CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice
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Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice.
We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing CishloxP/loxP mice with Pdx1-CreEarly mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR.
There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice.
Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.
KeywordsBeta cell proliferation CISH Pregnancy Prolactin SOCS
Cytokine-inducible SH2-domain containing protein
Glucose tolerance test
Janus kinase 2
Suppressor of cytokine signalling 2
Signal transducer and activator of transcription 5
An insufficient number of insulin-producing beta cells is a hallmark of both type 1 and type 2 diabetes. Therefore, one therapeutic strategy is to increase functional beta cell mass in order to overcome insulin deficiency . The majority of postnatal beta cell mass expansion is caused by replication of pre-existing beta cells, rather than by differentiation from progenitors or other cells, at least in rodents . Under normal physiological conditions, the proliferation rate of beta cells in adult mammals is very low (less than 1%) . However, beta cells have the capacity to expand by proliferation when metabolically challenged, such as during pregnancy, diet-induced insulin resistance and experimental beta cell ablation, as shown in rodent models . During pregnancy in rodents, the rate of beta cell proliferation increases dramatically as an adaptation to insulin resistance and peaks two-thirds through the gestational period, which in mice corresponds to day 14.5 of pregnancy [5, 6]. Beta cell mass in rodents increases threefold to fourfold during pregnancy, driven by both beta cell hyperplasia and replication .
Beta cell proliferation during pregnancy is regulated by many factors, including hormonal signals, chiefly lactogens [6, 7]. There are two types of lactogens in mammals: prolactin (PRL) and placental lactogen (PL), the levels of both of which are elevated during pregnancy . Both PRL and PL bind to the PRL receptor (PRL-R), which is a member of the cytokine receptor superfamily . Lactogens have been shown to enhance beta cell proliferation and insulin secretion both in vitro and in vivo , acting through a complex signalling network. The most important mediator of lactogen signalling is the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) pathway . Upon ligand binding to PRL-R, JAK2 kinase is activated and PRL-R is phosphorylated at specific tyrosine residues. STAT5 is recruited to phosphorylated PRL-R and is phosphorylated in turn by JAK2. p-STAT5 then dimerises and translocates into the nucleus, where it regulates gene expression as a transcription factor . STAT5 is critical for several cytokine-signalling pathways, including those involving PRL/PL, growth hormone, IL-2 and IL-3 . STAT5 phosphorylation and nuclear translocation is upregulated in islets during pregnancy in response to PRL/PL signalling .
Many known STAT5 targets are also upregulated during pregnancy, including Prlr, which forms a positive feedback loop of the PRL/PL signalling pathway ; Glut2 (also known as Slc2a2), which transports glucose into beta cells; and cyclinD2 (also known as Ccnd2), which drives beta cell proliferation [14, 15, 16, 17, 18]. As an apparent limit to unbridled replication, two of the negative feedback regulators of PRL/PL/STAT5 signalling, Cish (encoding cytokine-inducible SH2-domain containing protein) and the closely related gene Socs2 (encoding suppressor of cytokine signalling 2) are also upregulated during pregnancy [6, 19, 20].
SOCS2 and CISH are both members of SOCS family of proteins, which constitutes eight members with similar structures that function as inhibitors of cytokine signalling . All family members contain a central SH2 domain for binding to phosphorylated tyrosines and a C-terminal ‘SOCS-box’ domain for directing targeted proteins to proteasomal degradation. SOCS1 and SOCS3 also contain an N-terminal kinase inhibitory region domain . Thus, different SOCS proteins inhibit JAK/STAT signalling by different mechanisms. For example, SOCS1 binds to JAK2 and inhibits its ability to phosphorylate cytokine receptors and STAT5. On the other hand, SOCS2 and SOCS3 bind to the phosphorylated tyrosine site on the receptor, which competitively blocks the recruitment of STAT5, thus inhibiting the phosphorylation and activation of STAT5 proteins . Different gene ablation models and transgenic mice for multiple Socs genes have been described, and show various phenotypes depending on which particular cytokine signal is being regulated .
