Mice with beta cell overexpression of glycogen synthase kinase-3β have reduced beta cell mass and proliferation
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- Liu, Z., Tanabe, K., Bernal-Mizrachi, E. et al. Diabetologia (2008) 51: 623. doi:10.1007/s00125-007-0914-7
Glycogen synthase kinase-3 (GSK3) has been implicated in the pathophysiology of several prevalent diseases, including diabetes. However, despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, the involvement of GSK3 in islet beta cell growth and function in vivo is unknown. We therefore sought to determine whether over-activation of GSK3β would lead to alterations in islet beta cell mass and/or function.
Transgenic mice overexpressing a constitutively active form of human GSK3β (S9A) under the control of the rat insulin promoter (RIP-GSK3βCA) were created. Studies using mouse insulinoma cells (MIN6) were conducted to investigate the regulation of GSK3β activity and its impact on pancreas/duodenum homeobox protein-1 (PDX-1) levels.
We demonstrated that phosphorylation of GSK3β was decreased, indicating increased GSK3β activity in two animal models of diabetes, Lepr −/− mice and Ins2 Akita/+ mice. In MIN6 cells, the activity of GSK3β was regulated by glucose, in a fashion largely dependent on phosphatidylinositol 3-kinase. RIP-GSK3βCA transgenic mice showed impaired glucose tolerance after 5 months of age. Histological studies revealed that transgenic mice had decreased beta cell mass and decreased beta cell proliferation, with a 50% decrease (p < 0.05) in the level of PDX-1.
We showed direct evidence that GSK3β activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. These results may ultimately facilitate the development of potential therapeutic interventions targeting type 2 diabetes and/or islet transplantation.
KeywordsGlycogen synthase kinase-3 Islet beta cells Islet mass Pancreas Proliferation PDX-1
forkhead box protein O1A
glycogen synthase kinase-3
intraperitoneal glucose tolerance test
pancreas/duodenum homeobox protein-1
The prevalence of obesity and diabetes mellitus is increasing with alarming rapidity . Insulin resistance and beta cell failure are the hallmarks of type 2 diabetes . The importance of insulin receptor (InsR) signalling in the development of insulin resistance and beta cell failure has been shown by studies of genetic mouse models deficient in InsR and its downstream signalling components . Knockout of Irs2 in mice resulted in overt diabetes due to increased insulin resistance in peripheral tissues and a concurrent loss of pancreatic beta cells . Recent studies of mouse models lacking functional receptors for both insulin and IGF-1 only in beta cells (βDKO) have provided compelling genetic evidence that InsR signalling is critical for beta cell growth and function . Stimulation of InsR signalling leads to activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt, which promote cell proliferation, growth and survival in various tissue types . βDKO mice developed diabetes 3 weeks after birth due to reduced beta cell mass with reduced level of phosphorylated Akt, indicating decreased Akt activity . Conversely, overexpression of Akt in beta cells resulted in a marked increase in beta cell mass and resistance to streptozotocin-induced diabetes . While it is known that the more proximal components of insulin signalling are critical for the regulation of beta cell mass and function, less is known about the downstream components that relay specific signals controlling beta cell mass and function.
Glycogen synthase kinase-3 (GSK3) was the first substrate shown to be phosphorylated by Akt . GSK3 has two isoforms, GSK3α and GSK3β, both of which are serine/threonine protein kinases whose activities are inhibited by Akt-mediated phosphorylation. An increasing body of literature has demonstrated the diverse functions of GSK3 in the regulation of cellular structure, mobility, proliferation, apoptosis and metabolism [9, 10]. Originally identified as a modulator of glycogen metabolism , GSK3 shows enhanced activity in insulin-resistant states [12, 13] and has been proposed as a potential therapeutic target for the treatment of diabetes [14–16]. GSK3 expression and activity were shown to be increased in skeletal muscle samples from patients with type 2 diabetes . In diabetic or insulin-resistant animal models, two studies have reported increased GSK3 activity in the epididymal fat tissue of mice on a high-fat diet and db/db mice [12, 17]. Although mice with knock-ins of constitutively active GSK3α and GSK3β showed no major metabolic phenotypes , transgenic mice with overexpression of GSK3β in skeletal muscle became glucose-intolerant . More importantly, administration of GSK3 inhibitors to experimental animals has been shown to improve glucose tolerance and reduce insulin resistance [20–23].
Despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, there have been virtually no reports of the in vivo phosphorylation state of GSK3 in pancreatic islets of diabetic animal models and of how it could regulate beta cell growth and function. The purpose of the current studies was to determine the level of phosphorylated GSK3 in pancreatic islets of diabetic animal models and to assess whether over-activation of GSK3β would lead to alterations in islet beta cell mass and/or function. The results of our studies indicate that GSK3 activity can contribute to beta cell dysfunction, possibly enabling identification of a new therapeutic target geared to enhancing islet beta cell growth and preventing type 2 diabetes.
Generation of transgenic mice
To create transgenic mice overexpressing a constitutively active form of human GSK3β(S9A) under the rat insulin promoter (RIP-GSK3βCA), the rat insulin-1 promoter in a pBS-KS vector was fused with human GSK3β(S9A) cDNA (provided by M. Birnbaum, Department of Medicine, University of Pennsylvania, PA, USA). The transgene was linearised with PvuI, purified and microinjected into fertilised eggs of B6/CBA mice by the Washington University Mouse Genetics Core Facility. Transgenic mice were identified by PCR genotyping from tail DNA. Two transgenic founders were established and crossed to wild-type C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine, USA). Ins2 Akita mice and Lepr −/− mice were obtained from Jackson Laboratories and analysed at 3 months of age with average fed glucose above 33.3 mmol/l. All animal procedures were approved by the Washington University Animal Studies Committee.
Islet immunohistological and morphometric analysis
Immunohistological and morphometric analysis was as previously described . Antibodies used in this study were as follows: insulin and Ki67 (Dako North America, Carpinteria, CA, USA), phospho-GSK3β (Cell Signaling Technology, Danvers, MA, USA), haemagglutinin (Covance, Emeryville, CA, USA). Beta cell mass was quantified using NIH ImageJ software version 1.37n .
Fed and fasted glucose and insulin measurements
Fed and fasted glucose were measured as previously described . Insulin levels were determined on 10 μl aliquots using rat insulin ELISA kits with mouse insulin standards (Alpco Diagnostics, Salem, NH USA). Total pancreatic insulin content was measured after acid-ethanol extraction by radioimmunoassay at the Washington University RIA Core.
Intraperitoneal glucose tolerance tests
After mice were fasted overnight (18–20 h), an intraperitoneal glucose tolerance test (IPGTT), consisting of 50% dextrose (2 g/kg body weight) injected intraperitoneally, was performed. Blood was collected from the tail vein at 0, 30, 60 and 120 min after injection for glucose measurements. Plasma for insulin measurements was also collected at 0 and 10 min after glucose injection and submitted to the Washington University RIA Core.
Glucose-stimulated insulin secretion and insulin content from isolated islets
Islets were isolated by collagenase distension/digestion of the pancreas as previously described . Handpicked islets were cultured overnight in RPMI media. Islets were then preincubated for 1 h at 37°C in KRB buffer containing 2 mmol/l glucose. Islets of similar size were handpicked into groups of ten islets in triplicate and incubated for 1 h at 37°C with 1 ml KRB buffer containing either 2 or 20 mmol/l glucose. The supernatant fractions were stored at −20°C prior to insulin measurements. After insulin secretion experiments, islets were washed twice with PBS and extracted with acid-ethanol overnight at 4°C. Insulin levels were measured by radioimmunassay by the Washington University RIA Core.
Assessment of islet beta cell proliferation and apoptosis
Beta cell replication was determined by immunostaining for Ki67 on pancreatic sections. Rate of proliferation was measured by percentage of Ki67-positive cells per total number of insulin-positive cells (about 1,000 cells were counted per animal, four animals per genotype). Cleaved caspase-3 staining (Cell Signaling Technology) and TUNEL (Chemicon, Temecula, CA, USA) were used to measure rate of apoptosis.
