of Surgery (S.H.L.) and Emergency Medicine (S.H.L.), National Taiwan University Hospital, Taipei 10043, Taiwan; and Department of Biomedical Engineering (F.M.H.), Chung Yuan Christian University, Chung Li 32023, Taiwan

Address all correspondence and requests for reprints to Shing-Hwa Liu, Ph.D., Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 10043, Taiwan. E-mail: shliu{at}ha.mc.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular events of hyperglycemia-triggered increase in adipogenic induction of lipid accumulation remain unclear. We examined the effects of hyperglycemia on adipogenic induction of lipid accumulation and its involved signaling molecules, such as phosphoinositide 3-kinase (PI3K), ERKs, and peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). Bone marrow-derived mesenchymal stem cells (MSCs) isolated from FVB/N mice were capable of differentiating into adipocytes in adipogenic medium. The effects of high glucose (HG) (25.5 mM) were assessed in vitro by RT-PCR, ELISA, flow cytometry, immunostaining, and immunoblotting. The in vivo effect of hyperglycemia was further studied in streptozotocin (STZ)-induced diabetic FVB/N mice. Exposure of MSCs to HG enhanced adipogenic induction of lipid accumulation as compared with 5.5 mM glucose. HG increased PPAR{gamma} expression and PI3K activity and its downstream effector Akt phosphorylation during adipogenesis. Inhibition of PI3K/Akt activity with PI3K inhibitor LY294002 or by expressing the dominant negative p85 or Akt prevented the HG-enhanced PPAR{gamma}-dependent adipogenic induction of lipid accumulation. Moreover, HG increased the phosphorylation of ERK1/2 during adipogenesis. MAPK/ERK inhibitor PD98059 inhibited the PI3K activity, Akt phosphorylation, and lipid accumulation triggered by HG. PI3K inhibitor LY294002 did not affect the HG-increased ERK1/2 phosphorylation during adipogenesis. We next observed that adipogenic induction of lipid accumulation of MSCs isolated from STZ-induced diabetic mice is enhanced. Moreover, triglyceride, PPAR{gamma} expression, phosphorylated Akt and ERK1/2, and marrow fat in bones of STZ-diabetic mice were also increased. These results suggest that hyperglycemia enhances the adipogenic induction of lipid accumulation through an ERK1/2-activated PI3K/Akt-regulated PPAR{gamma} pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIABETES MELLITUS IS a disease in which the body cannot produce or properly use insulin. Hyperglycemia may result in many changes in both gene expression and protein function, which together contribute to the pathogenesis of diabetic complications (1). Diabetic osteopenia and osteoporosis are two of the long-term complications of insulin-dependent diabetes mellitus (IDDM) (type 1), which may cause the increased fracture rate and delayed fracture healing (2, 3). Bone marrow-derived mesenchymal stem cells (MSCs) are the common progenitors for both adipocytes and osteoblasts (4, 5, 6, 7). Adipogenesis involves two major stages. The early stage contains the recruitment and proliferation of preadipocytes. The late stage is the differentiation of preadipocytes into adipocytes, including lipid accumulations (8). The increase in marrow adipogenesis associated with osteoporosis and age-related osteopenia is well known clinically (9). In a streptozotocin (STZ)-induced IDDM mouse model, it has recently been shown that IDDM contributes to bone loss through changes in marrow composition, resulting in decreased mature osteoblasts and increased adipose accumulation (10). However, the subsequent molecular events of IDDM-triggered increase in adipogenic induction of lipid accumulation remain unclear.