Because lactogen signalling is critical for beta cell proliferation and beta cell function during pregnancy and Cish and, to a lesser extent, Socs2 are induced during pregnancy , we hypothesised that the CISH and SOCS2 proteins negatively regulate beta cell proliferation and beta cell function. In this study, we derived a novel mouse model with conditional ablation of the Cish gene in the pancreas to test the hypothesis that removing this negative feedback inhibitor could be exploited to stimulate beta cell replication.
A 19.0 kb DNA fragment containing the entire Cish coding sequence was retrieved from the C57BL/6J mouse bacterial artificial chromosome clone RP24-146L13 via bacterial recombination and subcloned into plasmid PL253, which contains a thymidine kinase cassette for negative selection. A targeting vector was engineered to contain a single loxP site and an FRT-tACE-FLP-neo-FRT-LoxP cassette  flanking exon 2 of the Cish gene. The targeting vector was electroporated into C57BL6 embryonic stem (ES) cells, stably transfected ES cells were selected for with G418 and correctly targeted ES cell clones were identified by Southern blot analysis with a 3′ external probe after digesting genomic DNA with EcoRI. Targeted ES cells were expanded and injected into albino C57BL/6J blastocysts. Germline transmission of the loxP allele in the chimeric pups was identified by crossing with albino C57BL/6J mice. Germline chimeras were then crossed to C57BL/6J mice to obtain heterozygous mice. The FRT-flanked neomycin resistance gene was self-excised by tACE-induced Flp expression in the male germline.
Cish loxP mice were crossed with Pdx1-CreEarly mice (kindly provided by Douglas A. Melton from Harvard University, Cambridge, MA, USA)  to induce pancreas-specific Cish ablation. CishloxP/loxP mice were used as controls and CishloxP/loxP; Pdx1-CreEarly mice constituted the mutant group. Pdx1-CreEarly mice were used as controls in the study of virgin mice in addition to the CishloxP/loxP controls, and no difference was observed in the insulin levels among the two control groups and the mutant group, consistent with previous studies  showing that the Pdx1-CreEarly transgene has no effect on beta cell proliferation or beta cell function. Therefore, we only used CishloxP/loxP mice as control mice in our further studies. In addition, CishloxP/loxP; Pdx1-CreEarly mice were crossed with the Mip-GFP mouse (purchased from The Jackson Laboratory, Bar Harbor, ME, USA) , which is a transgenic mouse with green fluorescent protein driven by mouse-insulin-promoter, labelling pancreatic beta cells, to generate CishloxP/loxP; Pdx1-CreEarly; Mip-GFP and CishloxP/loxP; Mip-GFP mice for the sorting of beta cells and non-beta cells in the islets. Mice were analysed between 3 and 5 months of age. All procedures involving mice were conducted in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee.
Twenty-four hours before killing the mice, 1 ml/100 g body weight of BrdU labelling reagent (Life Technologies, Grand Island, NY, USA) was injected i.p. Pancreases were dissected, flattened by forceps, fixed in 4% wt/vol. paraformaldehyde for 24 h and paraffin embedded so that tissues with the maximum pancreatic footprint were sectioned. Tissues were sectioned to 5 μm thickness. Deparaffinised and rehydrated slides were subjected to antigen retrieval by pressure cooker in 10 mmol/l pH 6.0 citric acid buffer. Simultaneous immunofluorescent staining was performed for BrdU and insulin. The primary antibodies used were guinea pig anti-insulin (1:1,000 dilution, Dako North America, Carpinteria, CA, USA) and rat anti-BrdU (1:500 dilution, AbD Serotec, Raleigh, NC, USA). Secondary antibodies were Cy2-anti-guinea pig (1:200) and Cy3-anti-rat (1:200). The beta cell proliferation rate was quantified as BrdU/insulin double-positive cells divided by insulin-positive cells. One section from each animal was manually counted, and nine animals from each genotype were analysed.
Beta cell mass
Three sections (40 μm apart) from each animal were immunostained for insulin using the standard diaminobenzidine (DAB) method without counterstaining. The entire tissue section was scanned with a PathScan Enabler IV Histology Slide Scanner and SilverFast PathScan 6.6 software (Meyer Instruments, Houston, TX, USA). The percentage of beta cell area relative to the total pancreatic area was measured and calculated using ImageJ (http://rsbweb.nih.gov/ij/). Beta cell mass was derived from the total pancreas weight multiplied by the percentage of beta cell area.