MIN6 cell culture and western blotting
MIN6 cells were maintained in DMEM . Cells with passage numbers of less than 35 were used in these experiments. Protein was extracted with a cell lysis buffer (Cell Signalling). Protein samples (20–40 μg) were separated by standard SDS-PAGE gels, transferred to polyvinylidene fluoride membranes and then immunoblotted. Antibodies used in this study were as follows: anti-phospho-Ser473 Akt, anti-phospho-GSK3, anti-GSK3β (Cell Signaling Technology), anti-phospho-glycogen synthase (Biosource, Camarillo, CA, USA), anti-beta catenin (BD Transduction Laboratories, San Jose, CA, USA), anti-cyclin D1 (Chemicon, Temecula, CA, USA), anti-IRS-1 (Upstate, Billerica, MA, USA) and anti-tubulin (Sigma, St Louis, MO, USA).
Unless otherwise indicated, all values are expressed as mean ± SEM and statistical analyses were carried out using an unpaired Student’s t test. Differences were considered to be statistically significant when p < 0.05.
Acute inhibition of GSK3β activity by glucose-induced insulin signalling in mouse insulinoma (MIN6) cells
Chronic activation of GSK3β in islets of diabetic mice with impaired insulin signalling
In animal models with reduced beta cell insulin signalling, either through reduction in InsR  or IRSs , reduction in beta cell mass and diabetes develops with age. Reduced insulin signalling in beta cells would be anticipated to lead to activation of the negatively regulated downstream targets of insulin/PI3K/Akt signalling, forkhead box protein O1A (FOXO1) and GSK3. While there is evidence for increased FOXO1 activity contributing to beta cell loss in animal models , the activity of GSK3 under conditions of impaired insulin signalling has not been studied. As insulin signalling results in suppression of GSK3β activity through Akt phosphorylation, we examined the level of phosphorylated GSK3β as a measure of activity in two diabetic animal models, Lepr −/− mice and Ins2 Akita/+ mice. As shown in Fig. 1d, the levels of phosphorylated GSK3β were markedly diminished in islets of Lepr −/− mice and Ins2 Akita/+ mice compared with those from control animals, suggesting increased GSK3β activity in these diabetic models. These results led to the hypothesis that a transgenic model of overexpression of GSK3β in beta cells would result in beta cell dysfunction and glucose intolerance.
Overexpression of a constitutively active form of GSK3β in pancreatic beta cells
RIP-GSK3βCA mice had normal growth and metabolism during early adult life. Body weight was measured weekly beginning on postnatal day 21. No significant body weight differences between RIP-GSK3βCA and control mice were observed (data not shown). Glucose and insulin levels and the various tolerance tests were performed upon fasted mice (16 h overnight fast) or random-fed mice at 6 weeks and 3 months after birth. Our studies indicated that these transgenic mice had normal glucose metabolism during these periods (data not shown).
RIP-GSK3βCA male mice showed impaired glucose tolerance and decreased beta cell mass at 5 months
To test whether insulin secretion was impaired in RIP-GSK3βCA pancreases in response to high glucose, we did glucose-stimulated insulin secretion assays on islets isolated from both RIP-GSK3βCA and control mice. No difference was observed between these two groups either in low or high glucose conditions (Fig. 3c). The difference in whole pancreatic insulin content was also not statistically significant, although in transgenic islets it was approximately 20% lower (Fig. 3d). Histological study revealed that RIP-GSK3βCA mice had a ~35% decrease in beta cell mass (p = 0.025) compared with that of wild-type control animals (Fig. 3e). Given that the reduction in islet beta cell mass was greater than the reduction in insulin content, we considered that the rate of insulin synthesis per islet may have been increased. However, measurement of insulin content in isolated islets revealed no significant difference (wild-type 1.2 ± 0.5 pmol/islet vs transgenic 1.0 ± 0.5 pmol/islet, p > 0.05). This suggested that the apparent decrease in insulin content was accounted for by decreased mass rather than an effect of GSK3β on insulin synthesis. Assessment of the rate of beta cell proliferation showed a ~25% decrease in the percentage of Ki67-positive cells (p = 0.010) (Fig. 3f). No difference was observed in the rate of beta cell apoptosis, measured by cleaved-caspase-3 and TUNEL assay, between the transgenic mice and littermate controls (data not shown). These studies indicate that constitutive activation of GSK3β in pancreatic beta cells impairs islet beta cell proliferation and subsequently beta cell mass.
