Signals in the pancreatic islet microenvironment influence β cell proliferation

Abstract

The progressive loss of pancreatic β cell mass that in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing, or restoring β cell mass. While factors that seem to influence β cell proliferation in specific contexts have been described, reliable stimulation of human β cell proliferation has remained a challenge. Importantly, β cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections, and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between β cells and these other components influences β cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on β cell proliferation may lead to the development of successful approaches to increase or restore β cell mass in diabetes.

1 Introduction

The progressive loss of functional β cell mass leading to disruption of glucose homeostasis is a hallmark of both type 1 and type 2 diabetes, and provides the rationale for efforts to restore or enhance β cell mass in hopes of re-establishing homeostatic glucose regulation and preventing the short- and long-term consequences of hyperglycemia. These efforts include strategies focused on introducing new β cells from exogenous sources, such as cadaveric islets or pluripotent stem cells; or generating β cells from exogenous sources, such as multipotent precursor cells, pre-existing β cells, or transdifferentiation of other adult endocrine or non-endocrine cells. Although significant strides have been made in identifying factors that influence β cell proliferation in specific contexts including growth factors, hormones, and other mitogenic compounds, efforts to translate findings from rodent β cells into humans have been largely disappointing and identification of factors or small molecules capable of promoting human β cell proliferation and thereby expanding β cell mass to treat patients with diabetes has remained elusive^1–3. Recently, new approaches to stimulate human β cell proliferation have been discovered^4,5.

Major challenges to these efforts include a limited understanding of signaling pathways relevant to human β cell proliferation, and the extremely low proliferative capacity of human β cells (near 0%), even upon stimulation^1–3,6–11. Therefore, moving forward we need a greater understanding of how mitogenic factors regulate β cell proliferation, and whether a single factor is sufficient to activate β cell proliferation or if combinations of coordinated signals are needed. Because β cells exist in a microenvironment and context of the pancreatic islet where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections, and immune cells, an understanding of the islet microenvironment will also be critical to these efforts.

Pancreatic islets are discrete, spherical clusters of cells embedded in the acinar tissue of the pancreas, that function as mini-organs, and make up only 1–2% of pancreatic mass^12,13. In addition to endocrine cells that sense nutrients such as glucose and respond by secreting hormones into the bloodstream, the islet is highly innervated and vascularized, containing a rich vascular network that produces extracellular matrix, and provides signals that can coordinate and support cellular differentiation, survival, and proliferation^12,14–17. Historically, studies on the role of immune cells in the islet microenvironment have primarily focused on the autoimmune destruction of β cells in type 1 diabetes. However several recent studies have demonstrated an important role for islet macrophages in promoting β cell regeneration^18–20. In this review, we highlight components of the pancreatic islet microenvironment and discuss current understanding of how these components influence β cell proliferation. Future studies further defining the microenvironment and the intrinsic and extrinsic signals that influence mature β cell replication will hopefully allow identification of compounds or combinations of compounds that can be developed to successfully regenerate human β cells in diabetes.

2 Pancreatic Islet Microenvironment

2.1 Endocrine Cells

Islets contain five types of hormone-secreting endocrine cells. The insulin-secreting β cell is the most abundant, followed by the glucagon-secreting α cell, then the somatostatin-secreting δ cell. Two rarer cell types produce pancreatic polypeptide (PP cells) and ghrelin (ε cells). The organization and relative abundance of the endocrine cell types in islets varies between species. In rodents, β cells are located in the center of the islet surrounded by a mantle of α and δ cells (Figure 1A). Adult human islets lack this clear core-mantle organization: β cells are found intermingled with α and δ cells in the center of the islet and on the periphery (Figure 1B)^21,22. Endocrine cell composition is also much more heterogeneous in humans, with β cells ranging anywhere from 28–75% of islet endocrine cells as opposed to 61–81% in mice. Similarly, the abundance of human α cells (10–65% versus 9–31% in mice) and δ cells (1.2–22% versus 1–13% in mice) displays a much wider variability than in mice (Figure 1C–D)²¹.

Figure 1. Islet morphology and composition varies between mice and humans.

(A) Mouse and (B) human islets labeled for insulin (green), glucagon (red), and somatostatin (blue). Endocrine cell composition of (C) mouse islets, n=28, and (D) human islets, n=32, determined by analysis of optical sections taken throughout the entire islets. Human islet composition differed significantly (p<0.0001) across all endocrine cell populations examined. Horizontal bar represents the mean of each islet cell population. Image adapted with permission from Brissova et al., 2005²¹.

