Differential regulation of mouse pancreatic islet insulin secretion and Smad proteins by activin ligands
Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is regulated by paracrine factors, the identity and mechanisms of action of which are incompletely understood. Activins are expressed in pancreatic islets and have been implicated in the regulation of GSIS. Activins A and B signal through a common set of intracellular components, but it is unclear whether they display similar or distinct functions in glucose homeostasis.
We examined glucose homeostatic responses in mice lacking activin B and in pancreatic islets derived from these mutants. We compared the ability of activins A and B to regulate downstream signalling, ATP production and GSIS in islets and beta cells.
Mice lacking activin B displayed elevated serum insulin levels and GSIS. Injection of a soluble activin B antagonist phenocopied these changes in wild-type mice. Isolated pancreatic islets from mutant mice showed enhanced GSIS, which could be rescued by exogenous activin B. Activin B negatively regulated GSIS and ATP production in wild-type islets, while activin A displayed the opposite effects. The downstream mediator Smad3 responded preferentially to activin B in pancreatic islets and beta cells, while Smad2 showed a preference for activin A, indicating distinct signalling effects of the two activins. In line with this, overexpression of Smad3, but not Smad2, decreased GSIS in pancreatic islets.
These results reveal a tug-of-war between activin ligands in the regulation of insulin secretion by beta cells, and suggest that manipulation of activin signalling could be a useful strategy for the control of glucose homeostasis in diabetes and metabolic disease.
KeywordsALK7 ATP GSIS Insulin secretion Islet Mitochondria Smad TGF-β
Glucose-stimulated insulin secretion
The TGF-β superfamily comprises a large group of structurally related ligands, including TGF-βs, growth and differentiation factors, bone morphogenetic proteins and activins. These factors signal via complexes of type I and type II receptor serine-threonine kinases, each binding to different classes of TGF-β ligands [1, 2]. The best known signalling pathway downstream of TGF-β superfamily receptors involves phosphorylation and nuclear translocation of Smad proteins, which in turn regulate gene transcription through interactions with transcription factors in a cell-type-specific manner [1, 2]. Cytoplasmic functions of Smad proteins have also been described, but their physiological significance is still unclear . TGF-β superfamily proteins are best known for their roles in cell differentiation, organogenesis and tissue homeostasis. In comparison, much less is known about their roles as acute mediators of adult metabolic functions.
Paracrine factors are known to regulate glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, but their identity and mechanisms of action are not well understood. TGF-βs 1 and 2 are expressed in islets and have been shown to acutely enhance GSIS [4, 5, 6]. Activins A and B are also expressed in islet cells, suggesting autocrine and/or paracrine roles in islet cell function and glucose homeostasis [7, 8, 9, 10, 11]. Immunohistochemical studies have localised activin A predominantly to alpha cells, while activin B was shown to be expressed by both alpha and beta cells . Earlier work showed that exogenous activin A can increase the cytoplasmic free Ca2+ concentration and stimulate insulin secretion in human and rat pancreatic islets, even at low levels of ambient glucose [12, 13, 14]. Those results have been challenged by more recent studies indicating that in mouse islets, activin A either has no effect  or may in fact decrease GSIS [15, 16]. It has been assumed in several of these studies that activin A and B signal in a similar fashion and thus have similar functions in pancreatic islets. However, the role of activin B in the paracrine control of insulin secretion has not been thoroughly investigated.
We have previously shown that the type I receptor ALK7 is a negative regulator of pancreatic beta cell function . ALK7 is expressed by all major islet cell types and, together with the type II receptor ACTRIIB, can signal in response to several members of the TGF-β superfamily, including activin B, but not activin A [18, 19, 20, 21, 22]. On the other hand, both activin proteins are able to signal through the related receptor ALK4. Mutant mice lacking ALK7 exhibit fasting hyperinsulinaemia, and pancreatic islets derived from Alk7 (also known as Acvr1c) mutants show enhanced GSIS . Similar to Alk7 knockouts, mutant mice lacking activin B display high serum insulin levels as adults , suggesting that activin B might function as an endogenous suppressor of insulin secretion in pancreatic islets.
In the present study, we examined glucose homeostatic responses in mice lacking activin B and in pancreatic islets derived from these mutants. We also compared the ability of the two activin proteins to regulate downstream signalling and GSIS in islets and beta cells. The results demonstrated that activins can elicit distinct acute signalling responses and differential regulation of insulin release in pancreatic islets, revealing a novel layer of paracrine control of insulin secretion based on antagonistic activin signalling.
The generation of InhβB −/− (also known as Inhbb −/−) mice has previously been described . They were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). All experiments in the present study were performed on mice back-crossed for several generations to a C57Bl/6 background. Male mice were used for all the studies. Animal protocols were approved by Stockholm’s Norra djurförsöksetiska nämnd in accordance with ethical guidelines of the Karolinska Institutet.