GSK3β modulates PDX-1 stability
To further assess the molecular mechanisms that might account for reduced PDX-1 protein level in RIP-GSK3βCA mice, we sought to confirm previous results implicating GSK3 post-translational effects on PDX-1. MIN6 cells were co-treated with cycloheximide to inhibit protein synthesis and observed 4 h after the following treatments. Application of LY294002, a PI3K inhibitor that mimics decreased insulin signalling with GSK3 activation, resulted in a marked decrease in PDX-1 protein compared with that in cells with no treatment (Fig. 4d,e). To specifically target GSK3, cells were treated with lithium, a well-known GSK3 inhibitor. Treatment with lithium resulted in a ~30% increase in total levels of PDX-1, along with a more than 50% reduction in GSK3 activity as shown by the level of pGS (Fig. 4d,f). Treatment with sodium as a control for lithium did not show any difference in either pGS or PDX-1 levels (Fig. 4d). Thus in summary the modulation of PDX-1 protein level by GSK3β activity provided a possible mechanism by which GSK3β overactivity in the RIP-GSK3βCA transgenic mice resulted in altered pancreatic beta cell growth and function.
While it is clear that insulin signalling is critical for maintaining normal beta cell function in the adult mouse and that impaired insulin signalling leads to islet dysfunction, its mediators have not been fully assessed. GSK3 is a well known negatively regulated target of the InsR/PI3K/Akt pathway in many tissues, a topic that has been reviewed [9, 10], yet the hypothesis that impaired insulin signalling and enhanced GSK3 activity result in beta cell dysfunction has not been explored. The current studies report three novel and potentially important observations relevant to InsR/Akt/GSK3 signalling in pancreatic islet beta cells. First is the novel observation that in cultured beta cells GSK3 activity, as shown by phosphorylation of its direct target glycogen synthase, is regulated by changing levels of glucose and that in diabetic islets phosphorylation of GSK3 is decreased, indicating increased activity. Second, overactivation of GSK3 in pancreatic beta cells resulted in glucose intolerance in male mice after 5 months of age with reduced beta cell mass and decreased beta cell proliferation. Third, these results illustrate in an in vivo model that GSK3β overexpression results in reduced levels of PDX-1 protein, while in vitro results further confirmed the role of GSK3 in regulating PDX-1 protein stability. These results thus highlight a new mechanism, whereby signalling through the InsR/Akt/GSK3 pathway regulates beta cell growth; they also provide an explanation for the beta cell dysfunction associated with impaired insulin signalling leading to beta cell failure and diabetes.
Insulin receptor signalling has been implicated in pancreatic beta cell growth, function and survival . Our observation of increased GSK3 activity in two of the well-studied diabetic mouse models suggests a possible mechanism for the reduced islet beta cell mass observed in these animals. Additionally, the current results suggest that the decrease in islet beta cell mass and the resultant diabetic phenotype observed in mouse models with deficiency in receptors for both insulin and IGF-1 (βDKO) or the IRS2 may be related to enhanced GSK3 activity in beta cells. Of course, GSK3 may not be the only mediator of this process, as other distal components of the InsR signalling pathway, e.g. the transcription factor FOXO1, have also been implicated in beta cell growth and function. FOXO1 haploinsufficiency reverses beta cell failure in Irs2 −/− mice through partial restoration of beta cell proliferation and Pdx1 expression . Overexpression of a constitutively nuclear Foxo1 in liver and pancreas prevented beta cell hyperplasia in two animal models . Taken together, these studies highlight the importance of InsR/Akt signalling in maintenance of beta cell growth and function, with two of the direct Akt targets, both GSK3 and FOXO1, contributing to these complex regulations.
The function of GSK3 in pancreatic beta cells may be more related to regulation of cell survival and proliferation than to its effects on carbohydrate metabolism seen in other tissues previously studied. In islet beta cells, previous studies from our group showed siRNA knock-down of GSK3β protected MIN6 cells from endoplasmic reticulum (ER)-stress-induced apoptosis . It has also been shown that inhibition of GSK3 protected beta cells from NEFA-induced apoptosis and this inhibition was also associated with increased beta cell proliferation in primary islets . These observations, along with the results of the current study, thus indicate a predominant role of GSK3 in beta cell proliferation and survival as opposed to an effect on glucose metabolism. The mild glucose intolerance phenotype of transgenic RIP-GSK3βCA mice with a moderate decrease in beta cell mass suggests that additional defect(s) in insulin secretion are possible.