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a member of the nuclear receptor superfamily of transcription factors, a large and diverse group of proteins that mediate ligand-dependent transcriptional activation and repression (11). PPAR{gamma} exists in two isoforms, PPAR{gamma}1 and PPAR{gamma}2, as a result of alternative promoter usage and alternative splicing (12). The PPAR{gamma}1 isoform is expressed in many cell types, including adipocytes, osteoblasts, muscle cells, and macrophages, whereas PPAR{gamma}2 expression is restricted primarily to adipose cells and is absolutely necessary for adipocyte differentiation in mice (13). Moreover, MAPKs are able to regulate adipogenesis at each step of the process, from stem cells to adipocytes; for example, ERKs would be necessary to initiate the pre-adipocyte into the differentiation process, and, thereafter, this signal transduction pathway needs to be shutoff to proceed with adipocyte maturation (14). Several growth factors, which inhibit adipocyte maturation, caused ERK-mediated phosphorylation of the dominant adipogenic transcription factor PPAR{gamma} and reduction of its transcriptional activity (15, 16). On the other hand, phosphoinositide 3-kinase (PI3K), a lipid kinase composed of a Src homology 2 domain-containing regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), catalyzes phosphorylation of the D3 position of phosphoinositides. This enzyme is important in a wide variety of cellular processes, including intracellular trafficking, organization of the cytoskeleton, cell growth and transformation, and prevention of apoptosis (17, 18). It has recently been demonstrated that PI3K was required for murine and human adipocyte differentiation (19). Wortmannin and LY294002, the specific PI3K inhibitors, have blocked the adipocyte differentiation of 3T3-L1 cells (20, 21, 22). However, the signaling relationship among hyperglycemia, PI3K, ERK/MAPK, and PPAR{gamma} in adipocyte formation and adipogenic induction of lipid accumulation in MSCs remains unknown.

Together, in the present study, we try to investigate the effect of hyperglycemia on adipocyte formation and adipogenic induction of lipid accumulation in MSCs. In addition, the role of the signaling pathways involving PI3K, ERK/MAPK, and PPAR{gamma} involved in the regulation of adipogenesis and lipid accumulation under hyperglycemia was also examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of mouse bone marrow-derived MSCs
Adult male FVB/N mice (6–8 wk old) are used for isolation of MSCs. Mice were purchased from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). The Animal Research Committee of College of Medicine, National Taiwan University, approved and conducted the study in accordance with the guideline for the care and use of laboratory animals. The animals were killed by cervical dislocation, and bone marrow was flushed out of tibiae and femora. After washing by centrifugation at 400 x g for 10 min, cells were resuspended in primary culture medium to initiate a MSC culture. Primary culture medium consisted of MEM {alpha} medium (MEM {alpha}; Life Technologies, Inc., Gaithersburg, MD) containing 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 52 mM NaHCO3. The culture was kept in a humidified 5% CO2 incubator at 37 C for 72 h, when nonadherent cells were removed by changing the medium. At d 7 in culture, MSCs were washed, harvested with 0.02% EDTA in PBS, and seeded at 1 x 104 cells/cm2 to another culture dish with adipogenic medium.

Adipogenic induction of lipid accumulation and oil red O staining
MSCs were cultured in adipogenic medium, which was primary culture medium supplemented with 10–8 M dexamethasone and 5 µg/ml insulin, a slight modification of a previously described protocol (23). MSCs were cultured on cover glass in six-well plates and incubated for various time courses in adipogenic medium. Adipocytes were easily discerned from the undifferentiated cells by phase-contrast microscopy. To confirm further their identity, cells on cover glasses were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature and stained with oil red O solution for 30 min. The oil red O solution was prepared by 0.5% oil red O diluted 3:2 with distilled water, allowed to stand for 5 min, and then filtered through 90-mm paper. Staining was quantified by collecting fixed, stained cells with 0.2 ml isopropyl alcohol; after being air dried, the absorbance at 490 nm was read (peak absorbance for oil red O).

Flow cytometry for the detection of adipogenesis
This method was performed using a protocol modified from Gimble et al. (24). MSCs in six-well plates were incubated in adipogenic medium for 12 d with high glucose (HG) (25.5 mM) or high mannitol (HM) (25.5 mM). Cells were carefully harvested by treatment with 0.25% trypsin/EDTA and centrifuged at 200 x g at 4 C for 5 min. After washing the pellet with PBS, cells were centrifuged as described previously and resuspended in PBS containing the lipophilic fluorescent dye Nile Red on ice for 30 min. Samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Nile Red fluorescence was measured on the FL2 emission channel through a 585 ± 21-nm band pass filter, after excitation with an argon ion laser source at 488 nm. For each sample, 104 cells were collected. To determine the number of adipocytes in each sample, a selection marker M1 was set in histograms. The percentage of adipocytes was assessed by determining the percentage of cells within the M1 region.

