Calcium/calmodulin-dependent kinase IV controls glucose-induced Irs2 expression in mouse beta cells via activation of cAMP response element-binding protein
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Irs2, which is upregulated by glucose, is important for beta cell plasticity. Cyclic AMP response element-binding protein (CREB) stimulates beta cell Irs2 expression and is a major calcium/calmodulin-dependent kinase (CaMK)IV target in neurons. We therefore hypothesised that CaMKIV mediates glucose-induced Irs2 expression in beta cells via CREB activation.
The functions of CaMKIV and CREB were investigated in MIN6 beta cells and mouse islets using the CaMK inhibitor KN62, the calcium chelator bapta-(AM) and the voltage-dependent calcium channel inhibitor nifedipine. Small interfering RNAs were used to silence endogenous CaMKIV production and expression vectors to overproduce constitutively active and dominant negative forms of CaMKIV and CREB. Irs1 and Irs2 expression were determined by quantitative PCR and Western blotting, and the role of CREB was also investigated by assessing its phosphorylation on serine 133.
Increasing the glucose concentration from 2.5 to 25 mmol/l stimulated CREB phosphorylation on serine 133 and specifically stimulated Irs2 but not Irs1 expression. Similarly, overproduction of a constitutively active form of CaMKIV promoted sustained CREB phosphorylation and a significant increase in Irs2 but not Irs1 expression. In contrast, these stimulatory effects of glucose were all suppressed by overproducing an inactive CaMKIV mutant. Inhibition of glucose-induced calcium influx with nifedipine or chelation of intracellular calcium with bapta-(AM), as well as silencing of CaMKIV or inhibition of its activity with KN62 resulted in similar observations. Finally, overproduction of a dominant negative form of CREB completely suppressed glucose and CaMKIV stimulation of Irs2 expression.
Our results suggest that the Ca2+/CaMKIV/CREB cascade plays a critical role in the regulation of Irs2 expression in beta cells.
KeywordsBeta cell CaMKIV CREB Irs2
Constitutively active form of CaMKIV
cAMP response element-binding protein
Constitutively active form of CREB
Dominant negative form of CREB
Dominant negative form of CaMKIV
Protein kinase A
Although it is generally accepted that type 2 diabetes mellitus is caused by a progressive decline in beta cell function  and beta cell mass [2, 3] following the development of insulin resistance [4, 5], several reports suggest that beta cell failure, rather than insulin resistance, is the primary defect, which occurs years before the onset of diabetes [1, 6]. This notion is supported by the general observation that type 2 diabetes mellitus does not develop in most obese individuals or in pregnant women, who can have severe insulin resistance [3, 7, 8], due to a compensatory process involving increased beta cell function and notably beta cell mass expansion [3, 9, 10, 11].
Short-term glucose administration promotes beta cell mass expansion [12, 13, 14], and recent observations implicate glucose-stimulated insulin secretion [15, 16] and Irs2 expression [17, 18] as the main upstream mechanisms. A strong correlation between IRS2 expression in beta cells with apoptosis, proliferation and type 2 diabetes suggests that IRS2 could become a target gene for future therapeutic intervention. Thus, Irs2 knockout mice exhibited a significant reduction in beta cell mass and developed the full phenotype of diabetes [19, 20], whereas targeted re-expression of Irs2 in beta cells increased their survival and promoted their growth through stimulation of proliferation . More recently, it was reported that exendin-4, a stable glucagon-like peptide-1 (GLP-1) receptor agonist known to stimulate Irs2 expression and beta cell mass expansion, failed to do so in Irs2 knockout mice, thus linking the cAMP signalling pathway with Irs2 expression and activity .
The role of the cAMP/protein kinase A (PKA) pathway in glucose-regulated Irs2 expression and beta cell mass expansion was recently investigated and experiments using PKA inhibitors indicated that glucose-stimulated Irs2 expression was reduced by only 20% to 25% [17, 18]. These observations indicate that GLP-1 and glucose do not share the same signalling cascade to increase Irs2 expression, and that activation of the cAMP/PKA pathway is not the major mechanism by which glucose stimulates Irs2 expression.
Interestingly, calcium-dependent stimulation of dendritic growth in neurons is mediated by calcium/calmodulin-dependent kinase (CaMK)IV-induced cAMP response element-binding protein (CREB) activation, independently of cAMP/PKA stimulation . Thus, since CamkIV (also known as Camk4) is expressed in beta cells , is activated by increases in intracellular Ca2+ levels as occurs following glucose metabolism  and CREB is known to stimulate Irs2 gene expression , it is possible that glucose regulates Irs2 levels in beta cells through a Ca2+/CaMKIV/CREB signalling cascade.