The hormones released by the endocrine pancreas regulate nutrient metabolism, specifically glucose homeostasis. Before secreting hormones into the bloodstream, islet endocrine cells sense and integrate a variety of signals including glucose, hormones, neurotransmitters, and other nutrients^23–26. Inter-endocrine cell interactions are critical to these functions, and include paracrine and autocrine signaling in addition to connections between endocrine cells that include cell adhesion molecules (e.g., N-CAM, cadherins), gap junctions, and ephrin (Eph) receptors and ligands. Cell adhesion molecules are important in the development of islet architecture. For example, blocking neural cell adhesion molecule (N-CAM) prevents endocrine cell types from segregating properly and leads to abnormalities in both insulin and glucagon secretion^27–29. During development, E-cadherin is required for β cell aggregation and islet formation, and is important for glucose sensing and insulin secretion in mature islets^30–32. Gap junctions connect the cytoplasm of cells together, and in β cells they allow exchange of ions and metabolites, including cytoplasmic calcium. This exchange of calcium is necessary to synchronize calcium oscillations across β cells and to maintain normal biosynthesis, storage, and insulin release; loss of gap junction proteins disrupts pulsatile glucose-stimulated insulin release^33–37. Ephs are receptor tyrosine kinases which initiate signaling upon binding their Eph ligands³⁸. Pancreatic β cells express both classes of Ephs (A and B), and EphA signaling plays an important role in regulating insulin secretion at both high and low glucose^15,39. While most of these inter-endocrine cell connections play vital roles in development of islet architecture and maintaining normal β cell function, for the most part there is little evidence that they play a role in β cell proliferation. One exception is E-cadherin which has been shown to mediate β cell survival and proliferation in mice by activating α- and β-catenins⁴⁰.

Similarly, paracrine and autocrine signaling between islet endocrine cells, which has been reviewed elsewhere^41,42, primarily serves to modulate endocrine cell function. However, neurotransmitters and neuropeptides that act as signaling molecules between islet cells in some cases also regulate β cell mass. Alpha cells release glutamate and acetylcholine, and while neither has been shown to influence β cell proliferation, glutamate-induced cell death may contribute to β cell loss in diabetes⁴³. Beta cells release γ-aminobutyric acid (GABA), serotonin, ATP, and dopamine. Both GABA and serotonin have been shown to promote proliferation in mouse β cells, with serotonin specifically implicated in β cell proliferation during pregnancy^44,45. Conversely, increased dopamine signaling decreased proliferation and increased apoptosis in rat β cells⁴⁶. Activation of purinergic and adenosine signaling by ATP metabolites has also been implicated in decreased apoptosis and preservation of mouse β cell mass and increased proliferation of zebrafish β cells, respectively^47–49. Translation of these findings to human β cells has been variable, with one study demonstrating increased human β cell graft survival and proliferation with GABA treatment in vivo, while another demonstrated no increase in human β cell proliferation upon treatment with GABA, serotonin, or activation of adenosine signaling in vitro^11,50. These findings highlight the need for further studies to better define the role of inter-endocrine cell interactions on human β cell proliferation, particularly as strategies for producing β cells ex vivo for transplant typically preclude these types of interactions.

2.2 Innervation

The pancreatic islet is richly innervated by the autonomic nervous system with both sympathetic and parasympathetic fibers. Signals from endothelial cells, which synthesize axon guidance molecules and basement membrane that functions as a scaffold for nerve ingrowth into islets during development are critical for islet innervation⁵¹. Neuronal projections follow blood vessels within the islet, however the degree and organization of these projections varies between species. In the mouse, autonomic axons innervate blood vessels and directly contact endocrine cells with equal parasympathetic input to both α and β cells and preferential sympathetic input to α cells. Conversely, in human islets sympathetic axons primarily innervate smooth muscle cells associated with blood vessels, with only rare parasympathetic axons penetrating the islets suggesting that functional regulation of endocrine cells in humans may occur indirectly by changing local islet blood flow^52,53.