Immunoblotting analysis
Cells were harvested by scraping in 50-µl ice-cold radioimmunoprecipitation (RIPA) buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Nonidet P-40, 1 mM orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 mM sodium fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin], incubated for 15 min at 4 C, and centrifuged at 12,000 x g for 20 min. In some experiments, bone samples were snap frozen in liquid nitrogen, pulverized, and also incubated with RIPA buffer for 15 min at 4 C, then centrifuged at 12,000 x g for 20 min. The supernatant was collected and stored at –80 C until use. The protein content of each lysate was measured using a commercial assay kit (BCA Protein Assay Kit; PIERCE, Rockford, IL) as described by the manufacturer. Equal amounts of cell lysate (40 µg proteins per lane) were subjected to 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore Co., Billerica, MA). The membranes were blocked with 5% fat-free milk in 0.1% PBS-Tween 20 (PBST) for 1 h. After blocking, blots were incubated with anti-PPAR{gamma}, anti-PI3K (p85), anti-{alpha}-tubulin, anti-ERK1/2, anti-phospho-ERK1/2 (Santa Cruz Biochemicals, Santa Cruz, CA), anti-Akt, anti-phospho-Akt, and anti-phospho-GSK3ß (New England BioLabs, Beverly, MA) antibodies in 0.1% PBST for 1 h, followed by three 10-min washes in 0.1% PBST. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biochemicals) for 1 h. Enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) were used to depict the protein bands on membranes.

PI3K activity assay
PI3K activity was measured using an ELISA kit (Echelon Biosciences, Inc., Salt Lake City, UT) according to the supplier’s protocol. In brief, cells were rinsed with buffer A [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, and 0.1 mM sodium orthovanadate] and harvested in lysis buffer (buffer A plus 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 4 C for 20 min at 12,000 x g, protein concentrations of the supernatants were measured, and equal amounts were incubated with immobilized anti-p85{alpha} antibody overnight. Immune complexes were washed three times with buffer A plus 1% Nonidet P-40; three times with buffer containing 100 mM Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate, and twice with buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate. The complexes were then incubated with a reaction mixture containing PtdIns(4,5)P2 substrate and ATP. The reaction mixtures were first incubated 3 h later with antibody to PtdIns(3,4,5)P3 and then added to the PtdIns(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary antibody and colorimetric detection were used to detect anti-PtdIns(3,4,5)P3 binding to the plate. The colorimetric signal was inversely proportional to the amount of PtdIns(3,4,5)P3 produced by activated PI3K.

RNA extraction and RT-PCR
Cells were seeded in a 10-cm dish. Total RNA was extracted using the TRIzol method as recommended by the manufacturer (Invitrogen, Carlsbad, CA). The yield and purity of RNA were estimated spectrophotometrically using the A260/A280 ratio. Five micrograms of RNA were reverse transcribed to cDNA by random hexamer primers (Promega Corp., Madison, WI) and murine leukemia virus reverse transcriptase (Invitrogen). Two micrograms of cDNA were amplified for 30 cycles using the specific primers. The primer pairs used were: PPAR{gamma} 1, forward primer (5'-TTCTGACAGGACTGTGTGACAG-3'), reverse primer (5'-ATAAGGTG-GAGATGCAGGTTC-3'); PPAR{gamma} 2, forward primer (5'-GCTGTTATGGG-TGAAACTCTG-3'), reverse primer (5'-ATAAGGTGGAGATGCAGGTTC-3'); osteocalcin, forward primer (5'-AGGTAGTGAACAGACTCCGGCG-3'), reverse primer (5'-GGAGCTGCTGT-GACATCCATA-C-3'); and ß-actin, forward primer (5'-TTGTAACCAACTGGGACG-ATAT-3'), reverse primer (5'-GATC-TTGATCTTCATGGTGCTA-3'). The cycling parameter was the following: 1 min at 94 C for denaturation, 1 min at 55 C for primer annealing, and 1 min at 72 C for polymerization. The PCR products were electrophoresed through a 1.5% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining.

