Effects of rosiglitazone and metformin on pancreatic beta cell gene expression
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- Richardson, H., Campbell, S.C., Smith, S.A. et al. Diabetologia (2006) 49: 685. doi:10.1007/s00125-006-0155-1
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Rosiglitazone and metformin are two oral antihyperglycaemic drugs used to treat type 2 diabetes. While both drugs have been shown to improve insulin-sensitive glucose uptake, the direct effects of these drugs on pancreatic beta cells is only now beginning to be clarified. The aim of the present study was to determine the direct effects of these agents on beta cell gene expression.
We used reporter gene analysis to examine the effects of rosiglitazone and metformin on the activity of the proinsulin and insulin promoter factor 1 (IPF1) gene promoters in the glucose-responsive mouse beta cell line Min6. Western blot and gel retardation analyses were used to examine the effects of both drugs on the regulation of IPF1 protein production, nuclear accumulation and DNA binding activity in both Min6 cells and isolated rat islets of Langerhans.
Over 24 h, rosiglitazone promoted the nuclear accumulation of IPF1 and forkhead homeobox A2 (FOXA2), independently of glucose concentration, and stimulated a two-fold increase in the activity of the Ipf1 gene promoter (p<0.01). Stimulation of the Ipf1 promoter by rosiglitazone was unaffected by the presence of the peroxisome proliferator activated receptor γ antagonist GW9662. No effect of either rosiglitazone or metformin was observed on proinsulin promoter activity. Metformin stimulated IPF1 nuclear accumulation and DNA binding activity in a time-dependent manner, with maximal effects observed after 2 h.
Metformin and rosiglitazone have direct effects on beta cell gene expression, suggesting that these agents may play a previously unrecognised role in the direct regulation of pancreatic beta cell function.
AMP-dependent protein kinase
electrophoretic mobility shift assay
forkhead homeobox A2
hepatocyte nuclear factor 1α
insulin promoter factor 1
peroxisome proliferator-activated receptor γ
Rosiglitazone and metformin are oral antihyperglycaemic drugs used in the management of type 2 diabetes. Rosiglitazone is a member of the thiazolidinedione class of drugs and acts largely through agonist action on the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) , through which it has been shown to increase insulin-dependent peripheral glucose disposal, decrease hepatic glucose output and alter adipocyte metabolism . Metformin is of the biguanide class of drugs, and also largely targets tissues of glucose storage and metabolism, predominantly the liver .
While both drugs improve insulin-sensitive glucose uptake [4, 5], their direct effects on pancreatic beta cells has not been fully clarified. Metformin has been shown to restore insulin secretion following chronic exposure of rat islets to non-esterified fatty acids or high glucose , an effect mediated through direct effects on beta cell glucose and fatty acid metabolism. The effects of metformin on glucose-induced insulin secretion have been linked to the activation of AMP-dependent protein kinase (AMPK) [7, 8]. Although not analysed in beta cells to date, in muscle cells rosiglitazone has also been shown to activate AMPK . The pathways involved in the activation of AMPK by metformin and rosiglitazone in muscle cells are reportedly distinct . The aim of the present study was to investigate the direct effects of rosiglitazone and metformin on pancreatic beta cell gene expression and the activity of the key beta cell regulatory transcription factors insulin promoter factor (IPF1) and forkhead homeobox A2 (FOXA2).
Regulation of the proinsulin gene in response to glucose is dependent in part on the activity of the beta cell transcription factor IPF1 (also known as pancreatic duodenal homeobox 1 [PDX1]) , which is central to the regulation of a complement of beta cell glucose-sensing genes [11, 12]. IPF1 accumulates in the beta cell nucleus in response to elevated glucose concentrations, events mediated by post-translational modification of the protein [13–15]. Expression of the Ipf1 gene is also regulated in response to glucose . The complex 4.5 kb Ipf1 gene promoter contains binding sites for many beta cell transcription factors, including hepatocyte nuclear factor 1α (HNF1α), IPF1 and FOXA2 [17–20]. FOXA2 is a member of the forkhead/winged helix family and has been identified as a key regulator of Ipf1 gene expression. FOXA2 is produced at the onset of pancreatic development in the foregut endoderm. Its expression pattern is similar to that of IPF1, and it has been shown to bind key regulatory elements within the Ipf1 gene promoter, regulating expression both during pancreas development and in adult islets [19, 20]. Although the key role of FOXA2 in regulating Ipf1 gene expression is becoming clear, at present little is known about the stimuli regulating FOXA2 production itself in adult beta cells.