While the functional significance of these differences in innervation are not well understood, neuronal input works to fine-tune hormone secretion and regulate blood flow in islets^24,54–56. There is also evidence, beyond the effects of neurotransmitters discussed above, that the nervous system plays a role in regulating β cell mass in rodents. For instance, during pancreatic development in mice, neural crest cells have been shown to negatively regulate β cell proliferation^57,58. Furthermore, disruption of vagal input into the pancreas led to reduction in β cell proliferation in rats and loss of compensatory β cell expansion in a mouse model of obesity, suggesting a role for these neuronal pathways in regulating β cell mass and proliferation^59,60. Currently there is no evidence that neuronal projections in the islet directly influence human β cell proliferation; however, as our understanding of human islet neuroanatomy and physiology continues to evolve, hopefully we can begin to investigate whether neuronal input plays a role in regulating β cell proliferation.

2.3 Vasculature

A characteristic feature of islets is their extensive vascularization (Figure 2). Although islets only represent 1–2% of pancreatic mass, they receive 6–20% of the direct arterial blood flow to the pancreas¹². Intra-islet capillaries are fenestrated and are thicker, denser, and more tortuous than capillaries in exocrine tissue^61,62. β cells directly communicate with these capillaries, suggesting that increased vascularization is important for β cells to rapidly respond to increases in blood glucose levels by secreting insulin into the bloodstream⁶³. Intra-islet capillaries connect endocrine cells to the blood supply to ensure proper gas exchange, nutrition, and waste removal. However, blood vessels also play an important role in providing non-nutritional signals to islets, creating a vascular niche in which cross-talk between β cells and endothelial cells is necessary to ensure proper β cell development and function⁶⁴.

Figure 2. Pancreatic islets are highly vascularized.

(A) Representative pancreatic islet from mouse immunolabeled for insulin (insulin), glucagon (blue), and endothelial cell marker, CD31 (red). (B) Mouse islet from an animal infused with FITC-conjugated tomato lectin (green) to label the functional vasculature. Islet capillaries (within dashed line) are thicker, denser, and more tortuous than vessels in the surrounding exocrine tissue. Images courtesy of Marcela Brissova, Vanderbilt University Medical Center.

Signaling between endothelial cells and the developing pancreatic epithelium throughout pancreatic development is critical to establish islet vasculature and β cell mass. During the specification of the pancreatic epithelium from the foregut, embryonic aortic endothelial cells are in direct contact with the dorsal pancreatic bud, and provide signals necessary for β cell differentiation; interrupting these signals prevents pancreatic differentiation¹⁶. These endothelial cell signals regulate expression of transcription factors in the developing pancreas that are required to maintain the multipotent progenitor population and induce lineage differentiation⁶⁵. After the early pancreatic epithelium remodels, it produces vascular endothelial growth factor A (VEGF-A), which binds VEGF receptors on endothelial cells, promoting endothelial migration and proliferation⁶⁶. Signals from these recruited blood vessels regulate pancreas branching and differentiation of exocrine and endocrine cells, and disrupting VEGF-A signaling either in the early pancreas or newly formed β cells leads to excessive exocrine differentiation and failure of the intra-islet plexus to form, causing significant defects in β cell proliferation, insulin secretion and glucose homeostasis^51,67,68. Conversely, overexpressing VEGF-A in developing β cells induces endothelial cell expansion and hypervascularization which disrupts islet formation and results in β cell loss^69–71. Therefore, precise control of VEGF-A is required for normal development of both the exocrine and endocrine pancreas.

In addition to regulating development of the pancreatic epithelium, VEGF-A is also required to establish and maintain normal islet vascularization, innervation, and function^{15,16,18,65,70–74} After development, the endocrine pancreas continues expressing VEGF-A at much higher levels than the exocrine pancreas⁷⁵. This continued VEGF-A expression is important in maintaining the distinctive microvasculature of the islet^12,15,61,62. A decrease in β cell-specific VEGF-A expression not only reduces islet vascularity 10-fold, but also leads to decreased innervation, impaired insulin secretion and reduced glucose tolerance, which indicates an important role for VEGF-A in normal β cell function^15,67,73. Conversely, inducible overexpression of VEGF-A in adult mice leads to increased islet vascularity, β cell loss, and recruitment of macrophages that promote β cell proliferation through coordinated interactions between endothelial cells and β cells following normalization of VEGF-A expression¹⁸.

In addition to VEGF-A signaling, the vasculature also produces several paracrine factors (i.e., hepatocyte growth factor, connective tissue growth factor) shown to regulate β cell proliferation and contributes key components to the islet extracellular matrix (ECM) that are critical for β cell differentiation, function, and proliferation (discussed below). New evidence has also suggested that islet endothelial cells may develop a dysfunctional phenotype that contributes to loss of β cell function in diabetes⁷⁶.