Transient transfection with dominant-negative vectors DN-p85 and DN-Akt
Cells were seeded in a six-well plate and transfected with 1 µg of plasmids containing the DN-p85 ({delta}-p85) or DN-Akt (Akt K179A), kindly provided by Dr. M. L. Kuo (Institute of Toxicology, National Taiwan University, Taiwan) (25, 26, 27), or pcDNA3 control vector using the Effectene Transfection Reagent (QIAGEN, Valencia, CA). Transfections were performed in triplicate. Twenty-four hours after transfection, the primary culture medium was replaced with adipogenic medium. The efficiency of transfection (~80%) was determined using an equal amount of a plasmid encoding the green fluorescent protein under the cytomegalovirus promoter.

STZ-diabetic mouse model
FVB/N mice were injected ip with 40 mg/kg STZ daily for d 1–5 (28). STZ was dissolved in sodium citrate buffer (pH 4.5) and injected within 15 min of preparation. One week after STZ injection, the blood glucose levels reached more than 400 mg/dl, and the blood insulin levels decreased to about 0.5 µg/liter. Age-matched mice were treated with vehicle. After 4 wk, the mice were killed to analysis of adipocyte differentiation of MSCs and bone or liver lipid accumulation.

Determination of bone triglyceride and marrow adipocyte and liver fat
Tibiae were harvested from the proximal metaphysis to the tibiofibular junction excluding all cartilaginous and soft tissues. The triglyceride levels of tibiae were determined by Triglycerides Liquicolormono (Human, Wiesbaden, Germany) after being snap frozen and pulverized. On the other hand, tibiae were decalcified in formic acid, embedded in paraffin, and sectioned at 5 µm as previously described (29). Histological sections were stained with hematoxylin and eosin. In some experiments, livers were collected immediately after the mice were killed, snap frozen, and sectioned. Sectioned livers of representative animals were fixed with formalin (37%), stained with oil red O for fat, and counterstained with methyl green. Fat cells present within the tissue area were shown at x200 magnification.

Statistical analyses
The values given in this article are presented as mean ± SEM. All analyses were performed by ANOVA followed by a Fisher’s least significant difference test. P < 0.05 was viewed as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HG enhances adipocyte formation and adipogenic induction of lipid accumulation and PPAR{gamma} expression in MSCs
MSCs were cultured in adipogenic medium contained dexamethasone and insulin for 12 d. A significant increase in adipocyte formation and adipogenic induction of lipid accumulation was detected as determined by oil red O staining and FACS analysis (Fig. 1Go). HG significantly enhanced adipogenesis of MSCs. Unlike HG, the addition of HM (25.5 mM) to the adipogenic medium did not enhance adipogenesis of MSCs, suggesting that the HG-enhanced adipogenesis was not the result of high osmolality within the medium (Fig. 1Go). GW9662 (20 µM), an irreversible inhibitor of PPAR{gamma}, alone did not affect basal adipogenesis but significantly depressed HG-enhanced adipocyte formation and adipogenic induction of lipid accumulation. These results indicate that the PPAR{gamma}-related pathway may be involved in the HG-enhanced adipocyte formation and adipogenic induction of lipid accumulation.


Figure 1
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  		FIG. 1. HG enhances adipogenesis of MSCs and adipogenic induction of lipid accumulation. The 6- to 8-wk-old FVB/N mice were killed, and MSCs were flushed out of tibiae and femora. Cells were treated with dexamethasone (10–8 M) and insulin (5 µg/ml) for adipogenesis. A, Oil red O-stained adipogenesis of MSCs on d 12 of differentiation in the presence or absence of HG (25.5 mM) or HM (25.5 mM) with or without GW9662 (GW) (20 µM) were quantified by measuring the absorbance at 490 nm. B-a, Adipogenesis of MSCs on d 12 of differentiation was detected by Nile Red flow cytometry. B-b, Quantification of Nile Red flow cytometry analysis. Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with control in adipogenic medium without any drug treatment. #, P < 0.05 as compared with HG group in adipogenic medium.