In the current study, the role of CaMKIV-induced CREB activation in mediating the glucose effects on Irs2 expression in MIN6 beta cells and mouse islets was examined through downregulation and overexpression studies that used small-interfering RNAs (siRNAs) and constitutively active or dominant negative forms of CaMKIV and CREB.
Experiments involving animals were approved by the local ethics committee.
Cells, plasmids and reagents
MIN6 cells were a gift from Y. Oka and J.-I. Miyazaki (Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan) and ICR mice were purchased from Harlan (Blackthorn, UK). Plasmids encoding the mouse constitutively active form of CaMKIV (ΔCaMKIV) and human kinase-dead form of CaMKIV (ΔK73ECaMKIV) were kindly provided by A. Ghosh (Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA). pcDNA3.1 plasmids encoding the following forms of CREB were a gift from Professor D. D. Ginty (Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, USA): wild-type; constitutively active forms of CREB (DIEDML); and dominant negative forms of CREB (CREBm1). Metafectene Pro was from Biontex (Martinsried/Planegg, Germany). Glucose-free DMEM and FBS were purchased from Invitrogen (Paisley, UK). KN62 was from Calbiochem (Nottingham, UK). The mouse monoclonal anti-α-tubulin antibody, culture media, nifedipine, bapta-(AM), Histopaque-1077, collagenase (type XI), penicillin/streptomycin and l-glutamine were from Sigma Aldrich (Poole, UK). The rabbit polyclonal anti-IRS1 and anti-IRS2 antibodies were from Millipore (Watford, UK), the mouse monoclonal anti-CaMKIV antibody was from Clontech (Oxford, UK), and mouse monoclonal anti-CREB and anti-(133phosphoserine) CREB antibodies were from New England Biolabs (Hitchin, UK). The siRNA duplexes were obtained from Dharmacon (Cramlington, UK). The mRNA purification kit (RNeasy) was from Qiagen (Crawley, UK). Enhanced chemiluminescent kits for Western blotting were from GE Healthcare (Little Chalfont, UK). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Pierce Biotechnology (Rockford, IL, USA).
Cell culture, treatment and transfection
MIN6 beta cells were maintained in culture at 37°C in DMEM (25 mmol/l glucose) supplemented with 2 mmol/l glutamine, 10% (vol./vol.) FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. To study the effect of glucose on Irs1 and Irs2 expression and on CREB phosphorylation, cells were maintained at 6 mmol/l glucose for 24 h followed by exposure to the experimental conditions described below (“Results”). Pre-treatment with the CaMK inhibitor KN62 (or DMSO vehicle, 45 min) and transient transfection with non-silencing RNA or siRNA duplexes designed to knock down CaMKIV levels were performed before final adjustment of the glucose concentration. High efficiency (~60–80%) transient transfection of MIN6 cells was achieved by electroporation (Nucleofector II; Amaxa, Cologne, Germany).
Mouse islets isolation, culture and siRNA transfection
Islets were isolated by collagenase digestion of mouse pancreas as described previously [27, 28] and maintained in culture for 24 to 48 h in RPMI 1640 medium (11 mmol/l glucose) supplemented with 10% (vol./vol.) FBS before use. To study the role of CaMKIV in primary tissue, mouse islets were transiently transfected with either a commercially available non-silencing RNA or with four siRNA duplexes designed to specifically reduce endogenous CaMKIV levels. The target sequences used were: 5′-gagauccucugggcgauuu-3′, 5′-ucaaggaaauauucgaaac-3′, 5′-ggugcuacauccauugugu-3′ and 5′-gggaugaagugucuuuaaa-3′. Transient transfection of mouse islets was performed using a two-step transfection protocol with Metafectene Pro as described .
Reverse transcription and quantitative polymerase chain reaction
Total RNA was isolated from ~5 × 105 MIN6 cells or ~150 mouse islets using RNeasy (Qiagen) according to the manufacturer’s instructions. Complementary DNAs were synthesised and quantitative PCR amplifications were performed as described previously . Each sample value was normalised to beta-actin copy numbers. In all quantitative PCR experiments, the presence of possible contaminants was checked by control reactions in which amplification was performed in reaction mixtures without cDNA templates. Specificity of each primer pair was confirmed by melting curve analysis and agarose-gel electrophoresis of PCR products.
Western blot analysis
MIN6 cell protein extracts (50–75 μg) were separated on 10% (wt/vol.) polyacrylamide gels and transferred to polyvinylidene fluoride membranes, which were incubated for 16 h with antibodies directed against IRS1 (1:166 dilution), IRS2 (1:1,000 dilution), α-tubulin (1:2,000 dilution), CaMKIV (1:750 dilution), CREB (1:750 dilution) or phospho-CREB(Ser133) (1:750 dilution). After three washes in Tris buffered saline (pH 7.4) containing 0.05% Tween 20, the polyvinylidene fluoride membranes were incubated for another hour with horseradish peroxidase-coupled anti-rabbit or anti-mouse IgGs as appropriate (1:5,000 dilution). Binding of secondary antibodies was revealed by chemiluminescence.