2.4 Extracellular Matrix

Pancreatic islets are separated from exocrine tissue by an incomplete peripheral capsule. This capsule is made up of a single layer of fibroblasts and collagen fibers sandwiched between two basement membranes (Figure 3A)¹⁷. The first basement membrane is located beneath the exocrine epithelium, and the other beneath the endocrine epithelium (peri-islet). Occasional breaks in the capsule allow direct exocrine-endocrine cell contact, and extensive membrane interdigitation occurs between the cell types in these areas⁷⁷.

Figure 3. Peripheral and internal extracellular matrix in the pancreatic islet.

(A) The peripheral extracellular matrix (ECM) is a discontinuous capsule composed of a layer of fibroblasts sandwiched between an exocrine cell-derived basement membrane and an endocrine cell-derived basement membrane (broken blue lines). (B) The internal ECM is made up of a perivascular basement membrane. Outline of the basement membrane components of the islet ECM include laminin (left; green) and collagen IV (right; green), which co-localize with endothelial cell marker CD31 (red). Adapted with permission from Reinert et al., 2014⁷³.

Studies in mice have demonstrated that unlike on the periphery, within the islet interior pancreatic endocrine cells lack a basement membrane, and directly interact with the vascular basement membrane surrounding islet capillaries (Figure 3B)^77–80. In addition to maintaining islet vasculature, this vascular basement membrane acts as a reservoir for the growth factors needed to maintain islet-specific phenotypes and promote β cell proliferation^15,81–83. Human islets were originally thought to have the same internal matrix architecture, but work by Virtanen et al. demonstrated that unlike mice, humans have two distinct layers of basement membrane. The vascular basement membrane is still present, but there is also a distinctive peri-islet basement membrane which invaginates into islets along vascular channels and expresses different laminin isoforms than the vascular basement membrane^84,85.

Integrin and non-integrin receptors

Integrins are heterodimeric cell surface receptors made up of both α and β subunits that can participate in both cell-cell and cell-matrix attachments. They enable cells to sense and respond to their surroundings using both outside-in and inside-out signaling. Many types of integrins can be formed by combining different α and β subunits. Depending on their specificity, integrins can bind various ECM ligands such as collagen, laminin, fibronectin, and vitronectin⁸⁶. Several integrin receptors are expressed in islets and have been found to influence islet development, β cell survival and function, and islet vascular remodeling^87–89. However, the exact composition of these islet integrins remains controversial⁹⁰. This controversy is partially due to the fact that expression of integrin receptors on islet cells is developmentally regulated, with the composition of integrins changing throughout development^91,92. During the secondary transition, αVβ3 and αVβ5 integrins are expressed in both the pancreatic ductal epithelium and the clusters of endocrine cells, and help regulate the delamination of these cells from the ducal epithelium^88,92. Integrins also promote the motility of these delaminated endocrine cells, which is necessary for the development of normal islet architecture and insulin secretion⁸⁹. During development, β1 integrin plays a crucial role in establishing β cell mass by regulating expansion of newly formed β cells, and mice without β1 integrin on β cells demonstrate a significant reduction in β cell mass and impaired β cell function^93,94. Furthermore, blocking β1 integrin signaling in human fetal islet epithelial cell cultures causes decreased differentiation and survival of islet cells⁹⁵. These integrins signal through Akt/β-catenin and ERK/MAPK pathways to promote β cell survival and proliferation^1,95–97. While expression of some of these developmentally important integrins is retained on adult islet cells, others are downregulated. However, treatment with connective tissue growth factor (CTGF) following diphtheria toxin-mediated ablation of β cells caused an increase in β1 integrin corresponding with improved β cell regeneration, suggesting that changes in β cell integrin expression may contribute to β cell regeneration in adult islets⁹⁸.

Although integrins appear to be the primary receptors involved in cell-matrix interactions in the islet, a few non-integrin receptors also play a role in these interactions. Discoidin domain receptors (DDRs) are receptor tyrosine kinases that bind collagens I-V and regulate ECM production, and cell adhesion, migration, and differentiation⁹⁹. Expression of DDR1 has been found in islet cells, but not in the surrounding exocrine tissue, suggesting that it may play a role in regulating endocrine cell development and/or function¹⁰⁰. Laminins can also bind several non-integrin receptors that are expressed in islets including, laminin receptor-1, dystroglycan protein complex, and Lutheran blood group glycoprotein (Lut), which is exclusively expressed on human islet cell membranes facing the basement membrane^84,85,100.