 
Moreover, we assessed the expression of PPAR{gamma} during HG-enhanced the late stage of adipogenesis of MSCs in adipogenic medium. HG significantly increased the expression of PPAR{gamma}, especially the PPAR{gamma}2 isoform [Fig. 2Go, A (protein expression) and B (mRNA expression)]. HM did not increase the expression of PPAR{gamma}. Treatment with GW9662 (20 µM) alone caused a small decrease in PPAR{gamma} expression but markedly decreased HG-increased PPAR{gamma} expression (Fig. 2Go).


Figure 2
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  		FIG. 2. HG up-regulates the PPAR{gamma} protein expression. Cells were treated with HG (25.5 mM) in adipogenic medium with or without GW9662 (20 µM) for 12 d, and detected the PPAR{gamma} protein expression by immunoblotting (A) or the PPAR{gamma}1 and PPAR{gamma}2 mRNA by RT-PCR (B), as described in Materials and Methods. A, Quantification of the PPAR{gamma} protein expression was performed by densitometric analysis. Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with control in adipogenic medium without any drug treatment. #, P < 0.05 as compared with HG group in adipogenic medium. B, Results shown are representative of at least four independent experiments.

 
PI3K/Akt is critical for HG-enhanced PPAR{gamma} expression
The PI3K/Akt signaling has been identified to promote the adipogenesis (22, 30, 31, 32). Therefore, we tested the role of PI3K/Akt signaling in the HG-enhanced adipogenesis and PPAR{gamma} expression. PI3K inhibitor LY294002 (7.5 µM) markedly depressed HG-enhanced adipogenesis (Fig. 3AGo). HG also increased the PI3K activity and Akt phosphorylation in MSCs under adipogenic medium for 12 d, which could be reversed by the treatment of LY294002 (7.5 µM) (Fig. 3Go, B and C). LY294002 could also inhibit the HG-enhanced PPAR{gamma} expression (Fig. 4Go).


Figure 3
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  		FIG. 3. The role of PI3K/Akt signaling in HG-enhanced adipogenesis. Cells were treated with HG (25.5 mM) in adipogenic medium with or without LY294002 (7.5 µM) or PD98059 (20 µM) for 12 d. PI3K inhibitor suppressed HG-enhanced adipogenesis (A). Adipogenesis of MSCs was measured by Nile Red flow cytometry analysis. HG activates PI3K activity (B) and Akt phosphorylation (C). Quantification of the phospho-Akt protein expression was performed by densitometric analysis. Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with control in adipogenic medium without any drug treatment. #, P ≤ 0.05 as compared with HG group in adipogenic medium.

 

Figure 4
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  		FIG. 4. PI3K inhibitor inhibits the HG-enhanced PPAR{gamma} expression during adipogenesis. Cells were cultured in adipogenic medium with HG (25.5 mM) or HM (25.5 mM) in the absence or presence of LY294002 (7.5 µM) for 12 d. Cell lysates were prepared to perform immunoblot analysis using the anti-PPAR{gamma} antibody (upper panel). Quantification of the PPAR{gamma} protein expression was performed by densitometric analysis (lower panel). Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with control in adipogenic medium without any drug treatment. #, P < 0.05 as compared with HG group in adipogenic medium.

 
The next aim of the investigation was to ascertain whether PI3K/Akt activity inhibition might affect the HG-enhanced PPAR{gamma} expression. To address this issue, MSCs were transfected with a dominant negative form of the regulatory subunit (p85) of PI3K (DN-p85) or a dominant negative form of Akt (DN-Akt). The results revealed that DN-p85 (Fig. 5AGo) and DN-Akt (Fig. 5BGo), but not the control pcDNA3 transfection, significantly inhibited the HG-enhanced PPAR{gamma} expressions. The expression levels of PI3K subunit p85 protein in DN-p85-transfected cells or Akt in DN-Akt-transfected cells were increased as compared with pcDNA3-transfected cells. The abilities of the transfected mutants to block PI3K and Akt activation were demonstrated by the decrease in Akt and GSK3ß phosphorylation, respectively (Fig. 5Go).


