Discussion
The insulin need increases during pregnancy and is met by increases in insulin production, release and sensitivity.1 Failure of this compensation may lead to glucose intolerance or even gestational diabetes, which has been shown to predispose the development of type 2 diabetes.3 Adequate gestational beta-cell growth is critical for maintaining gestational glucose metabolism, which relies on proper production and secretion of pregnancy-associated growth factors.38
Beta cells release many factors directly to pancreas in a paracrine manner, and these factors may act as chemoattractants to recruit the cells that express the corresponding receptors.39 In the current study, PlGF is such a chemoattractant ligand that is produced and secreted exclusively by beta cells in pancreas, while pancreatic macrophages express VEGFR1, the unique receptor for PlGF.20 Although the levels of PlGF in beta cells increased less than twofolds, it is known that the physiological level of a VEGF family member is maintained in a relatively small range,17 40 and a modest change may exert significant biological changes.41 42 VEGF-A may also play a role in the PlGF-associated effects since VEGF-A regulates vessel permeability, binds VEGFR1 similarly like PlGF, and even forms heterodimers with PlGF.43 44 A delicate interaction between VEGF-A and PlGF may coordinate the recruitment of pancreatic macrophages into the islet niche in a tightly organized manner, likely involving multidirectional crosstalk among beta cells, islet endothelial cells and pancreatic macrophages.27 Of note, we have previously shown that VEGFR1 is also expressed in islet endothelial cells.18 Hence, PlGF/VEGFR1 signaling may also regulate the restructure of islet endothelia necessary for circulated monocytes/macrophages to enter the islet niche and to join to the tissue-resident macrophages.
The major source of PlGF during pregnancy is the placenta.45 It is well known that beta cells locate in a niche where they directly contact with islet endothelial cells.15 The placenta-derived PlGF in circulation needs to cross the vascular endothelial cells to enter pancreatic parenchyma and attract VEGFR1-expressing macrophages in pancreas. Due to the expression of VEGFR1 on vascular endothelial cells, the majority of placenta-derived PlGF will be ‘consumed’ in the endothelial cells before entering the pancreatic zone. On the other hand, the beta cell-derived PlGF is likely released into pancreatic parenchyma in a paracrine manner, which attracts pancreatic macrophages that harbor the VEGFR1.
The basal expression of PlGF is much lower than VEGF-A in beta cells. PlGF does not likely play a critical role in baseline postnatal beta-cell homeostasis, since infusion of AAV–RIP–shPlGF did not alter beta-cell mass and glucose response of the mice. However, PlGF did play a critical role in postnatal beta-cell growth in response to increased metabolic need during pregnancy. The increase in PlGF levels in beta cells during pregnancy is only about 50%. However, previous studies have shown that the biological effects of the angiogenic factors are extremely narrow.17 40–42 For example, inactivation of only one allele of VEGF-A resulted in embryonic lethality at midgestation,46 47 while a twofold increase in VEGF-A levels also led to embryonic lethality.48 Therefore, the seemingly modest increase in PlGF levels in beta cells can exert a significant biological effect. The reduction in gestational growth in beta-cell mass by shPlGF was significant but not huge. Nevertheless, it did result in impaired glucose response, suggesting that the typical growth in beta-cell mass during pregnancy may be immediately enough for maintaining proper metabolic requirement without much reserve, which is consistent with the clinical reports showing that some women with gestational diabetes became normoglycemic after pregnancy.2
The intraductal infusion technique allows the pancreas-specific delivery of AAVs, as we have demonstrated in previous reports.19 21 24 25 31 49 The AAV-mediated beta cell-specific depletion of PlGF has an advantage over use of beta cell-specific PlGF knockout mice, which are even not available worldwide. Using RIP-Cre to knock out PlGF in beta cells will developmentally alter beta-cell phenotype. On the other hand, using RIP-creERT to knock out PlGF cannot bypass using tamoxifen, which binds to and signals through estrogen receptor highly expressed in beta cells to cause off-target effects.50 51 Although here we have shown that PlGF is exclusively produced by beta cells among all pancreatic cells, some inflammatory cells in the pancreas also express certain levels of PlGF.52 Thus, our use of the insulin promoter to drive expression of shPlGF assured the absence of effects from the knockdown of PlGF in non-beta cells.
The beta-cell-trophic factors that are produced and secreted by macrophages will be interesting targets to be studied in the future. Previous studies have suggested that WNT ligands,32 33 VEGF-A,16 and combined epidermal growth factor and transforming growth factor β121 may be such candidates. These studies also highlighted M2 macrophages, an alternatively polarized subtype of macrophages (as opposed to the classical M1 macrophages), as a very important regulator of beta-cell proliferation. It would thus not be surprising if the recruited macrophages to the beta cells during pregnancy are predominantly M2 macrophages.
MIP-GFP (mouse insulin promoter driving a green fluorescent protein reporter) mice have been commonly used for purification of beta cells.53 However, recent studies showed that the presence of human growth hormone (hGH) in the transgenic mice, including MIP-GFP, appeared to affect beta-cell function and proliferation through prolactin receptor signaling.54 55 Thus, INS1cre; Tomato mice, with the absence of hGH in either parental strain, were used to purify beta cells and non-beta islet cells in the current study.
Here, we presented a novel model for studying cell–cell communication in beta-cell proliferation. Our study suggests that increased expression of PlGF in beta cells may trigger gestational beta-cell growth through recruited macrophages. It may be intriguing to examine a possible contribution of insufficient beta cell-derived PlGF to the development of gestational diabetes in future studies.