Tan, Hui Yee and Tan, Sik Loo and Teo, Seow Hui and Roebuck, Margaret M. and Frostick, Simon P. and Kamarul, Tunku (2020) Development of a novel in vitro insulin resistance model in primary human tenocytes for diabetic tendinopathy research. PEERJ, 8. ISSN 21678359,
Full text not available from this repository.Abstract
Background. Type 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-alpha) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy. Methods. hTeno were isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-beta) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 mu M of TNF-alpha, with and without insulin supplement. Outcome measures include 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-alpha. Apoptosis assay for hTeno induced with TNF-alpha was conducted using Annexin-V FITC flow cytometry analysis. Results. Immunofluorescence imaging showed the presence of INSR-beta on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-alpha significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 mM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 mM of TNF-alpha. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-alpha treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF-alpha treated cells. TNF-alpha progressively increased the apoptotic cells at 48 and 72 h. Conclusion. At 0.008 mu M of TNF-alpha, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.
| Item Type: | Article |
|---|---|
| Funders: | None |
| Uncontrolled Keywords: | Tendon; Tenocyte; Insulin resistance; Obese; Orthopaedics; Cellular biology; Tumor necrosis factor-alpha (TNF-alpha); Glucose uptake; Type II diabetes; Hyperglycemia |
| Subjects: | R Medicine R Medicine > R Medicine (General) > Medical technology |
| Divisions: | Faculty of Medicine > Orthopaedic Surgery Department |
| Depositing User: | Ms Zaharah Ramly |
| Date Deposited: | 13 Aug 2024 02:48 |
| Last Modified: | 13 Aug 2024 02:48 |
| URI: | http://eprints.um.edu.my/id/eprint/36618 |
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