Insulin measurements and glucose tolerance test
A glucose tolerance test was performed as described  after overnight fasting. Serum insulin was measured with an ultrasensitive mouse insulin ELISA kit (Mercodia, Stockholm, Sweden) from tail blood at the indicated time points before and after i.p. injection of 2 g/kg (body weight) glucose. RAP-435 was injected i.p. at 10 mg/kg twice a week for 4 weeks, followed by examination of fasting glucose and insulin levels and a glucose tolerance test.
Quantification of islet mass
Pancreases were removed from three different mice for each genotype (2 months old and weight-matched) and immediately fixed in 4% paraformaldehyde overnight at 4°C. They were then immersed in 30% sucrose overnight at 4°C and cryopreserved in optimum cutting temperature compound (Histolab, Stockholm, Sweden) at −80°C. Sections (10 μm) were cut on a cryostat and stained with haematoxylin and eosin. Three sections per animal, 140 μm apart, were analysed. For each section, images were acquired with an Axiovert 200 inverted microscope (Carl Zeiss, Stockholm, Sweden) using ×1.25 and ×5 objectives to determine, respectively, pancreas sections and islet areas. An islet was defined as a cluster of at least four cells. Each image was calibrated to calculate the area in μm2 using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The total islet mass was calculated as the total area of islets expressed as a percentage of the total pancreas area.
Islet insulin release, perfusion and ATP production
For each experiment, pancreatic islets were isolated from three or four pancreases by collagenase dispersion and pooled . Isolated islets were left to recover overnight at 37°C (5% CO2) in RPMI medium containing 11 mmol/l glucose and supplemented with 100 μg/ml streptomycin, 100 μg/ml penicillin, 2 mmol/l glutamine and 10% FBS. After overnight incubation, groups each containing ten size-matched islets were glucose-starved by incubation at 37°C for 45 min in HEPES buffer (pH 7.4) containing (in mmol/l) 125 Nalco, 5.9 KCl, 1.2 MgCl2 and 1.28 CaCl2, supplemented with 3 mmol/l glucose and 1 mg/ml BSA.
For stimulation, islets were switched to a solution of the same buffer containing either 3 mmol/l (control) or 11 mmol/l (stimulated) glucose for 60 min at 37°C, with or without activin treatments. Although some studies have used higher glucose concentrations to stimulate islets (e.g. 16 mmol/l or higher), we chose an intermediate concentration to increase the dynamic range and allow for potential positive as well as negative effects of activins on GSIS. The supernatant fraction was assayed for secreted insulin by ELISA and the islets were collected for measurements of DNA content for normalisation. Independent experiments showed no significant differences in insulin content between wild-type and knockout islets or after treatment with activins. Where indicated, activin A or activin B (R&D Systems, Stockholm, Sweden) was added to the final incubation medium at 30–100 ng/ml, a concentration range used in previous islet studies. The inhibitor SB-431542 (Sigma, Stockholm, Sweden)  was used at a final concentration of 10 μmol/l. ATP was measured in lysates using the ATPlite 1step Luminescence Assay System (Perkin-Elmer, Stockholm, Sweden). ATP values were normalised to total islet protein content. For perfusion experiments, groups of 80 isolated islets were transferred to 0.27 ml columns containing Bio-Gel P4 polyacrylamide beads (Bio-Rad, Stockholm, Sweden) and perfused at a flow rate of 0.2 ml/min at 37°C as previously described . HEPES-based buffer (as above) was used as a perfusion solution supplemented with 3 mmol/l glucose, 11 mmol/l glucose or 25 mmol/l KCl, as indicated. Effluent samples were collected every 1 min for the duration of the experiments and assayed for insulin concentration by ELISA. Islet DNA was isolated from each perfusion column and quantified for normalisation.
Total RNA from islets was extracted using the RNeasy Mini Kit (Qiagen, Stockholm, Sweden). First-strand cDNA was obtained from 500 ng total RNA using SuperScript II RNase H− Reverse Transcriptase (Invitrogen, Stockholm, Sweden). Real-time quantitative PCR was carried out to assess the expression of InhβA and InhβB mRNAs and 18S rRNA (as an endogenous normalisation control) using the specific primers (see electronic supplementary material [ESM] Table 1). PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems, Stockholm, Sweden) on a StepOnePlus real-time PCR system (Applied Biosystems). For a quantitative comparison of InhβA and InhβB mRNA levels, each quantitative PCR experiment was run alongside a standard curve for each target amplicon created by serial dilutions of a known quantity of InhβA and InhβB cDNAs derived from islet RNA and subsequently purified by gel electrophoresis and quantified. InhβA and InhβB mRNA levels were normalised to that of 18S rRNA.