Interactions between endocrine cells and the matrix are critical for normal development, continuing β cell survival and function, and β cell proliferation and appear to be mediated by both integrin and non-integrin receptors. However, further work is still needed to determine which specific receptor-ligand interactions and signaling pathways are involved in promoting β cell proliferation.

Collagen

The fibroblasts in the peripheral capsule produce some fibrillar collagens (I, III, and V) and collagen VI, which forms beaded filaments^{77,79,101,102}. While these collagens may contribute to maintaining the architecture of the capsule, it is not known whether they play additional roles in islet development or function. The capsule is disrupted by collagenase during isolation of islets for transplantation, and some have speculated that disruption of these capsular collagens may contribute to the low survival rate of transplanted islets⁹⁰.

Because both the peripheral and internal ECM of islets is primarily composed of basement membrane molecules, collagen IV (Col-IV) is the collagen most often associated with islets¹⁰¹. Cell-matrix interactions typically occur by Col-IV binding to integrins located on the cell surface. There is evidence that Col-IV binds α1β1 integrin on β cells⁸⁹. This interaction is important during human islet development where it enables fetal β cells to attach and migrate to form normal islet architecture, and enhances insulin secretion⁸⁹. However, in adult islets there is no clear Col-IV/integrin binding pathway, and Col-IV interactions with endocrine cells appear to be limited⁸⁹. The influence of Col-IV on β cell survival, proliferation, and function is also unclear. Culturing islets on Col-IV improves islet survival compared to culture with Col-I, but also appears to decrease insulin secretion^78,103. This conflicting evidence has caused some to conclude that cell-matrix interactions with laminin may be more significant than interactions with collagens.

Laminin

Laminins (LMs) are cross-shaped trimeric glycoproteins that contain an α-chain, β-chain, and γ-chain connected by disulfide bonds¹⁰⁴. Along with Col-IV, laminins are a major component of basement membranes, and are therefore abundant in both the peripheral capsule and within islets^{77,79,84,102,105}. The specific expression and distribution of laminin isoforms in islets, and which cells produce the different isoforms is still unclear. However, some studies have found temporal and spatial differences in expression. LM-111 is the primary isoform expressed in the developing pancreas, and promotes β cell differentiation¹⁰⁶. Laminin binding to either dystroglycan or α6 integrin may play a role in this laminin-mediated β cell differentiation^91,107. As islets mature, LM-511 completely replaces LM-111, and other isoforms found in islets include LM-411 which is primarily located in the vascular basement membrane, and LM-332 which is associated with α cells^105,108,109. The internal ECM in human islets also contains the laminin β2 chain, a component of both LM-421 and LM-521, which is typically only found in specialized tissue such as kidney glomeruli⁸⁴. Laminins work through several different integrin and non-integrin receptors on β cells including β1 integrins, aV integrins, α6β4, Lut, dystroglycan, and laminin receptor 1^17,84,100. These interactions are important for enhancing insulin secretion and gene expression, and for promoting β cell survival and proliferation, but further work is needed to characterize laminin-receptor interactions that promote β cell proliferation^17,103,110.

Glycoproteins

Fibronectin is a large dimeric glycoprotein that plays an important role in binding matrix components together as well as binding cells to the matrix by interacting with several integrins and non-integrin receptors. Vitronectin, another matrix glycoprotein, has a similar function and is only expressed during pancreas development. Both fibronectin and vitronectin are necessary for normal islet development, and are thought to play a role in endocrine cell motility during islet formation^91,92,102. Blocking these fibronectin or vitronectin-integrin interactions in the fetal pancreas results in a significant decrease in the number of β cells⁹². In adult islets, fibronectin is located in the peripheral capsule and perivascular areas within the islets, and is often associated with collagens and laminins^{79,102,105,111}. It is unclear what contributions fibronectin makes to the function, survival, and proliferation of adult β cells⁹⁰.