Glucose tolerance test and insulin assay
Animals were fasted overnight and fasting glucose levels determined by glucometer. Glucose (2 g/kg body weight; Sigma-Aldrich, St Louis, MO, USA) was injected i.p. Glucose levels were measured at 15, 30, 60, 90 and 120 min postinjection. Glucose levels were measured by Glucometer Breeze2 (Bayer AG, Leverkusen, Germany). To determine plasma insulin levels during glucose tolerance tests (GTTs), blood was collected from the tail vein of mice before and after overnight fasting. Plasma insulin concentration was measured by ELISA.
Islet isolation and real-time PCR
Islets were isolated using standard collagenase procedures followed by purification through a Ficoll gradient (Ficoll PM 400, Sigma-Aldrich), as previously described . Islets were handpicked under a light microscope. Total RNA was isolated in TRIzol (Life Technologies) and reverse transcribed using 1 μg oligo(dT) primer, SuperScript II Reverse Transcriptase and accompanying reagents (Life Technologies). PCR reaction mixes were assembled using the Brilliant III SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA). PCR reactions were performed on an Mx4000 Multiplex Quantitative PCR System (Agilent Technologies). All reactions were performed in triplicate with reference-dye normalisation. Median cycling threshold values were used for analysis. Expression values were normalised to those of β-actin as internal standard.
A total of 150 islets were handpicked under a light microscope and placed into a perifusion chamber (EMD Millipore, Billerica, MA, USA). A computer-controlled fast-performance HPLC system (625 LC System, Waters Corporation, Huntingdon Valley, PA, USA) allowed programmable rates of flow and concentrations of the appropriate solutions held in a 37°C water bath. Islets were perifused with Krebs bicarbonate buffer (2.2 mmol/l Ca2+, 0.25% wt/vol. BSA, 10 mmol/l HEPES and 95% O2/5% CO2 equilibration [pH 7.35]) plus 2 mmol/l glucose and 4 mmol/l AAM-19 and glutamine to reach baseline hormone secretion values before the addition of the appropriate secretagogues. Samples were collected at regular intervals with a fraction collector (Waters Corporation). Insulin content was determined using radioimmunoassay.
For western blotting, islets were isolated and lysed in lysis buffer containing 50 mmol/l Tris (pH 8.0), 5 mmol/l EDTA, 150 mmol/l NaCl, 1% vol./vol. Triton, 1% vol./vol. SDS, 0.5% wt/vol. sodium deoxycholic acid and Complete Protease Inhibitor Cocktail Tablets (Genentech Roche, Newtown, PA, USA). Protein concentrations were measured by Bradford Assay using SpectraMax Plus 384 (Molecular Devices, Sunnyvale, CA, USA). Total lysate (6 μg) was heated at 95°C for 10 min and loaded on 4–12% Bis-Tris gel (Novex, Wadsworth, OH, USA). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes by iBlot Dry Blotting System (Life Technologies) and detected by antibodies against p-STAT5 (Santa Cruz Biotechnology, Dallas TX, USA) and rat anti-beta-actin (EMD Millipore Corporation, Billerica, MA, USA). The ECL-Plus detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA) was used to detect the signal.
Islets from two CishloxP/loxP; Pdx1-CreEarly; Mip-GFP mice and two CishloxP/loxP; Mip-GFP mice were isolated and pooled by the same genotype. Dissociated cells from isolated islets were sorted into GFP-positive cells for highly enriched beta cells and GFP-negative cells for non-beta cells. Total RNA was extracted from the sorted cells and subjected to further analysis.
Data are presented as means ± SEM. The statistical significance of differences was determined by Student’s t tests or multivariate ANOVA (MANOVA). p < 0.05 was considered statistically significant.
