The Effect of In Vitro Diabetes-mimicking Environment on Tenocyte Differentiation and Metabolism
Date Issued
2016
Date
2016
Author(s)
Wu, Yu-Fu
Abstract
Background: Previous studies have shown strong evidence that diabetes is associated with higher risk of tendinopathy. Diabetic tendinopathy is a complex pathology that reduces tolerance to exercise and functional activities affecting lifestyle and glycemic control. However, the molecular mechanisms of diabetic tendinopathy remained to be investigated. The study aims to investigate the pathomechanisms of diabetic tendinopathy using in vitro diabetes-mimicking culture condition, and to further investigate the effects of glucose concentrations on the regulation of tendon-related genes and tenocyte metabolism. Methods: Tenocytes harvested from Achilles tendons of Sprague-Dawley rats (8 weeks old, weighing 250–300g) were used in this study. Two glucose concentrations [5.5 mM (LG) or 25 mM (HG)] and insulin (30 μg/mL) in culture medium were administered to monolayer culture of tenocytes. Glucose uptake and consumption by tenocytes was measured by 2-NBDG fluorescent indicator and medium glucose meter, respectively. Tenocytes viability and proliferation were assayed by colorimetric MTT (Tetrazolium) assay. Tenocytes apoptosis was analyzed by JC-1 assay, caspase activity assay, and TUNEL assay. The gene expression level of tenogenic transcription factors Scx, Mkx and Egr1, transforming growth factor-βs (TGF-βs), and matrix proteins Col1a1, Col1a2, Tnmd, Tn-C, Dcn, and Bgn were assayed by qRT-PCR. Mkx and Egr1 were knocked down by siRNA to analyze the functions of Mkx and Egr1 in TGF-β1 expression. Western blotting was performed to measure the activity of AMPK. Compound C was used to block AMPK activation, and changes in mRNA expression of target genes were examined. Results: Neither glucose nor insulin altered tenocyte proliferation or apoptosis during two-week culture. It was found that glucose uptake into tenocyte and medium glucose consumption were both increased in high glucose condition (HG), where the medium glucose consumption was further enhanced by insulin. HG significantly attenuated Mkx and Egr1 expression, but not Scx, and further decreased TGF-β1 and Bgn expression at day 14. Similar effects on Mkx, Egr1, TGF-β1, and Bgn were found in HG with insulin. Down-regulation of Egr1 by siRNA decreased Scx, Mkx, TGF-β1, Col1a1, Col1a2, and Bgn expression. In addition, blocking AMPK activation with Compound C down-regulated the expression of Egr1, TGF-β1, Col1a1, Col1a2, and Bgn in LG condition. Discussion and Conclusion: Our study demonstrated that high glucose concentration altered tendon homeostasis through down-regulation of Egr1, and downstream TGF-β1 and Bgn expression. The AMPK signaling pathways may be involved in the progression of diabetic tendinopathy. The present study may help understand the pathomechanisms and further develop preventive and therapeutic strategies for diabetic tendinopathy.
Subjects
diabetes
glucose
insulin
tendinopathy
tenocyte
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-105-R02b22012-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):bfadb5919df6da0480b5e079b9fa16d8