Islet Studies BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2019-000921 on 5 March 2020. Downloaded from Open access Original research Placental growth factor in beta cells plays an essential role in gestational beta- cell growth Weixia Yang,1,2 Yinan Jiang,2 Yan Wang,2 Ting Zhang,2 Qun Liu,2,3 Chaoban Wang,2,4 Grant Swisher,2 Nannan Wu,2,5 Chelsea Chao,2 Krishna Prasadan,2 George K Gittes,2 Xiangwei Xiao 2 To cite: Yang W, Jiang Y, ABSTRACT Wang Y, et al. Placental Objective Pancreatic beta cells proliferate in response to Significance of this study growth factor in beta cells metabolic requirements during pregnancy, while failure of plays an essential role in this response may cause gestational diabetes. A member What is already known about this subject? gestational beta- cell growth. of the vascular endothelial growth factor family, placental ► The expression and function of placental growth fac- BMJ Open Diab Res Care growth factor (PlGF), typically plays a role in metabolic tor (PlGF) in the endocrine pancreas have not been 2020;8:e000921. doi:10.1136/ studied before. bmjdrc-2019-000921 disorder and pathological circumstance. The expression and function of PlGF in the endocrine pancreas have not What are the new findings? been reported and are addressed in the current study. ► PlGF is exclusively expressed by beta cells in adult Additional material is ► Research design and methods PlGF levels in beta cells pancreas. published online only. To view, were determined by immunostaining or ELISA in purified please visit the journal online ► Increased beta cell- derived PlGF promotes gesta- beta cells in non-pregnant and pregnant adult mice. An tional beta- cell growth through macrophages. (http:// dx. doi. org/ 10. 1136/ adeno- associated virus (AAV) serotype 8 carrying a shRNA bmjdrc- 2019- 000921). for PlGF under the control of a rat insulin promoter (AAV– How might these results change the focus of rat insulin promoter (RIP)–short hairpin small interfering research or clinical practice? Received 24 September 2019 RNA for PlGF (shPlGF)) was prepared and infused into ► The contribution of insufficient beta cell- derived Revised 28 January 2020 mouse pancreas through the pancreatic duct to specifically PlGF to the development of gestational diabetes de- Accepted 31 January 2020 knock down PlGF in beta cells, and its effects on beta-cell serves further investigation in the clinic. growth were determined by beta- cell proliferation, beta- cell mass and insulin release. A macrophage-depleting reagent, clodronate, was coapplied into AAV- treated mice type 2 diabetes, which occurs later in the life to study crosstalk between beta cells and macrophages. of pregnant women.3 Hence, understanding Results PlGF is exclusively produced by beta cells in http://drc.bmj.com/ the adult mouse pancreas. Moreover, PlGF expression in the etiology and pathogenesis of gestational beta cells was significantly increased during pregnancy. diabetes is extremely important, given that Intraductal infusion of AAV–RIP–shPlGF specifically this disease affects up to 10% of pregnant knocked down PlGF in beta cells, resulting in compromised women.4 beta- cell proliferation, reduced growth in beta- cell Studies on pregnant women and rodents mass and impaired glucose tolerance during pregnancy. have suggested that the gestational increases Mechanistically, PlGF depletion in beta cells reduced islet in insulin production and secretion by beta on September 24, 2021 by guest. Protected copyright. infiltration of trophic macrophages, which appeared to be cells may be largely attributable to beta- essential for gestational beta-cell growth. 5–10 Conclusions Our study suggests that increased cell growth during this period. Indeed, expression of PlGF in beta cells may trigger gestational numerous studies have shown that gesta- beta- cell growth through recruited macrophages. tional increase in beta-cell mass primarily stems from beta- cell proliferation.11–14 Failure to adequately increase beta-cell mass may © Author(s) (or their INTRODUCTION cause glucose intolerance or even gestational employer(s)) 2020. Re- use During pregnancy, the increase in maternal diabetes. To date, the molecular mechanisms permitted under CC BY-NC. No commercial re- use. See rights blood glucose requires the pancreas to underlying gestational beta-cell growth are 5–10 and permissions. Published produce more insulin to keep normal glucose not fully understood. by BMJ. homeostasis,1 and failure of this compen- The intimate relationship between beta cells For numbered affiliations see sation may cause gestational diabetes.2 and endothelial cells starts during embryonic end of article. Although the high blood sugar in gestational pancreatic organogenesis15 and plays a crit- Correspondence to diabetes usually returns to normal after preg- ical role in both endocrine pancreas develop- 16 Dr Xiangwei Xiao; nancy, previous studies have shown that gesta- ment and beta- cell function in adults. This xiangwei. xiao@ chp. edu tional diabetes is a predisposing factor for interaction is mainly mediated by vascular BMJ Open Diab Res Care 2020;8:e000921. doi:10.1136/bmjdrc-2019-000921 1 BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2019-000921 on 5 March 2020. Downloaded from Islet Studies endothelial growth factor A (VEGF- A), the best studied Harbor, Maine, USA). Tamoxifen induction of Tomato ligand from the vascular endothelial growth factor expression in acinar cells in Ela-CreER T; Tomato mice (VEGF) family.17 We have previously shown that VEGF-A has been described before.19 INS1cre knock- in mice were plays a role in the control of beta-cell mass in response bred with Tomato mice to generate INS1cre; Tomato mice to glucose alterations in the circulation,18 while the two for purification of beta- cells based on red fluorescence. major sources of VEGF-A in pancreas are beta cells and Fasting blood glucose monitoring and intraperitoneal duct cells, which differentially regulate VEGF- A release.19 glucose tolerance testing were performed as described These earlier studies contributed to our understanding before.21 Pancreatic intraductal virus infusion was of the role of VEGF- A in beta- cell biology. However, performed as described.24 Injection of 200 µL chlodro- VEGF family members other than VEGF- A biology have nate and control liposome (clodronate liposomes, Neth- been minimally studied in beta- cell biology. erlands) from the tail vein of the mice was performed Placental growth factor (PlGF) is another member of twice per week, starting 1 day before time breeding until the VEGF family and is thought to play a role in altered the end of the experiment, as previously described.21 metabolic states or under some pathological circum- stance.20 The expression and function of PlGF in the RNA isolation and quantitative reverse transcription PCR (RT- endocrine pancreas have not been examined. Here, we qPCR) investigated PlGF expression in beta cells, as well as its Total RNA was extracted using the RNeasy mini kit role in gestational beta- cell growth. (Qiagen, Valencia, California, USA) and then quanti- fied with the Nanodrop 1000 (Thermo Fisher Scien- tific, Waltham, Massachusetts, USA), followed by cDNA MATERIALS AND METHODS synthesis (Qiagen) and RT- qPCR. Primers for CycloA Production of AAV8 expressing shPlGF under a rat insulin (QT00247709) and PlGF (QT00103222) were both promoter (RIP) purchased from Qiagen. Relative values of mRNA levels A PlGF shRNA sequence ( GCTG TTCA CTTG CTTC TTAG were obtained by sequential normalization against the TCGA GTAA GAAG CAAG TGAACAGC) or a scramble ( housekeeping gene cyclophilin A and the experimental GCTG AGTA CTTC GAAA TGTC GTCG AGGA CATT TCGA control. AGTA CTCAGCG) was designed using Block- IT RNAi PlGF ELISA designer and was cloned into pAAV-mcherr y- flex- dtA PlGF levels were determined with a PlGF ELISA kit (R&D (dTA) (Addgene, No 58536, Cambridge, Massachusetts, Systems, Los Angeles, California, USA). All reagents, stan- USA) by restriction sites BsrG1 and SalI. A RIP described dard dilutions and samples were prepared as directed in before21 was swapped into the vector by restriction sites the product insert. First, 100 µL of Assay Diluent was added FseI and XbaI. Adeno- associated virus (AAV) serotype 8 to each well, followed by 100 µL of standard, control, or vectors were generated by cotransfection with the shPlGF sample to each well. The microplate was then covered or the scrambled plasmid, a packaging plasmid carrying with a plate sealer and incubated at room temperature rep and cap from the AAV serotype 8, and a helper for 2 hours. Each well was aspirated and washed three http://drc.bmj.com/ plasmid carrying the adenovirus helper in human embry- times, after which 200 µL of conjugate was added to each onic kidney 293 cells, as described before.22 AAV viruses well. The microplate was then covered with a new plate were purified by polyethylene glycol/aqueous two-phase sealer and incubated at room temperature for 1 hour partitioning and then stored at −80°C for later use. before another four times of aspiration and wash. Finally, Titration of viral vectors was determined by an AAVpro 200 µL substrate solution was added to each well, and Titration kit (TaKaRa Bio, Mountain View, California, the microplate was incubated at room temperature for on September 24, 2021 by guest. Protected copyright. USA). Recombinant mouse PlGF was purchased from 30 min before 50 µL of stop solution was added to each Abcam (Ab207150, Cambridge, Massachusetts, USA) well. The microplate was read at 450 nm within 30 min. and was delivered to mice via pancreatic duct at 20 µg/ The wavelength correction was set to 540 or 570 nm. mouse. The control mice received saline. Transfection of a beta- cell line Min6 (American Type Culture Collection, Pancreatic digestion and FACS Rockville, Maryland, USA) was performed using Lipo- Pancreatic duct perfusion, pancreas digestion, islet isola- fectamine 3000 (Invitrogen, Carlsbad, California, USA).