Derivation of CishloxP mice
The targeting vector was electroporated into mouse C57BL6-strain ES cells, stably transfected ES cells were selected for with G418 and correctly targeted ES cell clones were identified by Southern blot. Genomic DNA was isolated from ES cells and digested with EcoRI, and fragments were detected by hybridisation with a 3′ external probe (Fig. 1b). The wild-type allele produced a 9.8 kb fragment and the targeted allele produced a 8.1 kb fragment. Targeted ES cells were expanded and injected into mouse blastocysts. The FRT-flanked FLP-neo cassette was self-excised by tACE-induced Flp expression after germline transmission in male chimeric pups. CishloxP/loxP mice, obtained by intercrossing CishloxP/+mice, were healthy and fertile, indicating that the CishloxP allele was functionally wild type.
Cish ablation in islets
To evaluate the efficiency of Cish gene ablation, we determined expression of Cish at the mRNA level because no CISH-specific antibody is available. PCR primers were designed within exon 2 of the Cish gene. Cish mRNA levels were reduced by more than 90% in pancreatic islets isolated from CishloxP/loxP; Pdx1-CreEarly mice as compared with control CishloxP/loxP mice (Fig. 2c). To ascertain that CISH was ablated in beta cells, we bred CishloxP/loxP; Pdx1-CreEarly to Mip-GFP mice to allow for the efficient isolation of GFP-labelled beta cells by fluorescent-associated cell sorting. In the islets of control mice, Cish mRNA was highly enriched in beta cells (CishloxP/loxP; Mip-GFP, GFP+) compared with islet non-beta cells (CishloxP/loxP; Mip-GFP, GFP−). Analysis of Cish mRNA levels in GFP+ cells sorted from CishloxP/loxP;Pdx1-CreEarly; Mip-GFP mice established that Cish was efficiently ablated in the mutant beta cells (Fig. 2d). Mutant islets maintained normal morphology and architecture, as shown by staining of insulin, glucagon and somatostatin (Fig. 2e–h). Thus, Cish was not required for maintenance of normal islet architecture.
Cish is not required for beta cell proliferation or glucose homeostasis in mice
Although we detected no difference in the rate of beta cell proliferation in Cish-deficient mice, it was still possible that beta cell function was enhanced without affecting proliferation. To answer this question, we performed GTTs on 11 CishloxP/loxP; Pdx1-CreEarly mice and 11 CishloxP/loxP mice, all on day 14.5 of pregnancy. No difference was observed in body weight between the control and mutant groups (Fig. 4e). Furthermore, no difference in glucose tolerance was observed between the two groups of mice (Fig. 4f). GTTs were also performed on pregnancy day 9.5 mice, and again we found no difference in glucose tolerance between control and mutant mice (data not shown). In addition, we observed no differences in resting and fasted insulin levels (Fig. 4g). To confirm these findings, we performed islet perifusion studies of insulin secretion on islets isolated from day 14.5 pregnant mice. There was no difference in glucose-stimulated insulin secretion between the two groups (Fig. 4h). Thus, Cish deficiency in beta cells did not alter glucose tolerance or beta cell function during pregnancy.
Cish ablation is not sufficient to increase activation of STAT5
The Socs2 gene is upregulated upon Cish ablation at pregnancy day 9.5
Since Cish ablation did not lead to elevated STAT5 activation, we hypothesised that other Cish/Socs family members might be upregulated and compensate for the loss of Cish. We had previously shown that Socs2 is also upregulated in islets during pregnancy, making it a prime candidate for a compensatory effect. We synthesised cDNA from islet RNA of control and mutant mice, both before and during pregnancy. At day 14.5 of pregnancy, expression of Cish and Socs2 was increased, consistent with published work (Fig. 6c) . The mRNA expression of other Socs genes was also determined, and none of them was significantly upregulated upon Cish ablation (Fig. 6c). The expression of Socs1 was undetectable in islets. Interestingly, Socs2 mRNA levels were higher in Cish mutant mice than control mice at pregnancy day 9.5 (Fig. 6d), suggesting that Socs2 might be compensating for Cish ablation during this phase of pregnancy. Glut2, Gck, and CyclinB1 (also known as Ccnb1) mRNA levels were the same in Cish control and mutant mice at day 9.5 of pregnancy (Fig. 6d), indicating normal glucose metabolism and beta cell proliferation.