Isolated islets were fixed using 2.5% paraformaldehyde (wt/vol.) and 0.1% glutaraldehyde (vol./vol.). Ultrathin sections of 60 nm were stained with 2% uranyl acetate and visualised with transmission electron microscopy. Three male mice from each genotype were analysed. For each mouse, three sections were chosen and seven pictures showing one beta cell were randomly taken from each section. For each pancreas, beta cells were analysed to determine the mitochondrial volume density (volume of mitochondria per unit of beta cell volume).
Recombinant adenoviruses for expression of β-galactosidase, Smad2 and Smad3 were provided by P. ten Dijke (Leiden University Medical Center, Leiden, The Netherlands) and have been described previously [27, 28]. Viral particles were produced and amplified in cultures of HEK293 cells (ATCC, Manassas, VA, USA), as previously described .
Islets were dissected as above and cultured overnight in RPMI-based medium as pools of 80–100 islets per 3.5 cm culture dish. Islets were serum-starved 2 h prior to ligand stimulation. They were then treated with activins at 100 ng/ml for 1 h in the same RPMI-based medium. MEPI and INS-1 cells were cultured as previously described [29, 30]. Islets and cell monolayers were lysed in lysis buffer (50 mmol/l Tris-HCl, 0.15 mol/l NaCl, 1% Triton-X100, pH 7.4) supplemented with protease inhibitors (1 mmol/l PMSF and 1 μg/ml aprotinin) and phosphatase inhibitors (NaPPi, phosphoglycerol and NaO4Va). Total protein was quantified by the BCA method. Each batch of 80–100 islets yielded approximately 50 μg protein. In each case, 20 μg protein was used for SDS/PAGE followed by immunoblotting. Membranes were probed with antibodies against total- and phospho-Smad2 (Cell Signaling, Stockholm, Sweden), total- and phospho-Smad3 (Epitomics, Stockholm, Sweden) or beta actin (Cell Signaling). Proteins were visualised using the ECL western blotting kit (Thermo Scientific, Stockholm, Sweden). Blots were imaged in an LAS 4000 unit using ImageQuant software (GE Healthcare, Stockholm, Sweden).
The Student’s t test and ANOVA were used for statistical analyses.
RAP-435 is a recombinant fusion protein derived from an optimised human activin receptor IIB extracellular domain (ACTRIIB) fused to a human IgG1 Fc domain . RAP-435 can block ACTRIIB signalling and shows higher affinity for activin B compared with other TGF-β superfamily ligands . In order to assess whether endogenous activin B can exert homeostatic functions in the regulation of glucose and insulin levels in naive animals, we treated adult wild-type mice with RAP-435 for 4 weeks and examined fasting insulin and glucose levels, as well as glucose tolerance. Treatment with RAP-435 resulted in higher fasting insulin levels and enhanced GSIS in wild-type mice (Fig. 2c). In addition, animals treated with RAP-435 showed partial glucose intolerance similar to that displayed by InhβB −/− mice (Fig. 2d). Treatment with RAP-435 did not have any significant effect on body weight (data not shown). Together, these results indicate a role for activin B as an endogenous regulator of adult glucose and insulin responses.
We report that activin B can function as an endogenous suppressor of GSIS from pancreatic islets. We have found that activin A has the opposite activity, representing a previously unappreciated functional discrepancy between these two ligands. These differential functions correlate with a differential selectivity for activating Smad2 and Smad3 in pancreatic islets and beta cells, and with the differential ability of the two Smads to regulate GSIS in pancreatic islets. To the best of our knowledge, this is the first instance of closely related members of the TGF-β superfamily being found to elicit differential activation of Smad proteins, and suggests that type I receptors have sophisticated signal decoding capabilities that help to preserve the specificity of ligand-encoded information. The predominant abundance of mRNA encoding the activin B subunit over that of activin A indicates that islet insulin secretion may normally be under tonic suppression by endogenous activin B signalling, a concept that could be exploited to develop next-generation therapeutics for diabetes and metabolic disease. In agreement with this, acute treatment with the small-molecule inhibitor SB-431542 potentiated GSIS in wild-type mouse islets. The importance of activin signalling for glucose homeostasis in humans is underscored by recent studies indicating a positive relationship between plasma levels of activins and clinical characteristics of human type 2 diabetes , as well as a significant association between single-nucleotide polymorphisms in the ACVR1C gene, encoding the activin B receptor ALK7, and the incidence of this disease in humans .