Proteoglycans

Proteoglycans are composed of a core protein covalently bound to one or more negatively charged glycosaminoglycan (GAG) chains. These GAG chains include heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate, and keratan sulfate. Proteoglycans typically form large complexes by binding to hyaluronan and other matrix proteins. They can also bind and sequester cations, water, and growth factors, which allows them to regulate the movement, stability, and availability of molecules in the matrix¹¹². Not much is known about the composition of proteoglycans in islet ECM and whether they play a role in islet development and function. However, HS and CS proteoglycans have been detected in islets¹⁰⁰. HS proteoglycans are known to bind and regulate the bioavailability of growth factors such as fibroblast growth factors, VEGFs, and hepatocyte growth factor which are important for islet development, function, and β cell proliferation and regeneration^{15,81,113–115}. Spatiotemporal differences in expression of HS proteoglycans in islets suggest that they may also play a role in regulating processes during pancreas development^{100,116–118}.

Although integrin signaling is an established mechanism for promoting β cell survival and proliferation, our understanding how the other ECM proteins discussed above as well as how changes in integrin expression and matrix remodeling in development and disease may influence β cell function and proliferation remains largely unknown.

2.5 Growth Factors

Identifying endogenous factors such as growth factors, hormones, and mitogenic signals capable of inducing β cell proliferation in hopes of developing treatments that promote regeneration of lost β cells in diabetes has been the focus of numerous studies and has been extensively reviewed elsewhere^1–3. Growth factors play important roles in pancreatic development and maintaining normal islet function, and loss of these protective factors can lead to decreased β cell survival and diabetes.

Insulin-like growth factor (IGF-1) is a ubiquitous hormone that stimulates growth of most tissue types in conjunction with insulin and is critical for maintaining normal glucose homeostasis^2,119. In β cells, IGF-1 works primarily through the IGF-1 receptor, although it is also able to bind and activate the insulin receptor. Downstream signaling through the PI3K/Akt and ERK/MAPK pathways promote cell survival and proliferation^1,2,119,120. Although IGF-1 is not required during early β cell development, increasing levels of IGF-1 in β cells leads to decreased β cell apoptosis and increased β cell proliferation following streptozotocin (STZ)-induced β cell damage^119,121. While IGF-1 may play a role in regenerating β cell mass following injury, deletion of the IGF-1 receptor in β cells does not alter β cell mass, suggesting that IGF-1 receptor signaling is not required to maintain β cell mass¹²². Human β cells express the IGF-1R and downstream signals, but it is unclear what role IGF-1 plays in human β cell function, mass regulation, and response to injury^2,119.

Platelet-derived growth factor (PDGF) plays a role in development, cell proliferation and migration, angiogensis, fibrosis, and other disease processes. Although it is stored and released by platelets, PDGF is also made by a number of other cell types. Recently it was found that PDGF also regulates the age-dependent reduction in β cell proliferation that occurs in islets¹²³. PDGF signaling promotes β cell proliferation through ERK activation^1,123. Over time, β cells lose expression of PDGF receptors and this reduction in PDGF-mediated signaling results in decreased ERK activation, which in turn decreases β cell replication. Conversely, stimulation of PDGF signaling in quiescent mouse β cells triggers proliferation¹²³. In humans, stimulation of juvenile β cells with PDGF causes increased β cell proliferation, but adult human β cells do not express the PDGF receptor, making them unresponsive¹²³.

Connective tissue growth factor (CTGF) is highly expressed in islet vasculature and interacts with several signaling pathways including transforming growth factor β (TGFβ) and Wnt^98,124. CTGF is required for allocating endocrine progenitor cells into different lineages and promoting proliferation of developing β cells^124,125. In adult β cells, increasing CTGF has no effect on β cell proliferation under normal conditions; however, CTGF does promote increased β cell proliferation following diphtheria toxin-mediated ablation of β cells expressing diphtheria toxin receptor⁹⁸. Enhanced β cell proliferation in this model occurs through CTGF upregulation of ERK/MAPK-dependent TGFβ signaling and other growth factors⁹⁸. CTGF is also required for pregnancy-induced β cell hyperplasia, and for normal β cell function¹²⁶. The role of CTGF in human β cell function and β cell mass regulation is unknown.

Fibroblast growth factors (FGFs) play important roles in wound healing, development, and disease. During pancreatic development, FGF-7 is required for trunk cell proliferation and β cell differentiation, and disrupting FGF signaling in β cells leads to decreased β cell number and diabetes^81,127. Although overexpressing FGF-7 in acinar tissue leads to islet hyperplasia and increased β cell mass, when overexpressed in β cells under the insulin promoter, islets become fibrotic, suggesting that tight control of islet FGF-7 levels is important for regulating β cell mass^128,129. While FGF receptors are known to work through PI3K/Akt and ERK/MAPK pathways, downstream signaling of FGF receptors in β cells has not been investigated. The feasibility of using FGFs to promote human adult β cell proliferation is unknown, but a study of engrafted human fetal tissue in rats demonstrated increased β cell number in response to elevated FGF-7¹³⁰.