To test this hypothesis, we generated mice with pancreas-specific Cish ablation. These mice represent the first reported mouse model for tissue-specific ablation of Cish. In our CishloxP/loxP; Pdx1-CreEarly model, Cish expression was reduced by more than 90%, demonstrating the efficiency of the system. Cish was also found to be highly enriched in the sorted beta cells compared with the other islet cell types, and was efficiently deleted in islet beta cells. The reason for the remaining 5–10% of Cish mRNA expression could be that Cre recombination does not occur in all beta cells. The efficacy of Cre-mediated gene ablation we observed for the Cish locus was comparable with that of other loxP-flanked targets . The Pdx1-CreEarly transgene is also expressed in a subset of cells in the hypothalamus, and because there is central input to glucose homeostasis , we performed ex vivo experiments to exclude any potential beta cell non-autonomous effect. We did not observe any difference between control and mutant islets in our insulin secretion studies of isolated islets, in which neuronal input has been excluded. Furthermore, in order to evaluate if the Pdx1-CreEarly transgene itself might impact the phenotype of our mutant mice, we determined insulin levels in two control groups (CishloxP/loxP and Pdx1-CreEarly mice). We observed no abnormal phenotypes in Pdx1-CreEarly mice, consistent with previous studies using the same mouse strain .
Cish-deficient mice exhibited normal islet architecture and normal glucose homeostasis before, during and after pregnancy. Surprisingly, we found no difference in pregnancy-induced beta cell proliferation or glucose homeostasis in Cish-deficient females compared with controls. Cish-ablation mice exhibited p-STAT5 levels comparable with those of control mice, and mRNA levels of Tph1 and Tph2 further demonstrated that the STAT5 signalling pathway was not affected. mRNA expression levels of other Socs gene family members were determined and Socs2 mRNA levels were upregulated even higher than in control mice in the absence of Cish on day 9.5 of pregnancy, suggesting that Socs2 might compensate for Cish ablation during pregnancy. Therefore, a mouse model with simultaneous, beta cell specific ablation of Cish and Socs2 might be required to uncover non-redundant functions of the two proteins in beta cell proliferation and function. Alternatively, another explanation is that STAT5 is maximally active during pregnancy and its capacity for phosphorylation is saturated. If this were true, then ablation of its negative regulator would not be able to increase STAT5 phosphorylation or affect beta cell function or proliferation. Furthermore, other mediators of the JAK2/STAT5 signalling pathway other than SOCS proteins might be compensating for the loss of Cish. Finally, it is possible that other pathways limit the proliferative capacity of beta cells, such as the cell-cycle inhibitor p16 .
In summary, in our pancreas-specific Cish-ablation mice, no difference was discovered in glucose homeostasis or beta cell function before, during or after pregnancy. p-STAT5 levels were not altered in Cish-deficient mice, indicating that other mechanisms compensate in the regulation of the STAT5 pathway during pregnancy.
We thank Wei Qin in the Islet Cell Biology Core of the University of Pennsylvania Diabetes Research Center for performing the islet perifusion experiments, Heather Collins in the Radioimmunoassay/Biomarkers Core of the University of Pennsylvania Diabetes Research Center for running the insulin assay, Jean Richa in the Transgenic Chimeric Mouse Facility of the University of Pennsylvania for blastocyst injection, and Daniela Budo and Roxana Hasan in the Molecular Pathology & Imaging Core of the University of Pennsylvania for all the histological services.
This research was supported by National Institutes of Health (NIH) grant R01-DK055342 and JDRF grant 17-2011-262 to KHK. The Islet Core, Transgenic & Chimeric Mouse Facility, and Radioimmunoassay and Biomarkers Core of the Diabetes Research Center at the University of Pennsylvania were supported by NIH grant P30-DK19525. The Morphology Core of the Center for Molecular Studies in Digestive and Liver Diseases was supported by NIH grants P30 DK050306 and P01 DK049210.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
SR and JLL performed experiments and data interpretation and derived the Cish conditional gene ablation model. YJ performed experiments, acquired data, and performed data analysis and interpretation for characterising the mouse. KHK designed the study and participated in interpretation and discussion of the data. YJ wrote the manuscript. SR, JLL and KHK carried out critical revisions of the manuscript. All authors approved the final version of the manuscript.
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