jnh Journal of Nutrition and Health J Nutr Health 2288-3886 2288-3959 The Korean Nutrition Society 10.4163/jnh.2014.47.1.167 jnh-47-167 Research Article Study of the mechanisms underlying increased glucose absorption in Smilax china L. leaf extract-treated HepG2 cells∗ KangYun Hwan 1 KimDae Jung 1 KimKyoung Kon 2 LeeSung Mee 2 ChoeMyeon 1 2 Well-being Bioproducts RIC, Kangwon National University, Chuncheon 200–701, Korea Department of Bio-Health Technology, Kangwon National University, Chuncheon 200–701, Korea 01 2014 20 01 2014 47 3 167 175 26022014 18032014 22042014 Copyright © 2014 The Korean Nutrition Society 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Purpose

Previous studies have shown that treatment with Smilax china L. leaf extract (SCLE) produces antidiabetic effects due to α-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-1α 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-1α, 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-1α.

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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.

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.

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).

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).

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).

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).

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).

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

PCR primer sequences

Gene Primer Sequence (5' → 3')
GLUT-2 Forward GAT GAA CTG CCC ACA ATC TC
Reverse CTG ATG AAA AGT GCC AAG TG
GLUT-4 Forward GTT AAT CGG CAT TCT GAT CG
Reverse GTG AAG ACT GTG TTG ACC AC
HNF-1α Forward GAA TGC ATC CAG AGA GGG GT
Reverse GTG GAC CTT ACT GGG GGA GA
β-actin Forward ACA GGA AGT CCC TTG CCA TC
Reverse AGG GAG ACC AAA AGC CTT CA

PCR condition of each primer

Gene Pre-denaturation Denaturation Annealing Extension Final extension
GLUT-2 94℃ 5min 94℃ 30sec 59℃ 30sec 72℃ 30sec 72℃ 5min
26cycle
GLUT-4 94℃ 5min 94℃ 30sec 59℃ 30sec 72℃ 30sec 72℃ 5min
30cycle
HNF-1α 94℃ 5min 94℃ 30sec 57℃ 30sec 72℃ 30sec 72℃ 5min
25cycle
β-actin 94℃ 5min 94℃ 30sec 55℃ 30sec 72℃ 30sec 72℃ 5min
18cycle