Diabetes & Metabolism Journal

Original Article

An In Vitro Model to Probe the Regulation of Adipocyte Differentiation under Hyperglycemia: Kusampudi Shilpa, Thangaraj Dinesh, Baddireddi Subhadra Lakshmi; Diabetes Metab J. 2013;37(3):176-180. Published online June 14, 2013; DOI: https://doi.org/10.4093/dmj.2013.37.3.176

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9 Crossref

Background

The aim of this study was an in vitro investigation of the effect of high glucose concentration on adipogenesis, as prolonged hyperglycemia alters adipocyte differentiation.

Methods

3T3-L1 preadipocytes differentiated in the presence of varying concentrations of glucose (25, 45, 65, 85, and 105 mM) were assessed for adipogenesis using AdipoRed (Lonza) assay. Cell viability and proliferation were measured using MTT reduction and [³H] thymidine incorporation assay. The extent of glucose uptake and glycogen synthesis were measured using radiolabelled 2-deoxy-D-[1-³H] glucose and [¹⁴C]-UDP-glucose. The gene level expression was evaluated using reverse transcription-polymerase chain reaction and protein expression was studied using Western blot analysis.

Results

Glucose at 105 mM concentration was observed to inhibit adipogenesis through inhibition of CCAAT-enhancer-binding proteins, sterol regulatory element-binding protein, peroxisome proliferator-activated receptor and adiponectin. High concentration of glucose induced stress by increasing levels of toll-like receptor 4, nuclear factor κB and tumor necrosis factor α thereby generating activated preadipocytes. These cells entered the state of hyperplasia through inhibition of p27 and proliferation was found to increase through activation of protein kinase B via phosphoinositide 3 kinase dependent pathway. This condition inhibited insulin signaling through decrease in insulin receptor β. Although the glucose transporter 4 (GLUT4) protein remained unaltered with the glycogen synthesis inhibited, the cells were found to exhibit an increase in glucose uptake via GLUT1.

Conclusion

Adipogenesis in the presence of 105 mM glucose leads to an uncontrolled proliferation of activated preadipocytes providing an insight towards understanding obesity.

Citations

Citations to this article as recorded by

Interaction of high lipogenic states with titanium on osteogenesis
T.S. Pinto, B.C. van der Eerden, M. Schreuders-Koedam, J. van de Peppel, I. Ayada, Q. Pan, M.M. Verstegen, L.J. van der Laan, G.M. Fuhler, W.F. Zambuzzi, M.P. Peppelenbosch
Bone.2024; 188: 117242. CrossRef
Adipogenesis-Related Metabolic Condition Affects Shear-Stressed Endothelial Cells Activity Responding to Titanium
Thaís Silva Pinto, Anderson Moreira Gomes, Paula Bertin de Morais, Willian F. Zambuzzi
Journal of Functional Biomaterials.2023; 14(3): 162. CrossRef
Chronic and Transient Hyperglycemia Induces Changes in the Expression Patterns of IL6 and ADIPOQ Genes and Their Associated Epigenetic Modifications in Differentiating Human Visceral Adipocytes
Adam Wróblewski, Justyna Strycharz, Ewa Świderska, Aneta Balcerczyk, Janusz Szemraj, Józef Drzewoski, Agnieszka Śliwińska
International Journal of Molecular Sciences.2021; 22(13): 6964. CrossRef
Effects of high glucose conditions on the expansion and differentiation capabilities of mesenchymal stromal cells derived from rat endosteal niche
Ahmed Makki A. Al-Qarakhli, Norhayati Yusop, Rachel J. Waddington, Ryan Moseley
BMC Molecular and Cell Biology.2019;[Epub] CrossRef
Inhibition of WNT/β-catenin signaling under serum starvation and hypoxia induces adipocytic transdifferentiation in human leiomyoma cells
Hiroshi Harada, Yojiro Tsuda, Kei Yabuki, Eisuke Shiba, Kazuyoshi Uchihashi, Atsuji Matsuyama, Yoshihisa Fujino, Toru Hachisuga, Masanori Hisaoka
Laboratory Investigation.2018; 98(4): 439. CrossRef
Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes
Sara Palacios-Ortega, Maider Varela-Guruceaga, J. Alfredo Martínez, Carlos de Miguel, Fermín I. Milagro
Adipocyte.2016; 5(1): 65. CrossRef
Pathophysiological role of enhanced bone marrow adipogenesis in diabetic complications
Meghan A Piccinin, Zia A Khan
Adipocyte.2014; 3(4): 263. CrossRef
Letter: AnIn VitroModel to Probe the Regulation of Adipocyte Differentiation under Hyperglycemia (Diabetes Metab J2013;37:176-80)
In-Kyung Jeong
Diabetes & Metabolism Journal.2013; 37(4): 296. CrossRef
Response: AnIn VitroModel to Probe the Regulation of Adipocyte Differentiation under Hyperglycemia (Diabetes Metab J2013;37:176-80)
Kusampudi Shilpa, Thangaraj Dinesh, Baddireddi Subhadra Lakshmi
Diabetes & Metabolism Journal.2013; 37(4): 298. CrossRef

Validation Studies

Transcription Factor Profile by Degenerate RT-PCR/SSCP: Application in 3T3-L1 Adipocyte Treated with TNF-alpha.: Yoo Lee Kim, Sang Hwa Lee, Young Kil Choi, Seo Yoon Chang, Yun Soo Kim, Soo Kyung Kim, Seok Won Park, Won Kun Park, Yong Wook Cho, Sang Jong Lee; Korean Diabetes J. 2007;31(5):410-420. Published online September 1, 2007; DOI: https://doi.org/10.4093/jkda.2007.31.5.410

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18 Download

Abstract PDF: BACKGROUND
Several high-throughput gene analysis techniques - differential display PCR, suppression subtraction hybridization (SSH), serial analysis of gene expression (SAGE), and DNA microarray - have permitted transcriptome profiling to understand the molecular pathogenesis of multifactorial diseases. But these techniques are of no great utility regarding feasibility, reproducibility, cost, and the amount of material required for analysis. To establish more practical method for transcription factor transcriptome profiling, we combined degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and single strand conformational polymorphism (SSCP) technique. METHODS: We categorized 417 human/mouse transcription factor mRNA into 92 small groups according to homology with ClustalW method and established 92 degenerate RT-PCR including common motives of the 92 small groups with the software program of CODEHOP, Primer Premier, Amplify 1.2. Further analysis on the amplified PCR products was performed by SSCP. This system was applied for the evaluation of changes on transcription factor transcriptome of differentiated 3T3-L1 adipocyte treated with TNF-alpha. RESULTS: 82 groups and 52 groups showed amplification of PCR before and after TNF-alpha treatment respectively and 24 groups showed significant amplification difference after TNF-alpha treatment. After TNF-alpha treatment for 48 hours, mRNA expressions of group 7, 30, and 33 which include adipocyte related transcription factors such as CEBP-alpha, RXR-alpha, PPAR-gamma were downregulated and mRNA expression of group 8 including preadipocyte abundant CEBP-beta was upregulated. These results are largely concordant with the results analyzed by oligonucleotide microarray. Randomly selected single PCR bands of group 28 and 75 on agarose electrophoresis displayed additional multiple bands by SSCP and necessitated addition of this technique to degenerate RT-PCR for further analysis. CONCLUSION: It could be suggested that degenerate RT-PCR/SSCP is practical method and could be used as a screening test for transcriptome profiling of various disease states with further validation study.

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