The results of the current studies suggest that a likely mechanism by which GSK3 regulates beta cell proliferation is through destabilisation of PDX-1 protein. GSK3 has been shown to phosphorylate PDX-1 leading to its proteasomal degradation in MIN6 cells . Results of our current studies confirmed this observation not only in cultured insulinoma cells (Fig. 4d,e) but also in RIP-GSK3βCA mice, an in vivo mouse model with GSK3β overexpression and PDX-1 levels reduced by 50 to 60% (Fig. 4a,b). The possibility that PDX-1 levels are critical for maintaining beta cell mass was observed in the Irs2 knockout mouse, where PDX-1 levels were reduced to this magnitude, along with reduction of beta cell proliferation and mass . Importantly, crossing these globally IRS2-deficient mice with mice expressing Pdx-1 specifically in beta cells maintained beta cell mass and rescued the diabetes phenotype . Interestingly, FOXO1, another target of InsR/Akt signalling, has also been shown to regulate PDX-1 levels, but apparently through its effects on suppression of transcription . Thus in beta cells with impaired insulin signalling increased GSK3 as well as increased FOXO1 activity may converge to suppress PDX-1 levels by different mechanisms. In addition to the modulation of PDX-1 stability, GSK3 could also potentially regulate beta cell proliferation through phosphorylation and degradation of cyclin D1 and/or p27. GSK3 has been shown to be an excellent in vitro kinase for cyclin D1 and p27 and also to regulate their stability [37, 38]. However, we did not detect a significant change in the levels of cyclin D1 or p27 in the transgenic islets (Fig. 4c and data not shown).
In transgenic mice, we noted a 20% reduction in pancreatic insulin content (NS). There is good reason to suspect the rate of insulin synthesis per beta cell is reduced, as PDX-1, an important transcription factor for insulin gene transcription, was also reduced in transgenic mice. Additionally, GSK3 could potentially regulate eukaryotic initiation factor 2B activity . Alternatively ER stress could reduce insulin synthesis; indeed, our previous studies have shown that GSK3 activity was enhanced by ER stress . However, GSK3 activation appeared to be downstream of ER stress, so any reduction of insulin synthesis that might be a consequence of increased GSK3 activity is unlikely to be the effect of GSK3 on ER stress.
The results of the current study suggest the possibility of GSK3β inhibition as a means of preserving beta cell mass. Because of the involvement of beta cell failure in common forms of diabetes, these observations have potential clinical implications. Interestingly, the recently approved diabetes treatment Exendin-4 has been shown to promote beta cell proliferation and survival through activation of the PI3K/Akt pathway, see review . FOXO1 was shown to mediate this effect by activating PDX-1 . Both the similar regulation of GSK3 by PI3K/Akt activation, and the modulation of PDX-1 stability by GSK3 suggest that GSK3 may serve as an additional mediator of the effects of Exendin-4 on beta cell function.
In conclusion, we showed direct evidence that GSK3β activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. Further studies will probably identify additional Akt/GSK3β downstream targets responsible for control of beta cell proliferation. Ultimately, our results may facilitate the development of therapeutic interventions targeting type 2 diabetes and/or islet transplantation.
We gratefully acknowledge C. Cras-Méneur for helpful suggestions and stimulating discussion, as well as assistance with the preparation of the figures and manuscript and their submission, and C. Welling for editorial assistance. We also would like to acknowledge M. Birnbaum for the gift of the human GSK3βS9A cDNA. This work was supported in part by a National Institutes of Health grant R37 DK16746 (M. A. Permutt), a Postdoctoral Fellowship Award from the Juvenile Diabetes Research Foundation International 3-2005-968 (Z. Liu), the Washington University Diabetes Research and Training Center (5P60 DK020579) and the Morphology Core of the Digestive Diseases Research Core Center at Washington University (5P30 DK052574).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.