Diabetes Metab J.  2021 Mar;45(2):260-269. 10.4093/dmj.2019.0191.

Umbilical Cord-Mesenchymal Stem Cell-Conditioned Medium Improves Insulin Resistance in C2C12 Cell

• Kim KS
• Choi YK
• Kim MJ
• Hwang JW
• Min K
• Jung SY
• Kim SK
• Choi YS
• Cho YW

• KMID: 2514202
• DOI: http://doi.org/10.4093/dmj.2019.0191

Abstract

Umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) has emerged as a promising cell-free therapy. The aim of this study was to explore the therapeutic effects of UC-MSC-CM on insulin resistance in C2C12 cell.

Insulin resistance was induced by palmitate. Effects of UC-MSC-CM on insulin resistance were evaluated using glucose uptake, glucose transporter type 4 (GLUT4) translocation, the insulin-signaling pathway, and mitochondrial contents and functions in C2C12 cell.

Glucose uptake was improved by UC-MSC-CM. UC-MSC-CM treatment increased only in membranous GLUT4 expression, not in cytosolic GLUT4 expression. It restored the insulin-signaling pathway in insulin receptor substrate 1 and protein kinase B. Mitochondrial contents evaluated by mitochondrial transcription factor A, mitochondrial DNA copy number, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha were increased by UC-MSC-CM. In addition, UC-MSC-CM significantly decreased mitochondrial reactive oxygen species and increased fatty acid oxidation and mitochondrial membrane potential. There was no improvement in adenosine triphosphate (ATP) contents, but ATP synthesis was improved by UC-MSC-CM. Cytokine and active factor analysis of UC-MSC-CM showed that it contained many regulators inhibiting insulin resistance.

UC-MSC-CM improves insulin resistance with multiple mechanisms in C2C12 cell.

Keyword

Culture media, conditioned; Diabetes mellitus; Insulin resistance; Mesenchymal stem cells; Mitochondria; Muscles; Umbilical cord; Wharton jelly

Figure

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  Fig. 1 The effect of umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) on glucose uptake in C2C12 myotubes. The results are expressed as the fold change of the mean±standard error (n≥3). 2-DG, 2-deoxy glucose. aP<0.05, vs. insulin-unstimulated group, bP<0.05, vs. insulin-stimulated group, cP<0.05, vs. insulin and palmitate treated group.

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  Fig. 2 The effect of umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) on protein expression of glucose transporter type 4 (GLUT4). (A) Western blot. (B) Protein expression of membranous GLUT4. (C) Protein expression of cytosolic GLUT4. The results are expressed as the fold change of the mean±standard error (n≥3). aP<0.05, vs. insulin-stimulated group, bP<0.05, vs. insulin and palmitate treated group.

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  Fig. 3 The effect of umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) on the insulin-signaling pathway. (A) Representative Western blots for figure B–E. (B, C) Protein expression of insulin receptor substrate 1 (IRS1) at Ser612 (B) and Ser307 (C). (D) Protein expression of phosphoinositide 3-kinase (PI3K). (E) Protein expression of phosphorylated protein kinase B (p-AKT). The results are expressed as the fold change of the mean±standard error (n≥3). aP<0.05, vs. insulin-stimulated group, bP<0.05, vs. insulin and palmitate treated group.

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  Fig. 4 The effect of umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) on mitochondrial contents and functions. (A) Protein level of mitochondrial transcription factor A (mtTFA). (B) The relative level of mitochondrial DNA (mtDNA) copy number. (C) Protein level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). (D) Mitochondrial reactive oxygen species (ROS). (E) Intracellular ROS. (F) Fatty acid oxidation. (G) Mitochondrial membrane potential. (H) Adenosine triphosphate (ATP) contents. (I) ATP synthesis. The results are expressed as the fold change of the mean±standard error (n≥3). CCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. aP<0.05, vs. insulin-stimulated group, bP<0.05, vs. insulin and palmitate treated group.

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  Fig. 5 Analysis of secreted factors in umbilical cord mesenchymal stem cell-conditioned medium. EDA-A2, ectodysplasin-A2; IGFBP, insulin-like growth factor binding protein; TIMP, tissfATCC, Manassas, VA, USA) and maintained in Dulbeccoue inhibitor of metalloproteinases; IL-6, interleukin-6; MIP2, macrophage inflammatory protein 2; SPARC, secreted protein acidic and rich in cysteine; GRO, growth related oncogene; MCP, monocyte chemoattractant protein; Dkk, Dickkopf-related protein; VEGF, vascular endothelial growth factor; GDF, growth differentiation factor; HGF, hepatocyte growth factor; MMP-1, matrix metalloproteinase-1; FGF, fibroblast growth factor; KGF, keratinocyte growth factor.

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