Figure 5
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  		FIG. 5. HG-enhanced increase of PPAR{gamma} expression requires the activation of PI3K/Akt signaling. A and B, Cells were transiently transfected with pcDNA3 (control vector) or DN-p85 (A) and DN-Akt (B), and then the cells were cultured with adipogenic medium contained HG (25.5 mM) for 12 d. Cell lysates were prepared to perform immunoblot analysis using the anti-PPAR{gamma}, anti-p85, anti-Akt, anti-phospho-Akt, or anti-phospho-GSK3ß antibodies. Quantification of the PPAR{gamma} protein expression was performed by densitometric analysis. Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with control in adipogenic medium without any drug treatment. #, P < 0.05 as compared with HG group in adipogenic medium.

 
HG enhances the adipogenic induction of lipid accumulation via the ERK1/2-mediated PI3K/Akt pathway
Next, we investigate the role of ERK/MAPK in the HG-enhanced adipogenic induction of lipid accumulation. MSCs were treated with HG in adipogenic medium in the presence or absence of PD98059 (20 µM), a specific inhibitor of ERK/MAPK, for 3 h to 7 d. The up-regulation of ERK1/2 phosphorylation was shown in the cells treated with HG, which could be reversed by the treatment of PD98059 (Fig. 6AGo). The ERK1/2 phosphorylation was not affected in cells treated with HM for 2–7 d (Fig. 6AGo, lower panel). Furthermore, the role of ERK in the HG-enhanced PI3K activation and adipogenic induction of lipid accumulation was elucidated. PD98059 markedly inhibited the HG-enhanced PI3K activity (Fig. 3BGo) and Akt phosphorylation (Fig. 6BGo), and adipogenic induction of lipid accumulation (Fig. 6DGo) of MSCs. However, LY294002 (7.5 µM) did not inhibit the HG-enhanced ERK phosphorylation (Fig. 6CGo).


Figure 6
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  		FIG. 6. The activation of ERK1/2 enhanced by HG increases Akt phosphorylation and adipogenic induction of lipid accumulation. Cells were treated with or without HG (25.5 mM) or HM (25.5 mM) in adipogenic medium in the presence or absence of PD98059 (20 µM). A, Cell lysates were prepared at the indicated times, and subjected to immunoblot analysis using the anti-ERK1/2 and anti-phospho-ERK1/2 antibodies. B, Cell lysates were prepared at d 12, and subjected to immunoblot analysis using the anti-Akt and anti-phospho-Akt antibodies. C, Cell lysates were prepared at d 7, and subjected to immunoblot analysis using the anti-ERK1/2 and anti-phospho-ERK1/2 antibodies. Results shown are representative of at least four independent experiments. D, PD98059 reversed HG-enhanced increase of adipogenesis on d 12 of differentiation. Adipocyte differentiation of MSCs was quantified in oil red O stain by measuring absorbance of light at 490 nm. Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with adipogenesis on d 12 without PD98059.

 
Effect of hyperglycemia on the adipogenic induction of lipid accumulation in vivo
We next investigate the adipogenic induction of lipid accumulation of MSCs isolated from STZ-induced diabetic mice in the presence or absence of 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2, as a positive control), which has been identified as an endogenous ligand of PPAR{gamma} and inducing adipogenesis in vitro (33), as compared with age-matched control mice. The results showed that adipogenic induction of lipid accumulation of MSCs was increased under STZ-diabetic hyperglycemic condition (Fig. 7AGo). 15d-PGJ2 could enhance adipogenic induction of lipid accumulation. On the other hand, for mimicking the condition of STZ diabetes (hyperglycemia and low insulin), the isolated MSCs were cultured in adipogenic medium with low insulin (0.5 µg/liter) in the presence of HG or rosiglitazone (a PPAR{gamma} agonist, as a positive control). Both HG and rosiglitazone could enhance adipogenic induction of lipid accumulation of MSCs in adipogenic medium with low insulin concentration (Fig. 7BGo). Moreover, the levels of PPAR{gamma}, phosphorylated Akt, phosphorylated ERK1/2, triglyceride, and marrow fat in the bones of STZ-induced diabetic mice were also increased (Fig. 7Go, C–E). Histological examination also revealed that fat liver content is significantly increased in STZ-diabetic mice compared with the age-matched control mice (Fig. 7EGo, lower panel).


