J Nutr Health.  2014 Jun;47(3):167-175.

Study of the mechanisms underlying increased glucose absorption in Smilax china L. leaf extract-treated HepG2 cells

• Kang YH
• Kim DJ
• Kim KK
• Lee SM
• Choe M

Abstract

PURPOSE
Previous studies have shown that treatment with Smilax china L. leaf extract (SCLE) produces antidiabetic effects due to alpha-glucosidase inhibition. In this study, we examined the mechanism underlying these antidiabetic effects by examining glucose uptake in HepG2 cells cultured with SCLE.
METHODS
Glucose uptake and glucokinase activity were examined using an assay kit. Expression of glucose transporter (GLUT)-2, GLUT-4, and HNF-1alpha was measured by RT-PCR or western blot.
RESULTS
Treatment with SCLE resulted in enhanced glucose uptake in HepG2 cells, and this effect was especially pronounced when cells were cultured in an insulin-free medium. SCLE induced an increase in expression of GLUT-2 but not GLUT-4. The increase in the levels of HNF-1alpha, a GLUT-2 transcription factor, in total protein extract and nuclear fraction suggest that the effects of SCLE may occur at the level of GLUT-2 transcription. In addition, by measuring the change in glucokinase activity following SCLE treatment, we confirmed that SCLE stimulates glucose utilization by direct activation of this enzyme.
CONCLUSION
These results demonstrate that the potential antidiabetic activity of SCLE is due at least in part to stimulation of glucose uptake and an increase in glucokinase activity, and that SCLE-stimulated glucose uptake is mediated through enhancement of GLUT-2 expression by inducing expression of its transcription factor, HNF-1alpha.

Keyword

Smilax china. L; anti-diabetic activity; glucose uptake; glucose transporter; glucokinase

MeSH Terms

Absorption*
alpha-Glucosidases
Blotting, Western
China*
Glucokinase
Glucose Transport Proteins, Facilitative
Glucose*
Hep G2 Cells*
Hepatocyte Nuclear Factor 1-alpha
Smilax*
Transcription Factors
Glucokinase
Glucose
Glucose Transport Proteins, Facilitative
Hepatocyte Nuclear Factor 1-alpha
Transcription Factors
alpha-Glucosidases

Figure

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  Fig. 1. Concentration-dependent effects of SCLE on HepG2 cell growth. Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8) assay kit. Each bar is the Mean ± SD, derived from three independent experiments.

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  Fig. 2. Induction of glucose uptake following Smilax china L. leaf extract (SCLE) treatment. HepG2 cells were incubated for 20 min in a KRPH buffer containing SCLE (0.25 mg/ml). The 2-deoxyglucose (2-DG) assay was performed 20 min later, as detailed in “Methods.” The 2DG uptake was expressed in pmol in normal and insulin conditions. Results are presented as the Mean ± SD of three independent experiments. ∗∗: p < 0.01, Normal control versus Normal + SCLE.

• 

  Fig. 3. Measurement of GLUT-2, −4 mRNA expression. A: Effect of a Smilax china L. leaf extract (SCLE) treatment on GLUT-2 mRNA expression in HepG2 cells. B: Effect of SCLE treatment on GLUT-4 mRNA in HepG2 cells. Values are the Mean ± SD of triplicate determinations. ∗: p < 0.05, N versus N + SCLE, ∗∗: p < 0.01, H versus H + SCLE. L: Low glucose levels (1 mM) N: Normal glucose levels (5 mM) H: High glucose levels (25 mM).

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  Fig. 4. Measurement of GLUT-2 protein expression. Time-dependent effects (at 0 h, 24 h, and 48 h) of treatment with a Smilax china L. leaf extract (SCLE) on GLUT-2 expression in HepG2 cells cultured with 5 mM or 25 mM glucose. Values are the Mean ± SD of triplicate determinations. ∗: p < 0.05, ∗∗: p < 0.01 compared with 0 h N: Normal glucose levels (5 mM) H: High glucose levels (25 mM).

• 

  Fig. 5. Measurement of HNF-1α mRNA and protein expression. A: Effect of a Smilax china L. leaf extract (SCLE) on HNF-1α mRNA expression in HepG2 cells. B: Effect of SCLE treatment on HNF-1α protein expression in HepG2 cells. Values are Mean ± SD of triplicate determinations. ∗: p < 0.05, N versus N + SCLE, ∗∗: p < 0.01, H versus H + SCLE N: Normal glucose levels (5 mM) H: High glucose levels (25 mM).

• 

  Fig. 6. Measurement of HNF-1α expression in nucleus fraction. Effect of Smilax china L. leaf extract (SCLE) on HNF-1α protein expression in HepG2 cell fractions. Values are Mean ± SD of triplicate determination. ∗∗: p < 0.01 comparing cytoplasm and nucleus. N: Normal glucose level (5 mM).

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  Fig. 7. In vitro assay to evaluate glucokinase activity. Effect of treatment with a Smilax china L. leaf extract (SCLE) on glucokinase (GK) from Bacillus stearothermophilus. Values are the Mean ± SD of triplicate determinations. ∗∗: p < 0.01, control versus SCLE (0.25 mg/ml).

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  Fig. 8. Treatment with SCLE stimulates glucose absorption and utilization by increasing GLUT-2 mRNA expression and GK activity in HepG2 cells.

Reference

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