In the present study, we used the glucose-responsive beta cell line Min6 and a series of reporter gene constructs containing sections of the proinsulin and Ipf1 gene promoters to analyse the direct effects of rosiglitazone and metformin on the regulation of these key beta cell genes. We also used Western blotting and electrophoretic mobility shift assay (EMSA) analysis to evaluate the production, nuclear accumulation and DNA binding activity of the key beta cell transcription factors IPF1, FOXA2 and HNF1α. Our data indicate that the established effects of rosiglitazone and metformin on beta cell functional activity may occur in part through direct effects on beta cell transcriptional regulation.
Materials and methods
Cell culture materials, LipofectAMINE and OptiMem were purchased from Invitrogen (Paisley, UK). Rosiglitazone (purity >99.5%) was provided by GlaxoSmithKline (Harlow, UK). Metformin (purity >98%) was purchased from Sigma-Aldrich (Dorset, UK). Luciferin was from Perbio (Newcastle, UK). Protein assay reagent was from Bio-Rad (Hemel Hempstead, UK). All other chemicals and materials were purchased from Sigma-Aldrich.
The construct pGL-Luc200 is derived from the pGL2 vector (Promega, Southampton, UK) and contains 200 bp of the human proinsulin gene promoter upstream of the firefly luciferase reporter gene, as previously described . The control construct Luc lacks the proinsulin gene promoter fragment. The construct pGL-IPF1 is based on the pGL3 vector (Promega) and contains a 4,531 bp fragment of the mouse Ipf1 promoter upstream of the firefly luciferase reporter gene, as previously described . Plasmid DNA was prepared using an endotoxin-free Maxiprep kit (Qiagen, Crawley, UK).
Min6 cells, a beta cell line derived from transgenic mice expressing the SV40 large T antigen under the control of the rat proinsulin gene promoter , were cultured in DMEM containing 5 mmol/l glucose, supplemented with 15% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 μmol/l β-mercaptoethanol. All of the present studies were performed using cells between passage numbers 28 and 32.
Cells at 80% confluence were transfected using LipofectAMINE (Invitrogen) as previously described . Forty-eight hours after transfection (including preincubation for 12 h in 0.5 mmol/l glucose prior to stimulation) the cells were incubated with either 10 μmol/l rosiglitazone (2 μl of 1,000× stock in DMSO added to each 2 ml well) or 15 μmol/l metformin (2 μl of 1,000× stock in the same media added to each 2 ml well) for the indicated time periods. Cells were harvested and lysates prepared as previously described . Luciferase reporter gene assays were performed and luciferase activity was normalised to protein content as previously described .
Islets were isolated from male Wistar rats, weighing 250–300 g, as previously described , aliquoted into batches of 150 in RPMI containing 5 mmol/l glucose, supplemented with 10% fetal calf serum, and maintained in a humidified 37°C incubator with 5% CO2.
Whole cell or nuclear extracts were prepared, and fractionated by SDS-PAGE. Western blotting was carried out as previously described . Protein concentrations were measured using Bio-Rad Protein Assay Kit reagent, titrated against known concentrations of bovine serum albumin. Whole-cell extract (10 μg) or 5 μg of nuclear extract was added per well. Equal loading of protein to each well was additionally confirmed by blotting with a specific HNF1α antibody. Levels of HNF1α remained constant under all experimental conditions tested (not shown). The IPF1 antibody was a kind gift from C. V. Wright (Vanderbilt University, Nashville, TN, USA). FOXA2 and HNF1α antibodies were purchased from Santa Cruz (Calne, UK). Secondary horseradish peroxidase (HRP)-conjugated antibodies were purchased from Amersham (UK).
Electrophoretic mobility shift assays
EMSA analysis was performed using the DIG Gel Shift Kit (Roche Molecular Biochemicals, Mannheim, Germany) using Probe B, containing the A3 site of the human proinsulin gene promoter . Five micrograms of each nuclear extract, 0.4 ng of digoxigenin-labelled probe, 1× binding buffer, 1 μg (poly-deoxy-inosinic-deoxy-cytidylic acid) and 0.1 μg poly-l-lysine were incubated at room temperature for 20 min, fractionated on 6% polyacrylamide gels, and blotted onto nylon membrane, blocked for 30 min. Chemiluminescent detection reaction was carried out all according to the manufacturer’s protocol.
