Nutr Res Pract.  2018 Jun;12(3):183-190. 10.4162/nrp.2018.12.3.183.

A Portulaca oleracea L. extract promotes insulin secretion via a K⁺(ATP) channel dependent pathway in INS-1 pancreatic β-cells

• Park JE
• Han JS

• KMID: 2442291
• DOI: http://doi.org/10.4162/nrp.2018.12.3.183

Abstract

BACKGROUND
/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic β-cells. MATERIALS/METHOD: INS-1 pancreatic β-cells were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of Ca2+ caused by POE treatment, the effect of POE on intracellular Ca2+ in INS-1 pancreatic β-cells was examined using Fluo-2 AM dye.
RESULTS
POE at 10 to 200 µg/mL significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the K+ ATP channel (blocking insulin secretion) and by verapamil (a Ca2+ channel blocker). The insulinotropic effect of POE was not observed under Ca2+-free conditions in INS-1 pancreatic β-cells. When the cells were preincubated with a Ca2+ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular Ca2+, which closely correlated with increases in the levels of insulin secretion.
CONCLUSIONS
These findings indicate that POE stimulates insulin secretion via a K+ ATP channel-dependent pathway in INS-1 pancreatic β-cells.

Keyword

Portulaca; insulin; diabetes mellitus; calcium channels

MeSH Terms

1-Methyl-3-isobutylxanthine
Adenosine Triphosphate
Alanine
Calcium Channels
Diabetes Mellitus
Diazoxide
Enzyme-Linked Immunosorbent Assay
Fluorescence
Glucose
Insulin*
Portulaca*
Tolbutamide
Verapamil
1-Methyl-3-isobutylxanthine
Adenosine Triphosphate
Alanine
Calcium Channels
Diazoxide
Glucose
Insulin
Tolbutamide
Verapamil

Figure

• 

  Fig. 1 Cytotoxic effect of POE in INS-1 pancreatic β-cellsINS-1 pancreatic β-cells were treated with various concentrations (0.1, 0.5, 1.0 and 2.0 mg/mL) of POE for 24 h, and cell viability was measured via MTT assay. Each value was expressed as mean ± SD in triplicate experiments. NS: non-significant. POE: Portulaca oleracea L. extract.

• 

  Fig. 2 Effects of the POE on insulin secretion from INS-1 pancreatic β-cellsCells were incubated with various concentration of POE for 1 h to induce insulin secretion in 1.1 mM (basal glucose level), 5.6 mM (normal glucose level) and 16.7 mM glucose (hyperglycemic condition). Each value was expressed as mean ± SD in triplicate experiments. a~f Values denoted by different letters are significantly different (P < 0.05). POE: Portulaca oleracea L. extract.

• 

  Fig. 3 Effect of POE on insulin secretion from INS-1 pancreatic β-cells in the presence of insulin secretion inhibitor; verapamil and diazoxideCells were incubated with or without POE (200 µg/mL) for 1 h in the presence of 1.1, 5.6 or 16.7 mM glucose. The inhibitory effect of verapamil (Vera, 50 mM) and diazoxide (Diaz, 300 µM) on POE-induced increase in insulin secretion was assessed. Each value was expressed as mean ± SD in triplicate experiments. A~B, a~c Values denoted by different letters are significantly different (P < 0.05). POE: Portulaca oleracea L. extract.

• 

  Fig. 4 Effect of POE on insulin secretion from INS-1 pancreatic β-cells in the presence of KClCells were incubated with or without POE (200 µg/mL) for 1 h in the presence of 1.1, 5.6 or 16.7 mM glucose, respectively. The stimulating activity of KCl (30 mM) on POE-induced increase in insulin secretion was examined. Each value was expressed as mean ± SD in triplicate experiments. Significantly different from control at * P < 0.05. POE: Portulaca oleracea L. extract.

• 

  Fig. 5 Effect of insulin secretagogues (L-alanine, IBMX, and tolbutamide) on POE-induced insulin secretion from INS-1 pancreatic β-cells.Cells were incubated with L-alanine (10 mM), IBMX, (100 µM) and tolbutamide (200 µM) for 1 h in the presence or absence of POE (200 µg/mL). (A) 1.1 mM was basal glucose levels. (B) 5.6 mM was used for normal glucose level. (C) 16.7 mM was for hyperglycemic conditions. Each value was expressed as mean ± SD in triplicate experiments. A~B, a~c Values denoted by different letters are significantly different (P < 0.05). POE: Portulaca oleracea L. extract.

• 

  Fig. 6 Effect of the POE on insulin secretion from INS-1 pancreatic β-cells in the presence and the absence of Ca2+(A) 1.1 mM was basal glucose levels. (B) 5.6 mM was used for normal glucose level. (C) 16.7 mM was for hyperglycemic conditions. Each value was expressed as mean ± SD in triplicate experiments. A~E, a~d Values denoted by different letters are significantly different (P < 0.05). POE: Portulaca oleracea L. extract.

• 

  Fig. 7 The effect of POE on intracellular Ca2+ in INS-1 pancreatic β-cellsCells were treated with POE (200 µg/ml) in various glucose concentration (1.1, 5.6 and 16.7 mM glucose). (A) The fluorecence of intracellular Ca2+ cells due to treatment. The fluorescence of the INS-1 pancreatic β-cells in the bright and dark field that shows changes in red, green and blue tone. Red and Blue correspond to high and low Ca2+ concentrations, respectively. (B) The concentration (%) of intracellular Ca2+. Each value was expressed as mean ± SD in triplicate experiments. a–e Values denoted by different letters are significantly different (P < 0.05). POE: Portulaca oleracea L. extract.

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