Several other growth factors and hormones are also known to play important roles in β cell development and proliferation. Hepatocyte growth factor (HGF) has been shown to promote fetal β cell proliferation and proliferation of mature β cells in pregnancy¹³¹. Prolactin (PRL) has been implicated in regulating β cell function and proliferation during pregnancy in rodents, when levels of circulating PRL and β cell PRL receptor expression are increased^132–135. PRL receptor signaling activates the JAK/STAT pathway, which then activates other signaling cascades such as PI3K/Akt and ERK/MAPK¹³³. Interestingly, PRL can also work synergistically with other growth factors to activate ERK/MAPK^136,137. Unlike in rodents, human β cells do not seem to proliferate in response to PRL, partially due to the lack of functional PRL receptors¹³⁸. Incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) signal through G protein-coupled receptors (GPCRs), activating PI3K/Akt and ERK/MAPK signaling, and promoting survival and proliferation of β cells in rodents; however, their role in human β cell proliferation is unclear^139,140.

2.6 Macrophages

Macrophages belong to the mononuclear phagocytic system and represent a phenotypically and functionally heterogeneous cell population. During development, macrophages originating from the yolk sac or hematopoietic tissues (i.e., fetal liver or bone marrow) seed tissues throughout the body where they establish self-renewing resident populations¹⁴¹. In addition to providing immune surveillance, these tissue-resident macrophages are highly specialized to perform tissue- and niche-specific functions, with their phenotype being dictated by signals they receive from their widely variable microenvironments¹⁴¹. In response to signals from infected or damaged tissue, circulating monocytes leave the bloodstream and infiltrate tissues, differentiating into macrophages, where they can function to phagocytize cell debris, recruit additional inflammatory cells, resolve inflammation, and restore tissue homeostasis, among other things¹⁴². Macrophages are often broadly classified into classically activated (M1) and alternatively activated (M2) phenotypic subtypes, corresponding to exposure to inflammatory signals (i.e., INFγ, LPS) and subsequent inflammatory phenotype (M1 macrophages) or exposure to anti-inflammatory signals (e.g., IL-4, IL-13, CSF-1, TGFβ) leading to a tissue reparative/regenerative phenotype (M2 macrophages)¹⁴³. However, debate over the utility of these limited categories is ongoing given our expanding knowledge of macrophage phenotype plasticity, with gene expression and resulting function being continuously dictated by the type, concentration, and duration of signals from their immediate surroundings¹⁴³.

Macrophage precursors are present in the pancreatic buds by E12.5 and mature by E14.5, when they are in close association with developing insulin-expressing endocrine progenitors¹⁴⁴. Expansion of these macrophage precursors with colony-stimulating factor (M-CSF) in cultured E12.5 fetal pancreas explants leads to an increased number of insulin-producing cells¹⁴⁴. Signaling between macrophages and developing E14.5–15.5 pancreatic epithelium in vitro regulates migration and cell cycle progression, and increasing macrophages at this stage leads to both increased delamination of endocrine cells from developing ducts and decreased proliferative capacity in these differentiated endocrine cells¹⁴⁵. Mice homozygous for a null mutation in the colony-stimulating factor 1 (CSF-1) gene are deficient in the entire mononuclear lineage, have decreased β cell mass from E18.5 throughout adulthood, and exhibit abnormal islet morphogenesis and impaired postnatal β cell proliferation¹⁴⁶. These studies suggest that macrophages may function to regulate β cell differentiation, survival, and/or proliferation during pancreatic development.

In mature islets, tissue-resident macrophages have been found to be distinct in origin and phenotype from those found in acinar tissue, with islet macrophages derived from definitive hematopoiesis and expressing high levels of Il1b and Tnfa, typically associated with classically activated (M1) macrophages¹⁴⁷. This distinction of islet-specific resident macrophages suggests that these macrophages may have homeostatic functions that could be critical for normal β cell function and/or β cell mass regulation. While inflammatory macrophages have been shown to play a role in the pathogenesis of diabetes where they contribute to islet damage and β cell loss, several recent studies have also demonstrated exciting new roles for macrophages in β cell survival, proliferation, and regeneration^{18–20,148–151}:

Macrophage-dependent β cell proliferation occurred in a model where inducible VEGF-A overexpression in β cells led to endothelial cell expansion, β cell loss, and macrophage infiltration into islets followed by the β cell proliferative response (Figure 4)¹⁸. Macrophage ablation prevents β cell proliferation, and macrophage phenotype characterized by transcriptome analysis shift through phases of quiescence, β cell loss and recovery. Ongoing work on this model has revealed evidence for a dynamically changing microenvironment where β cell proliferation is mediated by coordinated interactions between recruited macrophages, intra-islet endothelial cells, and β cells.