Numerical data are expressed as means ± SEM. Differences between two groups were analysed by unpaired Student’s t test and considered significant at p < 0.05. Differences between several groups were analysed by one-way analysis of variance followed by Tukey’s honestly significant differences test.
Glucose stimulates Irs2, but not Irs1 mRNA expression in beta cells
Glucose-induced IRS2 production is calcium-dependent
Glucose stimulation of Irs2 production is mediated by a CaMK pathway
The data in Figs 1 and 2 indicate that glucose stimulates Irs2 mRNA expression and IRS2 protein levels in a concentration-dependent manner in MIN6 beta cells and in mouse islets. We therefore used MIN6 beta cells to further examine the molecular mechanisms downstream of calcium influx, which mediate this effect. Thus, knowing that CREB has the ability to induce beta cell Irs2, but not Irs1 expression , and that CaMKI and CaMKIV mediate the calcium effect on CREB activation in the GH3 growth hormone-secreting cell line , we tested the hypothesis that glucose-stimulated Irs2 expression in beta cells is regulated by a CaMK pathway. This was achieved by assessing the effect of the non-selective CaMK inhibitor KN62 (30 μmol/l) on glucose-stimulated CREB phosphorylation at serine 133 and on IRS2 protein levels.
Glucose-stimulated Irs2 expression is controlled by CaMKIV
CaMKIV mediates glucose-induced Irs2 expression via CREB activation
To establish whether a direct link between glucose, CaMKIV, CREB and IRS2 production exists in beta cells, CREBm1 and the constitutively active form of CREB (CREBDIEDML) were transiently overexpressed in MIN6 cells and the resulting effects on glucose- and CaMKIV-induced stimulation of IRS2 protein levels were analysed by Western blotting. As shown in Fig. 6b, IRS2 production was stimulated in a glucose- and CaMKIV-dependent manner, confirming the results obtained in our stable transfection studies (Fig. 6a). The data also show that excess levels of CREBm1, as with excess ΔK75ECaMKIV, suppressed the stimulatory effect of high glucose concentrations (12 and 25 mmol/l) on IRS2 production. As expected, Fig. 6c shows that overproduction of CREBDIEDML at a non-stimulatory glucose concentration of 6 mmol/l resulted in a marked increase in IRS2 protein in MIN6 cells. In addition, whereas simultaneous excess of ΔCaMKIV and the wild-type form of CREB generated an additive effect on IRS2 production, excess levels of CREBm1 completely abolished the stimulatory effect of ΔCaMKIV.
Reduction of CaMKIV levels decreases glucose-induced Irs2 expression
CaMKIV is a multifunctional enzyme whose function is best understood in neurons, where it inhibits apoptosis and stimulates growth in a calcium- and CREB-dependent manner [23, 33, 34, 35, 36, 37]. CamkIV is also expressed by pancreatic beta cells , but its roles in beta cells have not been fully defined. Earlier reports that glucose and GLP-1 receptor agonists regulate beta cell mass through CREB- and IRS2-dependent inhibition of apoptosis and stimulation of proliferation [17, 18, 21, 26, 38, 39, 40, 41] led us to examine the role of the CaMKIV–CREB cascade in the regulation of Irs2 expression by beta cells.
Our results demonstrate that glucose stimulates Irs2 expression in islets and MIN6 cells in a calcium-dependent manner and provide evidence for the first time that this is mediated by the CaMKIV–CREB pathway. Indeed, glucose-dependent Irs2 mRNA and protein upregulation were substantially reduced when either ΔK75ECaMKIV or CREBm1 were overabundant in MIN6 cells or following siRNA-induced reduction of endogenous CaMKIV content. In contrast, excess levels of the constitutively active forms of CaMKIV (ΔCaMKIV) or of CREB (CREBdiedml) resulted in enhanced Irs2 expression, while the stimulatory effect of ΔCaMKIV was suppressed by coproduction of the dominant negative CREB mutant (CREBm1 ). These novel observations in MIN6 cells are consistent with previous in vitro [23, 42] and in vivo  reports in other tissues, demonstrating that CaMKIV controls CREB transcriptional activity. The observation that only Irs2, but not Irs1 expression levels were modified when glucose concentration was increased or the constitutively active form of CaMKIV was overproduced also confirms previous observations of selective upregulation of Irs2 by glucose [17, 18], as well as demonstrating the specificity of this glucose–CaMKIV effect. We also observed that stable ΔCaMKIV production by MIN6 cells produced significant elevations of Irs2 mRNA at 2.5 and 12 mmol/l glucose compared with native MIN6 cells, but this did not occur at 25 mmol/l glucose. These observations suggest that production of the constitutively active ΔCaMKIV in MIN6 cells by-passed the requirement for glucose-stimulated calcium entry and enabled the cells to maximally stimulate Irs2 expression independently of a glucose stimulus.