Figure 7
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  		FIG. 7. Effects of hyperglycemia on the adipogenic induction of lipid accumulation in vivo. Hyperglycemia was induced by STZ treatment in mice. A, MSCs were flushed out of tibiae and femora for analysis of adipogenesis in the presence or absence of 15d-PGJ2 (1 µM). B, Isolated MSCs were cultured in adipogenic medium with low insulin in the presence or absence of HG (25.5 mM) and rosiglitazone (5 µM). C and D, The tibial bones were harvested from the proximal metaphysis to the tibiofibular junction, excluding all cartilaginous and soft tissues. After being snap frozen in liquid nitrogen and pulverized, the specimens were detected for expressions of PPAR{gamma}, phosphor-Akt, and phosphor-ERK1/2 (C) or determined the amount of triglyceride (D). A and C, Data are presented as mean ± SEM from three to five independent experiments. *, P < 0.05 as compared with age-matched control mice. C, Results shown are representative of at least four independent experiments. E, Histological sections of bone and liver of representative animals were performed for analysis of adipocytes. Marrow adipocytes present in the white globular areas were recognized by the specific appearance of the space previously occupied by them. Sectioned livers were fixed with formalin, stained with oil red O for fat. Magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrated for the first time that HG enhances an ERK-mediated PI3K/Akt-dependent up-regulation of PPAR{gamma} expression, as well as an increase of adipocyte formation and adipogenic induction of lipid accumulation in MSCs under adipogenic medium. These findings are supported by functional studies of MSCs in adipogenic medium, showing that the activities of PI3K and Akt are enhanced by HG, and the inhibition of PI3K/Akt activity with LY294002 or by expressing the dominant negative p85 or Akt is capable of preventing the HG-enhanced PPAR{gamma} expression and adipogenic induction of lipid accumulation. Moreover, HG increased the activation of ERK1/2 signaling, and MAPK/ERK inhibitor could inhibit the PI3K/Akt activities and adipogenic induction of lipid accumulation of MSCs in adipogenic medium. Therefore, activation of the ERK-mediated PI3K/Akt pathway may represent a proximal node in the intracellular signaling, leading to hyperglycemia-enhanced adipogenic induction of lipid accumulation.

MSCs are capable of differentiating into a variety of lineages, including cartilage, bone, fat, muscle, and other connective tissues, depending on culture conditions, which include supplementation of lineage-specific inducing agents, as well as hormones and growth factors (34, 35, 36). Adipogenesis of MSCs could be induced by an adipogenic hormonal cocktail containing dexamethasone, insulin, 3-isobutyl-l-methylxanthine, and indomethacin (36). The inducing effects of dexamethasone and 3-isobutyl-l-methylxanthine are still not well understood and are controversially discussed (36, 37). Moreover, indomethacin has been demonstrated to bind and activate PPAR{gamma}, a ligand-activated transcription factor known to play a pivotal role in adipogenesis (38). In the present study, we used only dexamethasone and insulin as inducers in adipogenic medium, which excluded the PPAR{gamma} ligand effect induced by indomethacin. Our results showed enhancing effects of HG on adipogenic induction of lipid accumulation in response to the activation of PPAR{gamma}.