Figure 4. Role for macrophage and endothelial cell interactions in β cell regeneration.

In a model of β cell specific-VEGF-A overexpression, upon VEGF-A induction, intra-islet endothelial cells proliferate, and circulating monocytes recruited to islets differentiate into CD45+CD11b+Gr1- macrophages. These recruited macrophages and endothelial cells produce effector molecules that directly and/or cooperatively induce β cell proliferation and regeneration. Image reproduced with permission from Brissova, et al., 2014¹⁸.

Criscimanna, et al. observed a large population of islet-associated macrophages expressing M1 markers TNFα and IL-6 following diphtheria toxin-mediated β cell injury. This macrophage population shifted phenotype with loss of M1 marker expression and gain of M2 markers IL-10 and CD206 during β cell regeneration. Ablation of macrophages impaired the β cell proliferative response.¹⁹

Using pancreatic ductal ligation (PDL), Xiao, et al. showed that subsequent β cell proliferation in this model is dependent on infiltration of macrophages expressing M2 markers and macrophage-mediated upregulation of SMAD7 expression in β cells. Based on their findings, the authors proposed a model whereby TGFβ1 and EGF secreted by recruited macrophages upregulates β cell SMAD7 expression, leading to β cell proliferation.²⁰

Collectively these findings demonstrate that macrophages play a critical role in several models of β cell proliferation, suggesting they may have a wider role in establishing and maintaining β cell mass. Given differences in macrophage phenotypes and mechanisms of β cell injury across the studies, these effects may occur through different mechanisms in mice. Significantly, the regenerative microenvironment in the VEGF-A overexpression model promoted human, as well as mouse, β cell proliferation¹⁸.

3 Summary and Conclusion

In order to develop new approaches for expanding and replacing functional β cell mass, it is important to understand the microenvironment and context in which β cells exist and how that environment affects β cell development, function, survival, and proliferation (Figure 5). Complex inter-endocrine cell interactions including paracrine and autocrine signaling not only dictate normal β cell function, but may also influence β cell proliferation. Neuronal projections modulate and fine-tune endocrine cell function, and some evidence suggests a role for neural input in regulating β cell mass. The islet vasculature and VEGF-A signaling are also important for β cell proliferation and the establishment of β cell mass during development in addition to ensuring normal β cell function. Islet endothelial cells also produce growth factors and extracellular matrix components shown to promote β cell proliferation in certain models. Although integrin signaling can lead to β cell proliferation, our knowledge is limited regarding what role different ECM proteins, signaling molecules, and integrin receptor ligands play in regulating β cell function and promoting β cell proliferation. Exciting new roles for macrophages in models of β cell regeneration and new insight into the islet resident macrophage population provide opportunities for furthering our understanding of how macrophages may influence islet homeostasis, β cell function, and β cell mass regulation.

Figure 5. Pancreatic islet microenvironment.

(A) The islet microenvironment is highly vascularized and innervated, with an extracellular matrix composed primarily of vascular and endocrine cell-derived basement membrane. Endocrine cell types include insulin-secreting β cells, glucagon-secreting α cells, somatostatin-secreting δ cells, and rare ghrelin-secreting cells and pancreatic polypeptide (PP)-secreting cells. Islet-specific resident macrophages are also present, and may play a role in normal β cell development and function. (B) Endocrine cells respond to circulating signals, such as glucose, and release hormones into intra-islet capillaries (dashed arrows). Intra-endocrine cell interactions include direct signaling through connections, such as gap junctions, in addition to autocrine and paracrine signaling (white arrows). Signaling between endocrine cells, macrophages, and endothelial cells also occurs and is important in islet development and function (grey arrows). These cell types also interact with, and in some cases produce, the extracellular matrix (black arrows). Nerve fibers that penetrate the islet interact with vascular smooth muscle cells to regulate blood flow, and may in some cases directly interact with endocrine cells as well. Legend applies to A and B.