Contributed equally
Polymethoxylated flavones (PMFs), which are compounds characteristic of citrus plants, possess a wide range of biological properties, particularly affecting glucose and lipid metabolism. However, the mechanism underlying the antidiabetic activity of PMFs has not been fully elucidated. In this study, we aimed to investigate the potential effect of PMFs on the biosynthesis and secretion of insulin, which are crucial in diabetes. We investigated whether PMFs are able to induce insulin secretion by pancreatic β-cells and observed that different concentrations (12.5, 25, 37.5 and 50 μg/ml) of PMFs exerted no effect on insulin synthesis and secretion in INS-1 cells, regardless of the glucose levels. To the best of our knowledge, this is the first study to demonstrate that the regulation of glucose and lipid metabolism by PMFs is not mediated by directly affecting insulin synthesis or secretion. Therefore, further studies are required to elucidate the role of PMFs in diabetes.
Citrus plants are rich sources of health-promoting substances (
PMFs were shown to possess antiatherosclerotic activity, inhibiting the formation of atheroma in several steps during its pathogenesis (
PMFs (the main component was nobiletin, with 98% purity) were extracted from
PMFs were extracted from dried
The rat INS-1 pancreatic β-cell line was cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mmol/l HEPES, 11.1 mmol/l glucose, 50 μg/ml ampicillin, 50 μg/ml kanamycin, 2.0 mmol/l glutamine, 1.0 mmol/l sodium pyruvate and 50 μmol/l β-mercaptoethanol. The cells were maintained at 37°C and in 5% CO2.
For the measurement of cell doubling time, the INS-1 cells were harvested by trypsinization and seeded into normal 96-well dishes (2.5×105/well). The optical density at 490 nm was detected at the indicated times by the VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
The INS-1 cells (2.5×104/well) were seeded into 96-well plates. The medium was changed after 40–50% confluence was reached. The cells were incubated with fresh medium containing different concentrations of PMFs (0, 12.5, 25, 50, 60, 70, 80 and 90 μg/ml) for 72 h and the medium was changed every 24 h. The control cells were treated with DMSO for the same time. After 72 h, cell viability was assessed using the MTT method as previously described (
The INS-1 cells (2.5×104/well) were seeded into 24-well plates. After cell confluence reached 60–70%, the medium was removed and the cells were treated with Krebs-Ringer bicarbonate buffer (KRB; 129 mmol/l NaCl, 5 mmol/l NaHCO3, 4.8 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4·7H2O and 2.5 mmol/l CaCl2) without glucose for 60 min to ensure that the cells were in a non-glucose metabolic state. Subsequently, KRB was removed and the cells were incubated with fresh KRB containing different concentrations of glucose (3, 11 and 20 mmol/l) and PMFs (12.5, 25, 37.5 and 50 μg/ml) for 60 min. After the 60-min incubation, the supernatants were collected for insulin measurement by ELISA, according to the manufacturer’s instructions.
The cells were lysed with TRIzol and RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The RNA samples were treated with DNase and used for RT-PCR according to the manufacturer’s instructions. The sequences of the primers used were as follows: β-actin: sense, 5′-AGAAGGATTCCTATGTGGGCG-3′ and antisense, 5′-CATGTCGTCCCAGTTGGTGAC-3′; insulin: sense, 5′-CAGCTCCACACTCCAGGTAC-3′ and antisense, 5′-CTTTCGCTGGGCTCTGAAGG-3′; pancreatic and duodenal homeobox 1 (PDX1): sense, 5′-ATGAATAGTGAG GAGCAGTACTACG-3′ and antisense, 5′-CCGGGGTTC CTGCGGTC-3′. For relative quantification, we calculated the n-fold differential expression with the ΔCt method (Ct, threshold cycle of PCR amplification at which the product is first detected by fluorescence), which compares the amount of target gene amplified normalized to the β-actin endogenous reference. The sizes of the amplified products were measured by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining (
The results are expressed as the means ± standard deviation. One-way analysis of variance (ANOVA) and the Student’s t-test were performed using SPSS software for Windows, version 12.0, 2003 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. All the assays were performed in triplicate.
In this study, PMFs were extracted from dried tangerine peel with ethyl acetate at room temperature and the extract was analyzed by HPLC (
To ensure that the INS-1 cells were in an optimal state for the subsequent experiments, the cell doubling time was determined using a test wavelength of 490 nm. As shown in
To investigate the cytotoxic effect of PMFs on INS-1 cells, the cell viability under treatment with different concentrations of PMFs was measured with the MTT assay. The cells were incubated with different concentrations of PMFs for 72 h. As shown in
To achieve a preliminary understanding of the molecular mechanism of PMF function in diabetes, the INS-1 cells were treated with different concentrations of glucose, mimicking a hyperglycemic environment. The insulin secretion by INS-1 cells treated with different concentrations of glucose and PMFs was assessed using ELISA. As shown in
Subsequently, we investigated whether PMFs affect the expression of insulin mRNA using RT-PCR. The INS-1 cells were treated with 25 μg/ml PMFs and were collected at indicated times for RNA isolation and RT-PCR. As shown in
We also analyzed the mRNA levels of PDX1, which is an insulin secretion-stimulating factor. Following incubation with 25 μg/ml PMFs for different time periods (
In the present study, PMFs were extracted from dried
PMFs are the characteristic flavones of tangerine and are of particular interest due to their anticancer, anti-inflammatory, antioxidant, antimutagenic and antimicrobial properties (
The secretion of insulin by pancreatic β-cells is characterized by two phases. In the first phase, pancreatic β-cells are stimulated by glucose or other inducers and the resulting increase in the ATP/ADP ratio closes the ATP-sensitive potassium KATP channel, causing plasma membrane depolarization, influx of Ca2+ and, finally, insulin secretion (
In conclusion, this study adds to our understanding of the antidiabetic properties of PMFs, which were found to be unrelated to insulin expression and secretion by pancreatic β-cells.
This study was supported by grants from the Research Project of Shaanxi Provincial Key Laboratory of Biotechnology (no. 11JS085), the Development Project of Science and Technology Research of Shaanxi Province (no. 2011K12-61) and the Natural Science Research Specialized Foundation of Department of Education of Shaanxi Province (no. 09JK754). We would like to thank Professor Dongmin Li for her technical assistance and supply of the INS-1 cells.
High-performance liquid chromatograms of polymethoxylated flavones (PMFs). (A) PMFs standards; (B) PMFs samples. AU, absorption units.
INS-1 cell doubling time measured with the MTT assay.
Insulin secretion by INS-1 cells treated with different concentrations of glucose and polymethoxylated flavones (PMFs).
Insulin and pancreatic and duodenal homeobox 1 (PDX1) mRNA expression in INS-1 cells treated with polymethoxylated flavones (PMFs) and control cells. (A and C) INS-1 cells treated with PMFs and control cells were collected for reverse transcriptase-polymerase chain raction to determine the insulin and PDX1 mRNA levels; (B and D) gray values of insulin and PDX1 cDNA.
Toxicity of different concentrations of polymethoxylated flavones (PMFs) on INS-1 cells.
Concentration of PMFs (μg/ml) | OD490 | Inhibition ratio (%) |
---|---|---|
0 | 0.478 | - |
12.5 | 0.468 | 2.1 |
25 | 0.456 | 4.6 |
50 | 0.47 | 1.6 |
60 | 0.461 | 3.5 |
70 | 0.432 | 9.6 |
80 | 0.321 |
32.8 |
90 | 0.33 |
30.9 |
P<0.05, significant difference compared to the control group;
OD, optical density.