Adipogenesis is a complex process involving differentiation of MSCs to mature adipocyte. Adipogenesis is regulated by two important transcription factors: CCAAT/enhancer binding proteins (C/EBPs), specifically C/EBP{alpha}, C/EBPß, and C/EBP{delta}; and PPAR, specifically PPAR{gamma}. Upon the addition of adipogenic cocktail to 3T3-L1 preadipocytes, there is a rapid and transient induction of C/EBPß and C/EBP{delta}, which precedes expression of the two master regulators of terminal adipogenesis, PPAR{gamma} and C/EBP{alpha} expression (39, 40, 41). Activated PPAR{gamma} controls the late-stage adipogenesis by inducing expression of C/EBP{alpha}, which is required for the production of specific adipogenic genes. Recently, it has also been demonstrated a role for MEK/ERK signaling and C/EBPß in regulating expression of PPAR{gamma} during adipogenesis (42). MAPK pathways are able to regulate adipogenesis at each step of the process, from stem cells to adipocytes, whereas the ERK pathway is involved throughout adipocyte differentiation of MSCs, displaying both positive and negative effects (14). The study of Bost et al. (14) has shown an essential and specific role for ERK pathway in the early proliferative stage of adipogenesis, thereby, extending the role of this pathway throughout adipogenesis. They have also found that PD98059 did not affect neurogenesis, myogenesis, or the formation of cardiomyocytes. On the other hand, it has been shown that insulin receptor substrate-1/PI3k/Akt activation was an essential requirement for insulin stimulation of lipid synthesis in brown adipocytes (43). Insulin and TNF-{alpha} have also induced expression of the forkhead transcription factor gene Foxc2 in 3T3-L1 adipocytes via PI3K and ERK1/2-dependent pathways (44). The activation of signaling pathways by hyperglycemia is not clear. Understanding the mechanism for this signaling activity is required to understand the changes in lipogenesis. In the present study, we found that hyperglycemia enhanced the up-regulation of ERK1/2 phosphorylation during adipogenesis in vitro and in vivo. PD98059 markedly inhibited the HG-enhanced PI3K/Akt activities and adipogenic induction of lipid accumulation, but LY294002 could not inhibit the HG-enhanced ERK1/2 activation. These results suggest that HG enhances the adipogenic induction of lipid accumulation through an increase of ERK-mediated PI3K/Akt-dependent PPAR{gamma} pathway. On the other hand, it has been reported that in vitro adipogenic differentiation is impaired in primary embryonic fibroblasts isolated from Akt1/Akt2 double knockout mice (45). However, the current data do not seem to suggest that PI3K activation is required for adipogenic differentiation under the experimental condition of this study (Fig. 3AGo) but is required for the hyperglycemia-dependent increase in adipogenic induction of lipid accumulation.

Thiazolidinediones are a new class of antidiabetic agents. They act by binding to PPAR{gamma}, and can improve different insulin-resistant states in both human and animal studies (46). It has been demonstrated that PPAR{gamma} activated with rosiglitazone acted as a dominant inhibitor of osteoblastogenesis in murine bone marrow in vitro and in vivo (47, 48). The adult animals receiving rosiglitazone have exhibited bone loss characterized by the increase in marrow adipocytes, and the decrease in the osteoblast to osteoclast ratio and bone formation rate (48, 49). IDDM has been recently demonstrated to contribute to bone loss through changes in marrow composition, resulting in decreased mature osteoblasts and increased adipose accumulation (10). In the present study, we also found that the expression of PPAR{gamma} and the levels of triglyceride and marrow fat in the bones of STZ-induced diabetic mice are increased. Therefore, the role of PPAR{gamma}-related signaling pathways in the hyperglycemia-enhanced osteoblastogenesis inhibition may be valuable to investigate in the future.

In conclusion, our findings suggest that hyperglycemia accelerates the adipogenic induction of lipid accumulation, through an ERK1/2-mediated PI3K/Akt pathway that results in an increase of PPAR{gamma} expression.


   Footnotes
 
This work was supported by research grants from National Science Council of Taiwan (NSC94-2314-B-002-096).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 31, 2007

1 C.C.C, R.S.Y., and K.S.T. contributed equally to this work. Back

Abbreviations: EBP, Enhancer binding protein; HG, high glucose; HM, high mannitol; IDDM, insulin-dependent diabetes mellitus; MSC, mesenchymal stem cell; PBST, PBS-Tween 20; PI3K, phosphoinositide 3-kinase; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; STZ, streptozotocin.

Received February 7, 2007.

Accepted for publication May 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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