However, whereas glucose-induced CREB phosphorylation at serine 133 was abolished by the calcium/calmodulin kinase inhibitor KN62 or in MIN6 cells stably transfected with ΔK75ECaMKIV (Figs. 3a and 4b), glucose-stimulated IRS2 protein production was only partially reduced (Figs 3b and 6a). This suggests that part of the stimulatory effect of glucose on IRS2 abundance may be independent of CaMKIV and that alternative mechanisms of action exist. One possible alternative signalling cascade might involve glucose-induced increases in intracellular cAMP levels , which have been shown to promote MIN6 cell CREB phosphorylation at serine 133 and CREB activation, but with a delayed time course compared with depolarising stimuli . This hypothesis is supported by previous observations showing that H-89 and KT5720, two PKA inhibitors, reduced glucose-stimulated Irs2 mRNA expression and protein levels by approximately 25% in rat islets .
MIN6 beta cells were used for many of the experiments presented in this study because they have several of the key functional characteristics of primary beta cells  and are readily amenable to stable transfection. However, MIN6 cells are a transformed beta cell line expressing the SV40 large T-antigen, which keeps them in a proliferative state, and they are also adapted to maintenance in media containing high glucose concentrations. Therefore, to ensure that the data obtained using MIN6 cells did not reflect signalling cascades present in cell lines but not in primary beta cells, key experiments were repeated using isolated mouse islets. We found that glucose stimulated a calcium-dependent upregulation of Irs2 in islets, with stimulatory profiles similar to those seen in MIN6 cells; glucose also stimulated CREB phosphorylation. In addition, our experiments in mouse islets, in which glucose-stimulated Irs2 upregulation was lost when CaMKIV production was transiently knocked down, confirmed the existence, in mouse islets, of the glucose/CaMKIV/Irs2 cascade that we had identified in MIN6 cells. Taken together, our data imply that CaMKIV plays a central role in the regulation of Irs2 expression in islets. Previous studies using the HIT-T15 and INS-1 insulin-secreting cell lines suggest that CaMKIV also mediates glucose-induced insulin gene expression and insulin secretion [24, 25], and that it stimulates glucokinase expression . In addition, CamkIV is a target gene for the canonical wingless-type MMTV integration site family (WNT)/β-catenin signalling pathway , whose signalling-associated transcription factor TCF7L2 is a diabetes susceptibility gene [46, 47] known to regulate beta cell proliferation , insulin gene expression and insulin secretion . Thus, a central role for CamKIV in islet function implicates it as a useful target gene for the development of future drug therapies to treat type 2 diabetes mellitus.
The results presented here establish a critical role for CaMKIV in Irs2 expression. This, together with previous observations showing a reduction in beta cell mass due to increased beta cell apoptosis in Irs2 knockout mice [19, 20, 21], suggests that CaMKIV might regulate beta cell survival and proliferation. Preliminary data, obtained in our laboratory and demonstrating that overexpression of the constitutively active form of CaMKIV in MIN6 cells stimulates proliferation and reduces caspase-3/7 activities, are consistent with this hypothesis (D. S. Muller, S. J. Persaud, B. Liu and P. M Jones, unpublished data). The precise role of Irs2 in these CaMKIV-mediated effects is now being investigated.
In conclusion, the current study demonstrates for the first time that CaMKIV has a central role in CREB-dependent mechanisms by which glucose regulates Irs2 expression in beta cells. Moreover, since Irs2 deficiency has been linked with the progressive development of type 2 diabetes mellitus, our results suggest that finding a mechanism to stimulate CamKIV expression and/or activity could have a significant clinical impact in the future for patients with type 2 diabetes mellitus.
We are grateful to J. I. Miyazaki (University of Osaka, Osaka, Japan) for the provision of MIN6 cells, to A. Gosh (University of California, San Diego, CA, USA) for CaMKIV plasmids, to D. D. Ginty (John Hopkins University, Baltimore) for CREB plasmids, to P. Marsh (King’s College London, London, UK) for helping with plasmid amplifications, and to J. Bowe and A. King (King’s College London, London, UK) for assisting with mouse islet isolation. We gratefully acknowledge The Eli Lilly International Foundation for grant support. B. Liu was supported by an Overseas Research Students Postgraduate Award. D. S. Muller was a Diabetes UK RD Lawrence Fellow.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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