Diabetes Metab J.  2013 Jun;37(3):176-180. 10.4093/dmj.2013.37.3.176.

An In Vitro Model to Probe the Regulation of Adipocyte Differentiation under Hyperglycemia

• Shilpa K
• Dinesh T
• Lakshmi BS

• KMID: 2174291
• DOI: http://doi.org/10.4093/dmj.2013.37.3.176

Abstract

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 kappaB and tumor necrosis factor alpha 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 beta. 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.

Keyword

3T3-L1 adipocytes; High glucose concentration; Hyperplasia; Obesity; p27

MeSH Terms

Adipocytes
Adipogenesis
Adiponectin
Blotting, Western
CCAAT-Enhancer-Binding Proteins
Cell Survival
Glucose
Glucose Transport Proteins, Facilitative
Glycogen
Hyperglycemia
Hyperplasia
Insulin
Obesity
Peroxisomes
Phosphotransferases
Proto-Oncogene Proteins c-akt
Receptor, Insulin
Thymidine
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha
Adiponectin
CCAAT-Enhancer-Binding Proteins
Glucose
Glucose Transport Proteins, Facilitative
Glycogen
Insulin
Phosphotransferases
Proto-Oncogene Proteins c-akt
Receptor, Insulin
Thymidine
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effect of 105 mM glucose on molecular markers involved in the process of adipogenesis and inflammation at gene level. (A) CCAAT-enhancer-binding proteins α (C/EBPα) (a); sterol regulatory element-binding protein 1c (SREBP 1c) (b); peroxisome proliferator-activated receptor γ (PPARγ) (c); tumor necrosis factor α (TNFα) (d); toll-like receptor 4 (TLR4) (e); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (f). Lane 1 to 4 indicates preadipocytes, adipocytes differentiated with 25, 105 mM glucose and negative control, respectively. GAPDH was used as internal control for the study. (B) The graph represents the ratio of integrated density value (IDV) data of genes to IDV of GAPDH. The 25 and 105 mM glucose expression levels were compared to preadipocytes. aP<0.001, bP<0.0001.

• 

  Fig. 2 Effect of 105 mM glucose on molecular markers involved in the process of proliferation, adipogenesis, inflammation and insulin signaling at protein level. (A) nuclear factor-κB (NF-κB) (a); Adiponectin (b); p27 (c); Akt/phosphoprotein kinase B (PKB) (d); phosphoinositide 3 kinase (PI3K) (e); insulin receptor β (IRβ) (f); glucose transporter 4 (GLUT4) (g); GLUT1 (h); and β-actin (i). Lane 1, 2, and 3 indicate preadipocytes, adipocytes differentiated with 25 and 105 mM glucose, respectively. β-Actin was used as internal control for the study. (B) The graph represents the ratio of integrated density value (IDV) data of proteins to IDV of β-actin. The 25 and 105 mM glucose expression levels were compared to preadipocytes. aP<0.05, bP<0.01, cP<0.001, dP<0.0001.

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