Resveratrol induces insulin gene expression in mouse pancreatic α-cells
- Sherwin Xie†1,
- Rohit Anthony Sinha†1,
- Brijesh K Singh1,
- Guo Dong Li2,
- Weiping Han3 and
- Paul M Yen1Email author
© Xie et al.; licensee BioMed Central Ltd. 2013
Received: 13 August 2013
Accepted: 1 November 2013
Published: 13 December 2013
Type 1 and type 2 diabetes are characterized by loss of β-cells; therefore, β-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood.
Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic β-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition.
In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express β-cell genes in α-cells.
Resveratrol has been shown to improve glycaemic control in humans . Animal studies have shown similar beneficial effects of resveratrol  by increasing insulin secretion or enhancing sensitivity to insulin in peripheral organs via activation of SirT1 . Recently, several reports described the ability of pancreatic α-cells to de-differentiate into insulin-producing cells after β-cell loss [4–6]. These findings raise the possibility for new diabetic therapies that exploit α-cell plasticity. In this study, we show that resveratrol can induce expression of several β-cell genes and insulin expression in pancreatic α-cells. Our results shed light on resveratrol action in α-cells and expand our understanding of its anti-diabetic effects.
Resveratrol induces re-expression of insulin and other pancreatic β-cell genes in a SirT1-dependent manner
Re-expression of insulin gene by resveratrol in α-cells is enhanced by HDAC inhibition
Resveratrol has emerged as a promising anti-diabetic agent that exhibits significant ability to lower serum glucose in diabetic patients . Recent experiments in genetically-manipulated mice have established that α-cells can directly trans-differentiate into β-cells under certain conditions such as β-cell loss in lineage-traced mice . While the induction of β-cell genes such as Pdx1 can lead to insulin expression in α-cells [8, 9], cell transformation leading to expression of β-cell genes is another potential strategy to increase insulin production . In this regard, several new drugs are being developed that modulate α-cell plasticity . Our observation that resveratrol was able to induce insulin synthesis in α-cells is germane since it currently is undergoing clinical trials for treatment of type 2 diabetes.
The insulin-inducing effect on α-cells by resveratrol was SirT1-dependent. Furthermore, the induction of Pdx1 by resveratrol and the accompanying epigenetic changes on the insulin promoter suggests that it may have a broader reprogramming action than mere stabilization of low abundance insulin mRNA in these cells. In this connection, using an HDAC inhibitor in combination with resveratrol further enhanced insulin induction at both the mRNA and protein levels. In summary, our findings demonstrating the effects of resveratrol on α-cell plasticity provide a new understanding of its anti-diabetic actions and point towards novel treatment strategies for diabetes.
Materials and methods
αTC9 cells, a mouse pancreatic α-cell line , were grown in DMEM containing 1 g/L glucose, supplemented with 10% FBS, 50 U/mL penicillin and 50 U/mL streptomycin. After adherence, cells were treated with 25 μM resveratrol for 24 hr. SirT1 knockdown was performed using Silencer Select duplex oligo-ribonucleotides targeting mouse SirT1 and a non-targeting control siRNA (Life Technologies, USA). In knockdown studies, resveratrol was added for 24 hr after 2 days of knockdown. Rat INS-1 cells were cultured using standard protocol.
RNA isolation and real-time PCR (qPCR)
Total RNA was isolated using Invitrap Spin Cell RNA Mini Kit (Stratec Molecular, Germany) and qPCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN, USA) according to the manufacturer’s instructions. Samples were normalised to actin. Fold changes were calculated using 2-ddCt.
Cells were lysed using Celytic M mammalian lysis buffer (Sigma-Aldrich, USA) and immunobloting was performed according to manufacturer’s instructions (Bio-Rad, USA). Densitometry analysis was performed using Image J software (NIH, USA).
Chromatin immunoprecipitation (ChIP)–qPCR analysis
ChIP assays using control rabbit IgG (Santa Cruz, USA), anti-acetylated histone H3 (Abcam, USA) and anti-acetylated histone H4 (Merck-Millipore, USA) were performed using Magna ChIP™ G - Chromatin Immunoprecipitation Kit (Merck-Millipore, USA) according to manufacturer’s instructions. 2 μL of immunoprecipitated DNA or 1% input DNA was used with QuantiFast SYBR Green PCR Kit (QIAGEN, USA) for 40 cycles of qPCR using Rotor-Gene® Q (QIAGEN, USA). Primers used amplify the Pdx1 binding region (−126 to −296) on the insulin promoter.
Insulin measurement by radioimmunoassay (RIA)
Cells were lysed and extracted by acid-ethanol and insulin content was assayed by RIA (Linco Research, USA).
Compound treatments were performed in triplicate and repeated at least three times independently using matched controls. The data were pooled and results were expressed as mean ± SEM. The statistical significance of differences (P < 0.05) was assessed by two-tailed student’s t-test.
This work is supported by Duke-NUS Graduate Medical School grant awarded to Paul M. Yen.
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