Lab Anim Res.  2013 Jun;29(2):84-95. 10.5625/lar.2013.29.2.84.

Red Liriope platyphylla stimulated the insulin secretion through the regulation of calcium concentration in rat insulinoma cells and animal models

• Lee HR
• Kim JE
• Lee YJ
• Kwak MH
• Im DS
• Hwang DY

• KMID: 1431339
• DOI: http://doi.org/10.5625/lar.2013.29.2.84

Abstract

The aim of this study was to investigate the effects of Red L. platyphylla (RLP) on calcium and glucose levels during insulin secretion. To achieve this, alteration of insulin and calcium concentrations was measured in rat insulinoma-1 (INS-1) cells and animal models in response to RLP treatment. In INS-1 cells, maximum secretion of insulin was detected upon treatment with 200 microg/mL of RLP for 20 min. Nifedipine, an L-type calcium channel blocker, effectively inhibited insulin secretion from INS-1 cells. Regarding calcium levels, the maximum concentration of intracellular calcium in INS-1 cells was obtained by treatment with 100 microg/mL of RLP, whereas this level was reduced under conditions of 200 microg/mL of RLP. Further, RLP-treated INS-1 cells showed a higher level of intracellular calcium than that of L. platyphylla (LP), Korea White Ginseng (KWG), or Korea Red Ginseng (KRG)-treated cells. This RLP-induced increase in intracellular calcium was abrogated but not completely abolished upon treatment with 40 microM nifedipine in a dose-dependent manner. Furthermore, the insulin level was dramatically elevated upon co-treatment with high concentrations of glucose and RLP, whereas it was maintained at a low level in response to glucose and RLP co-treatment at low concentrations. In an animal experiment, the serum concentration of calcium increased or decreased upon RLP treatment according to glucose level compared to vehicle treatment. Therefore, these results suggest that insulin secretion induced by RLP treatment may be tightly correlated with calcium regulation, which suggests RLP is an excellent candidate for diabetes treatment.

Keyword

Red Liriope Platyphylla; diabetes; insulin; calcium; glucose

MeSH Terms

Animal Experimentation
Animals
Calcium
Calcium Channels, L-Type
European Continental Ancestry Group
Glucose
Humans
Insulin
Insulinoma
Korea
Models, Animal
Nifedipine
Panax
Rats
Calcium
Calcium Channels, L-Type
Glucose
Insulin
Nifedipine

Figure

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  Figure 1 Effects of Red L.Platyphylla (RLP) on insulin secretion ability. (A) Time-dependent effects of RLP. After 100 µg/mL of RLP treatment to INS-1 cells for different time, the concentration of insulin in the supernatant was measured using an anti-insulin ELISA kit. (B) Dose-dependent effects of RLP for 20 min. INS-1 cells were cultured with one of four different RLP concentrations for 20 min. Concentration of insulin in the supernatant was measured using an anti-insulin ELISA kit. (C) Inhibitory effects of nifedipine. INS-1 cells were treated with RLP and different concentrations of nifedipine for 20 min. Data values represent the means±SD of three experiments. a, P<0.05 is the significance level compared to vehicle-treated cells.

• 

  Figure 2 Dose-dependent effects of Red L. Platyphylla (RLP) on intracellular calcium concentration in INS-1 cells. Fura-2-loaded INS-1 cells were challenged with four different concentrations of RLP (25, 50, 100, and 200 µg/mL) or vehicle at the time indicated by the arrow. Relative level of intracellular calcium concentration was expressed as fluorescence ratio (340:380 nm). Data are representative of three independent experiments.

• 

  Figure 3 Comparison of intracellular calcium regulation abilities of Red L. Platyphylla (RLP) in INS-1 cells. Fura-2-loaded INS-1 cells were challenged with 100 µg/mL of four different samples (LP, RLP, KWG, and KRG) or vehicle at the time indicated by the arrow. Relative level of intracellular calcium concentration was expressed as fluorescence ratio (340:380 nm). Data are representative of three independent experiments.

• 

  Figure 4 Inhibitory effect of Red L. Platyphylla (RLP) on intracellular calcium concentration after blockage of L-type calcium channel. Fura-2-loaded INS-1 cells were sequentially challenged with three different concentrations of nifedipine (10, 20, or 40 µM) and RLP (100 µg/mL) at the time indicated by the arrow. Relative level of intracellular calcium concentration was expressed as fluorescence ratio (340:380 nm). Data are representative of three independent experiments.

• 

  Figure 5 Effects of Red L. Platyphylla (RLP) on insulin secretion under different glucose concentrations. (A) Glucose dose-dependent effects of RLP (100 µg/mL) on cell viability. INS-1 cells were cultured with one of four different glucose concentrations and dH2O or RLP for 24 h. Cellular morphology was viewed at 200x magnification. Cell viability was measured via MTT assay. (B) Glucose dose-dependent effects of RLP on insulin secretion. Concentration of insulin was measured using an anti-insulin ELISA kit under the same RLP and nifedipine co-treatment conditions. Data values represent the means±SD of three experiments. a, P<0.05 is the significance level compared to non-glucose cells. b, P<0.05 is the significance level compared to vehicle-treated cells. c, P<0.05 is the significance level compared to cells incubated under non-nifedipine conditions.

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  Figure 6 Effects of Red L. Platyphylla (RLP) on calcium concentration in STZ-induced diabetic model animals. Blood was collected from the abdominal veins of STZ-induced diabetic model mice after steaming RLP treatment for 7 days. Concentration of glucose was measured at three different times using the sensitive strip of the Blood Glucose Monitoring System (A). Insulin (B) and calcium (C) concentrations were detected in the serum of STZ-induced diabetic mice on the final day. Data values were expressed as the means±SD of three experiments. a, P<0.05 is the significance level compared to non-treated group. b, P<0.05 is the significance level compared to vehicle-treated cells.

• 

  Figure 7 Changes in glucose, insulin, and calcium concentrations in OLETF rats after Red L. Platyphylla (RLP) treatment. (A) Glucose concentration was measured in blood collected from the abdominal veins of rats. (B) Insulin concentration was measured in the serum using an ELISA kit with 0.1 ng/mL of sensitivity, and the inter-assay coefficient of variation ranged from 2.86-5.17. (C) Calcium concentration was measured in the serum of OLETF rats on the final day using a Colorimetric Calcium Assay kit. Data are reported as the mean±SD from three replicates. a, P<0.05 is the significance level compared to LETO rats. b, P<0.05 is the significance level compared to vehicle-treated OLETF rats.

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