Statistics and densitometry
The data are expressed as mean±SD. Data were compared using two-tailed Student’s t-test for paired data. Densitometry was performed using the Tina v2.09 g software (Raytest, Straubenhardt, Germany). A p value of less than 0.05 was considered significant.
Rosiglitazone stimulates Ipf1 gene promoter activity in a concentration-dependent manner
Metformin has no effect on the activity of either the Ipf1 or the proinsulin gene promoter in Min6 beta cells
To investigate the effects of rosiglitazone and metformin on proinsulin promoter activity, the same experiment was performed using the construct Luc200, containing a 200 bp section of the human proinsulin gene promoter upstream of the firefly luciferase gene, or the control construct Luc, which lacks the proinsulin gene promoter fragment (Fig. 2b). Incubation in 5 mmol/l glucose resulted in a two-fold increase in Luc200 activity compared with that observed in 0.5 mmol/l glucose. However, no effect of rosiglitazone or metformin was observed on Luc200 activity, or on the activity of the control construct Luc (not shown).
Rosiglitazone stimulation of the Ipf1 promoter is unaffected by the presence of GW9662
Metformin and rosiglitazone both stimulate IPF1 protein levels at 5 and 25 mmol/l glucose
Metformin promotes the nuclear accumulation of IPF1 and FOXA2 in a glucose-dependent manner over 24 h
Rosiglitazone promotes the nuclear accumulation of IPF1 and FOXA2 in a glucose-independent manner over 24 h
Metformin rapidly (2 h) stimulates IPF1 nuclear accumulation and DNA binding activity
The results of the present study show that both metformin and rosiglitazone have marked effects on transcriptional regulation in the pancreatic beta cell line Min6 and in freshly isolated rat islets of Langerhans. Our initial concentration-response analysis indicates that rosiglitazone has maximal effects on the Ipf1 gene promoter over 24 h at a concentration of 10 μmol/l. In agreement with our previous studies, increased glucose concentrations (0.5–5 mmol/l) stimulated a marked increase in the activity of the Ipf1 promoter . A further two-fold increase was observed upon addition of 10 μmol/l rosiglitazone, indicating that glucose and rosiglitazone have an additive effect on Ipf1 promoter activity. Comparable results were observed at 25 mmol/l glucose.
In the present study, rosiglitazone had no effect on the 200 bp section of the human proinsulin gene promoter analysed. This section of the promoter has been shown in previous studies to contain key regulatory elements essential for the control of proinsulin gene transcription [10, 13, 23]. Although our data do not exclude the possibility that further elements which may respond to rosiglitazone lie outside this region, our findings are consistent with previous studies which show 100 μmol/l rosiglitazone has no effect on proinsulin mRNA levels in INS1 cells over a 24-h period . In contrast to the marked effects of rosiglitazone, metformin had no effect on the activity of either the Ipf1 or the proinsulin promoter over the same 24-h incubation period. The effects of rosiglitazone and metformin on Ipf1 promoter activity have not been reported previously. It is intriguing that metformin did not affect Ipf1 promoter activity, but did stimulate IPF1 protein levels over 24 h. This suggests that the observed effects of metformin on IPF1 production occur post-transcriptionally.
Since metformin and rosiglitazone are often prescribed in combination for the treatment of insulin-resistance in type 2 diabetes [25, 26], we tested the combined effect of these agents on our selected targets, both in Min6 cells and in freshly isolated rat islets. No additive effect of rosiglitazone and metformin was observed. Several recent studies indicate that these agents stimulate distinct intracellular signalling pathways [27, 28]. Recent studies in muscle cells indicate that although both agents are able to activate AMP kinase, even activation of the same target may occur through distinct signalling pathways . These pathways remain to be fully elucidated; however, phosphatidylinositol 3-kinase, mitogen-activated protein kinase and p38 do not appear to be involved .