We note that, as in our previous ALK7 study , the InhβB −/− mice used here were back-crossed to a C57/Bl6 background. On the other hand, a recent study performed with outbred InhβB −/− mice of mixed background failed to find differences in fasting insulin levels or glucose tolerance compared with wild-type controls . Interestingly, InhβB −/− mice have been shown to display some defects (e.g. failure of eyelid fusion) in the C57/Bl6 background but not in Sv129 or hybrid backgrounds , suggesting that the genetic background can influence the effects of this mutation. C57/Bl6 is a widely used strain in metabolic studies. These mice are prone to insulin resistance and obesity, and are known to differ from other mouse strains in their insulin and glucose responses . Hence, it is possible that this strain offers a more sensitive background on which to assess the effects of genetic mutations affecting molecular components that regulate insulin and glucose homeostasis.
Previous studies on the effects of activin A on insulin secretion by islet beta cells have reported somewhat conflicting results. Earlier studies indicated that activin A enhances insulin secretion [12, 13, 37]. However, more recent work reported that activin A has either no effect  or in fact decreases [15, 16] GSIS by islets. One difference between the older studies and the new work is the onset and duration of activin stimulation. While the earlier work initiated activin A treatment at the same time as glucose stimulation, the later work preincubated islets in activin A for several hours prior to glucose treatment and prolonged the stimulation for an additional 2 h [4, 15] to 3 days . In line with the earlier work, we have treated islets with activins acutely and concomitantly with glucose stimulation. It is possible that the effects of activins on islet GSIS fade over the course of a few hours, thus explaining how later studies may have missed these activities. This would be in agreement with a rapid, non-transcriptional effect of activin signalling in islet cells, as discussed below.
In line with our observation that Smad3 overexpression suppresses GSIS, a previous study reported that pancreatic islets isolated from Smad3 knockout mice showed enhanced insulin secretion in response to glucose as well as altered expression of several genes involved in beta cell function . On the other hand, overexpression in adult islets of Smad7, which inhibits Smad signalling by all TGF-β superfamily proteins, has been shown to reduce GSIS by affecting islet insulin content and the expression of several key genes for beta cell function, such as Mafa and Menin (also known as Men1) . These effects may have been due to the ability of Smad7 to interrupt bone morphogenetic protein signalling, which is known to be required for the maintenance of the beta cell phenotype . However, we note that some of the effects that we have observed in response to activins are difficult to reconcile with the typical time course of a transcriptional response. It is unclear how Smad proteins may affect acute GSIS responses in islets and it is possible that Smad-independent mechanisms are also at play. Smad proteins are also thought to have transcription-independent functions. For example, Smad3, but not Smad2, has been found to specifically interact with and activate protein kinase A in response to TGF-β but independently of cAMP . The ability of activin ligands to regulate glucose-dependent ATP production in islets, together with the normal response to KCl-mediated depolarisation of InhβB −/− islets, suggests that activin signalling may impact mitochondrial function in the beta cell. In addition, our observation that InhβB −/− beta cells contain more mitochondria then their wild-type counterparts suggests roles for activin B signalling in mitochondrial biogenesis or maintenance.
In summary, our results indicate that activin A and B elicit unexpected differential signalling responses and biological activities in pancreatic islets that contribute to the regulation of glucose and insulin homeostasis in the adult organism. Understanding how activin signalling connects to mitochondrial function and insulin secretion in pancreatic islets will reveal novel insights into TGF-β superfamily signalling and open new opportunities for therapeutic intervention in metabolic diseases.
We thank G. Li (National University of Singapore, Singapore, Republic of Singapore) for providing MEPI cells, C. Wollheim (Centre Médical Universitaire, Geneva, Switzerland) for providing INS-1 cells and R. Kumar (Acceleron Pharma, Cambridge, MA, USA) for providing RAP-435 protein.
This work was supported by grants from the European Research Council, Swedish Research Council, Strategic Research Programme in Diabetes of Karolinska Institutet and Swedish Cancer Society (to CFI), and Berth von Kantzow’s Foundation, Family Erling–Persson Foundation, Knut and Alice Wallenberg Foundation, Novo Nordisk Foundation, Skandia Insurance Company, Stichting af Jochnick Foundation, Strategic Research Program in Diabetes at the Karolinska Institutet, Swedish Diabetes Association, Swedish Research Council, Torsten and Ragnar Söderberg Foundation, Consortium of In Vivo Imaging of Beta-cell Receptors by Applied Nano Technology (Grant FP7-228933-2) and Diabetes Wellness Foundation (to POB).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
HW, KM, PM, TG, AM and SNY designed experiments, acquired, analysed and interpreted data, and contributed to the draft of the manuscript. POB conceived and designed experiments and provided critical revisions to the final version of the manuscript. CFI conceived and designed experiments, interpreted data and wrote the manuscript. All authors approved the final version of the manuscript.
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