J Menopausal Med.  2015 Aug;21(2):93-103. 10.6118/jmm.2015.21.2.93.

Effect of Glucagon-like Peptide-1 on the Differentiation of Adipose-derived Stem Cells into Osteoblasts and Adipocytes

• Lee HM
• Joo BS
• Lee CH
• Kim HY
• Ock JH
• Lee YS

• KMID: 2200986
• DOI: http://doi.org/10.6118/jmm.2015.21.2.93

Abstract

OBJECTIVES
Glucagon-like peptide-1 (GLP-1) is an intestinally secreted hormone and it plays an important role in the regulation of glucose homeostasis. However, the possible role of GLP-1 in the differentiation of adipose-derived stem cells (ADSCs) remains unknown. Therefore this study investigated the effect of GLP-1 on the differentiation of ADSCs into osteoblasts and adipocytes.
METHODS
ADSCs were isolated from human adipose tissues of the abdomens, cultured and characterized by flow cytometry and multi-lineage potential assay. ADSCs were induced in osteogenic and adipogenic media treated with two different doses (10 and 100 nM) of GLP-1, and then the effect of GLP-1 on differentiation of ADSCs into osteoblast and adipocyte was examined. The signaling pathway involved in these processes was also examined.
RESULTS
Isolated human ADSCs expressed mesenchymal stem cell (MSC) specific markers as well as GLP-1 receptor (GLP-1R) proteins. They also showed multiple-lineage potential of MSC. GLP-1 was upregulated the activity and mRNA expression of osteoblast-specific marker, alkaline phosphatase and the mineralization of calcium. In contrast, GLP-1 significantly suppressed the expression of adipocyte-specific markers, peroxisome proliferator-activated receptor gamma (PPAR-gamma), lipoprotein lipase (LPL) and adipocyte protein 2 (AP2). This decreased expression of adipocyte specific markers caused by GLP-1 was significantly reversed by the treatment of extracellular signal-regulated kinase (ERK) inhibitor, PD98059 (P < 0.05).
CONCLUSION
This result demonstrates that GLP-1 stimulates osteoblast differentiation in ADSCs, whereas it inhibits adipocyte differentiation. The ERK signaling pathway seems to be involved in these differentiation processes mediated by GLP-1.

Keyword

Adipocytes; Adipogenesis; Adipose tissue; Cell differentiation; Glucagon-like peptide 1; Osteogenesis

MeSH Terms

Abdomen
Adipocytes*
Adipogenesis
Adipose Tissue
Alkaline Phosphatase
Calcium
Cell Differentiation
Flow Cytometry
Glucagon-Like Peptide 1*
Glucose
Homeostasis
Humans
Lipoprotein Lipase
Mesenchymal Stromal Cells
Osteoblasts*
Osteogenesis
Phosphotransferases
PPAR gamma
RNA, Messenger
Stem Cells*
Glucagon-Like Peptide-1 Receptor
Alkaline Phosphatase
Calcium
Glucagon-Like Peptide 1
Glucose
Lipoprotein Lipase
PPAR gamma
Phosphotransferases
RNA, Messenger

Figure

• 

  Fig. 1 Characterization of adipose-derived stem cells (ADSCs). (A) Flow cytometry analysis of ADSCs. Cells were stained with fluorescein fluorescein isothiocyanate (FITC)-labeled CD73, CD90, CD105, CD31, CD34 and CD45 antibodies. Fluorochrome-conjugated nonspecific mouse IgG1 was used as isotype controls (dotted red lines). ADSCs were stained positively for surface markers characteristic of mesenchymal stem cell, CD73, CD90 and CD105. The cells were negative for markers of the endothelial cells CD31, hematopoietic lineage CD45 and of hematopoietic stem cells CD34. (B, C) Osteogenic differentiation of ADSCs. Seven days of osteogenic induction, mRNA expressions of osteogenic markers were observed by reverse transcriptase-polymerase chain reaction (RT-PCR). Fourteen days of osteogenic induction, the deposition of calcium precipitates was stained by alizarin red S staining. (D, E) Adipogenic differentiation of ADSCs. Seven days of adipogenic induction, mRNA expressions of adipocyte markers were detected by RT-PCR. Fourteen days of adipogenic induction, the lipid droplets were stained by Oil red O staining. Original magnification, × 100. Original magnification, × 100. FITC: fluorescein isothiocyanate, OIM: osteogenic induction media, ALP: alkaline phosphatase, RUNX2: runt-related transcription factor 2, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, AIM: adipogenic induction media, AP2: adipocyte protein 2, LPL: lipoprotein lipase, PPAR-γ: peroxisome proliferator-activated receptor-gamma.

• 

  Fig. 2 Glucagon-like peptide-1 (GLP-1) receptor expression in adipose-derived stem cells (ADSCs) was detected by immunofluorescence staining (green). Nuclei were labeled with DAPI (blue). Original magnification, × 200.

• 

  Fig. 3 Effects of glucagon-like peptide-1 (GLP-1) on the osteogenic differentiation of adipose-derived stem cells (ADSCs). The ADSCs were cultured osteogenic induction medium (OIM) treated with various concentration of GLP-1 (0, 10 nM and 100 nM). The media containing GLP-1 was renewed every day. (A) The mRNA level of alkaline phosphatase (ALP) was determined by real time polymerase chain reaction (PCR) and it was normalized by the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *P < 0.05 (vs. OIM). (B, C) On day 10, ALP activity was measured by ALP staining and enzyme-linked immunosorbent assay (ELISA). ALP was indicated by the red staining. Original magnification, × 100. ALP activity was determined using a colorimetric end point assay measuring the enzyme p-nitrophenol (pNP) in the presence of ALP. (D) On day 14, cells were stained with alizarin red S and calcium deposition nodules were stained as dark red areas. Original magnification, × 100. (E) Bar graph shows quantitative results of (D). †P < 0.05 (vs. OIM).

• 

  Fig. 4 Effects of glucagon-like peptide-1 (GLP-1) on the adipogenic differentiation of adipose-derived stem cells (ADSCs). The ADSCs were induced to with adipogenic induction medium (AIM) with or without GLP-1 (0, 10 nM and 100 nM). (A, B) On day 14, cells were stained with oil red O to visualize lipid droplets. Original magnification, × 100. *P < 0.05 (vs. AIM). (C) On day 3 and 7, the mRNA levels of aP2, peroxisome proliferator-activated receptor (PPAR) and lipoprotein lipase (LPL) were determined by real time polymerase chain reaction (PCR). †P < 0.05 (vs. AIM).

• 

  Fig. 5 The treatment of extracellular signal-regulated kinase (ERK) inhibitor in adipogenic differentiation of adipose-derived stem cells (ADSCs) by glucagon-like peptide-1 (GLP-1). ADSCs were induced with adipogenic induction medium (AIM) for 7 days in the presence or absence of GLP-1 and then they were treated with ERK inhibitor PD98059. Real time polymerase chain reaction (PCR) for indicated genes were performed. *P < 0.05 (vs. GLP-1(+) / PD98059(-) group).

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