Nutr Res Pract.  2017 Jun;11(3):180-189. 10.4162/nrp.2017.11.3.180.

Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

• Kim DJ
• Kang YH
• Kim KK
• Kim TW
• Park JB
• Choe M

• KMID: 2407765
• DOI: http://doi.org/10.4162/nrp.2017.11.3.180

Abstract

BACKGROUND/OBJECTIVES
Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism.
MATERIALS/METHODS
Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement.
RESULTS
CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose.
CONCLUSION
CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment.

Keyword

Cordyceps militaris; antidiabetics; glucose transporter; glucokinase; glycogen

MeSH Terms

Acarbose
alpha-Glucosidases*
AMP-Activated Protein Kinases
Blotting, Western
Cordyceps*
Food Habits
Glucokinase
Glucose Transport Proteins, Facilitative
Glucose*
Glycogen
Glycogen Synthase Kinase 3
Glycolysis
Hep G2 Cells*
Hepatocyte Nuclear Factor 1-alpha
Hypoglycemic Agents
Liver
Metabolic Diseases
Metabolism*
Motor Activity
Obesity
Oxidoreductases
Phosphatidylinositol 3-Kinase
Phosphoenolpyruvate
Phosphorylation
Proto-Oncogene Proteins c-akt
Pyruvic Acid
Social Conditions
Water*
AMP-Activated Protein Kinases
Acarbose
Glucokinase
Glucose
Glucose Transport Proteins, Facilitative
Glycogen
Glycogen Synthase Kinase 3
Hepatocyte Nuclear Factor 1-alpha
Hypoglycemic Agents
Oxidoreductases
Phosphatidylinositol 3-Kinase
Phosphoenolpyruvate
Proto-Oncogene Proteins c-akt
Pyruvic Acid
Water
alpha-Glucosidases

Figure

• 

  Fig. 1 Concentration-dependent effects of Cordyceps Militaris water extract (CMW) on HepG2 cell growth. Cell viability was analyzed using MTT assay. Data were expressed as mean values ± SD, derived from three independent experiments.

• 

  Fig. 2 Measurement of glucokinase (GK) activity of Cordyceps Militaris water extract (CMW) in HepG2 cell. (A) GK activity of CMW treatment according to concentration dependent manner. (B) GK activity CMW treatment (1.0 mg/mL) on time dependent manner. Data were expressed as mean values ± SD and comparisons of data were carried out using one-way ANOVA, as appropriate. * P < 0.05, *** P < 0.001 compared 0 mg/mL, * P < 0.05, *** P < 0.001 vs. 0h.

• 

  Fig. 3 Measurement of pyruvate dehydrogenase (PDH) activity of Cordyceps Militaris water extract (CMW) in HepG2 cell. (A) PDH activity of CMW treatment according to concentration dependent manner. (B) PDH activity CMW treatment (1.0 mg/mL) on time dependent manner. Data were expressed as mean values ± SD and comparisons of data were carried out using one-way ANOVA, as appropriate. * P < 0.05 compared 0 mg/mL, * P < 0.05 vs. 0h.

• 

  Fig. 4 Measurement of glucose uptake and glucose consumption following Cordyceps Militaris water extract (CMW) treatment. HepG2 cells were incubated for 20 min in a KRPH buffer containing insulin (1uM) and CMW (1 mg/mL). The 2-deoxyglucose (2-DG) assay was performed 20 min later, as detailed in “Methods”. Data were expressed as mean values ± SD and comparisons of data were carried out using one-way ANOVA, as appropriate. ** P < 0.01, *** P < 0.001 vs. control.

• 

  Fig. 5 Measurement of glucose transporter 2 (GLUT2) mRNA and protein and hepatocyte nuclear factor 1 alpha (HNF-α) protein expression. (A) Effect of Cordyceps Militaris water extract (CMW) on GLUT2 mRNA expression in HepG2 cells. (B) Effect of CMW treatment on GLUT2 protein expression in HepG2 cells. (C) Effect of CMW treatment on HNF-1α protein expression in HepG2 cells. Data were expressed as mean values ± SD and comparisons of data were carried out using Student's unpaired t-test, as appropriate. * P < 0.05, *** P < 0.001 vs. control.

• 

  Fig. 6 Measurement of glycolysis related enzymes protein expression. (A) Effect of Cordyceps Militaris water extract (CMW) on phosphorylated AMP-activated protein kinase (pAMPK) protein expression in HepG2 cells. (B) Effect of CMW on glucokinase (GK) protein expression in HepG2 cells. (C) Effect of CMW on phosphorylated pyruvate dehydrogenase (pPDH) protein expression in HepG2 cells. (D) Effect of CMW on phosphoenolpyruvate carboxykinase (PEPCK) protein expression in HepG2 cells. Data were expressed as mean values ± SD and comparisons of data were carried out using Student's unpaired t-test, as appropriate. * P < 0.05, *** P < 0.001 vs. control.

• 

  Fig. 7 Measurement of glucose storage related enzymes protein expression and glycogen contents. (A) Effect of Cordyceps Militaris water extract (CMW) on phosphorylated phosphatidylinositol 3-kinase (pPI3K) protein expression in HepG2 cells. (B) Effect of CMW on phosphorylated protein kinase B (pAkt) protein expression in HepG2 cells. (C) Effect of CMW on phosphorylated glycogen synthase kinase 3 beta (pGSK-3β) protein expression in HepG2 cells. (D) Effect of CMW treatment on glycogen contents in HepG2 cells. Data were expressed as mean values ± SD and comparisons of data were carried out using Student's unpaired t-test, as appropriate. *** P < 0.001 vs. control.

• 

  Fig. 8 α-Glucosidase inhibitory activity of Cordyceps Militaris water extract (CMW). Acarbose was used as positive control. Data were expressed as mean values ± SD and comparisons of data were carried out using one-way ANOVA, as appropriate. *** P < 0.001 vs. control.

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