In the present study, rosiglitazone had a marked effect on the expression level and nuclear accumulation of the IPF1 protein. This is consistent with the observed effects on Ipf1 promoter activity. Nuclear accumulation of IPF1 was independent of glucose concentration and was evident at each of the 0.5, 5 and 25 mmol/l glucose conditions tested over 24 h. We and others have previously shown that stimuli such as glucose and glucagon-like peptide-1 (GLP-1) can promote the translocation of IPF1 from the cytoplasm to the nucleus in pancreatic beta cells [23, 29, 30]. Further studies have shown that nuclear export of IPF1 in response to oxidative stress is dependent on signalling events regulated through the activity of c-JNK . Interestingly, thiazolidinediones have previously been shown to decrease oxidative stress  and to suppress c-fos/c-Jun . Further investigation is required to determine the intracellular signalling events regulating the effects of rosiglitazone in the present study.
Although the effects of rosiglitazone on insulin target tissues have been largely attributed to selective activation of PPARγ, recent studies have begun to reveal that members of the thiazolidinedione family of drugs can also have marked effects on cell function through PPARγ-independent mechanisms [34, 35]. In the present study, addition of GW9662 had no effect on rosiglitazone stimulation of the Ipf1 gene promoter, indicating that these events are not dependent on the activity of PPARγ. This is consistent with bioinformatic analysis of the Ipf1 promoter sequence, which does not reveal the presence of any consensus PPARγ response elements. The transcription factor targets mediating the effects of rosiglitazone on the Ipf1 promoter remain to be delineated.
We have shown that rosiglitazone promotes elevated nuclear levels of FOXA2 over 24 h. FOXA2 is a known regulator of the Ipf1 gene promoter and plays a key role in Ipf1 gene expression both during pancreas development and in adult islets [19, 20]. Nuclear/cytosolic localisation of FOXA2 in hepatocytes is regulated through Akt-mediated phosphorylation events . This study is the first to describe changes in the nuclear accumulation of FOXA2 in response to a specific stimulus in pancreatic beta cells. The exact role of FOXA2 in mediating the effects of rosiglitazone on the Ipf1 promoter has yet to be more fully explored.
The effects of metformin on protein levels, nuclear accumulation and the binding activity of IPF1 appear to be time- and glucose-dependent. The effects of stimuli such as glucose and GLP1 on IPF1 binding activity are known to occur through post-translational modification of the protein over relatively short periods [23, 30]. In the final part of the current analyses, we examined the temporal activation of IPF1 binding activity. EMSA analysis indicates that metformin rapidly activates IPF1 DNA binding activity. Consistent with this observation, nuclear accumulation of IPF1 was observed after 2 h. However, time course analysis of nuclear accumulation confirmed that this effect is not prolonged, with nuclear IPF1 returning to basal levels within 24 h. EMSA analysis of nuclear extracts at 24 h confirms that IPF1 binding activity is unaffected by metformin at this later time point (not shown).
It is perhaps surprising that metformin stimulates IPF1 DNA binding activity but has no effect on the activity of the Luc200 construct, containing the proinsulin gene promoter section. However, two key factors may have contributed to these observations: firstly, the promoter experiments were performed over 24 h, by which time nuclear IPF1 levels have dropped and DNA binding activity has decreased. Secondly, it has become increasingly clear that regulation of the proinsulin promoter is controlled through the synergistic activities of a number of beta cell transcription factors [37–39]. Hence, activation of IPF1 alone may not be sufficient to effect proinsulin gene transcription in this case. Further studies would be necessary to clarify this.
The effects of rosiglitazone in the present study are of particular importance since they add to a growing body of evidence suggesting that thiazolidinediones can have a direct positive impact on beta cell function. Along with improved glycaemic control and improvements in insulin resistance, beta cell ‘rejuvenation’ and improvements in beta cell function have been reported in several recent thiazolidinedione trials . In parallel with in vitro data indicating that thiazolidinediones can protect beta cells not only from apoptosis but also from loss of function , the role of thiazolidinediones in directly targeting and preserving beta cell function is only now becoming clear .
In summary, the present study indicates that both metformin and rosiglitazone have profound effects on transcriptional regulation in pancreatic beta cells, these effects being dependent on both exposure time and glucose concentration. These data are the first to indicate that both metformin and rosiglitazone have direct effects on beta-cell gene expression, and suggest that these widely prescribed antidiabetic drugs may play a previously unrecognised role in the direct regulation of pancreatic beta cell function. Further studies are now required to fully delineate the cell signalling mechanisms regulating these events.
H. Richardson was supported by a Biotechnology and Biological Sciences Research Council CASE studentship with GlaxoSmithKline. W. M. Macfarlane was supported by a Career Development Award from the Juvenile Diabetes Research Foundation. S. C. Campbell was supported by Diabetes UK.