Abstract
Historic and emerging studies provide evidence for the deterioration of pancreatic α cell function and identity in diabetes mellitus. Increased access to human tissue and the availability of more sophisticated molecular technologies have identified key insights into how α cell function and identity are preserved in healthy conditions and how they become dysfunctional in response to stress. These studies have revealed evidence of impaired glucagon secretion, shifts in α cell electrophysiology, changes in α cell mass, dysregulation of α cell transcription, and α-to-β cell conversion prior to and during diabetes. In this review, we outline the current state of research on α cell identity in health and disease. Evidence in model organisms and humans suggests that in addition to β cell dysfunction, diabetes is associated with a fundamental dysregulation of α cell identity. Importantly, epigenetic studies have revealed that α cells retain more poised and open chromatin at key cell-specific and diabetes-dysregulated genes, supporting the model that the inherent epigenetic plasticity of α cells makes them susceptible to the transcriptional changes that potentiate the loss of identity and function seen in diabetes. Thus, additional research into the maintenance of α cell identity and function is critical to fully understanding diabetes. Furthermore, these studies suggest α cells could represent an alternative source of new β cells for diabetes treatment.
Introduction
α cells are one of four major endocrine cell types that reside in adult pancreatic endocrine clusters called the Islets of Langerhans. The islet α cells secrete the hormone glucagon in response to hypoglycemic conditions to normalize blood sugar by stimulating the release of glucose into the bloodstream. The insulin-producing β cells – the most abundant pancreatic endocrine cell population – have traditionally been a focal point of research for the islet field, primarily because of their causal association with diabetes mellitus. For this reason, α cell research has been somewhat neglected, leaving many unanswered questions about α cell biology. However, in recent years, α cell research has gained momentum due to the deepening understanding of its dysfunction in diabetes; some of the most compelling findings have supported an independent association of α cell dysfunction as a contributor to diabetes pathology. Furthermore, there is growing evidence of inherent α cell plasticity that is unique among the pancreatic islet cell populations that may contribute to their potential as a novel source of β cells.
Since the mid-twentieth century, researchers have observed impaired glucagon secretion and a corresponding expansion of α cell mass in both type 1 (T1D) and type 2 diabetes (T2D) (Maclean & Ogilvie 1959, Müller et al. 1970, Gerich et al. 1973, Bolli et al. 1983, Brissova et al. 2018, Marchetti et al. 2000, Dai et al. 2022). However, molecular understanding of α cell development and function has been historically challenging due to a paucity of immortalized mouse and human cell lines, the relatively low α cell number in mouse islets, and the availability of few efficient and specific glucagon-driven Cre systems for mouse genetic models. Together, these hurdles have relegated α cell experiments to a single immortalized rodent cell line or complicated inducible genetic mouse models. More recently, α cell research has gained prominence in large part due to the development and growth of advanced single-cell sequencing technologies and unbiased molecular techniques, along with the increased availability of human islets that harbor a greater proportion of α cells. Recent work has revealed substantial dysregulation of the α cell transcriptome in α cells from mouse models of diabetes and from islet tissue collected from human donors with diabetes (Segerstolpe et al. 2016, Xin et al. 2016, Brissova et al. 2018, Avrahami et al. 2020, Dai et al. 2022). In addition, studies done in mice with major β cell loss have identified alterations in α cell function and numbers and α-to-β cell interconversion (Thorel et al. 2010, Oropeza et al. 2021).
As we are gaining more of a holistic view of α cell dysfunctions in diabetes, new areas of research related to α cell biology and diabetes are emerging. In particular, there is renewed interest in investigating whether α cell dysfunction could be less of a bystander event and more integral to islet health and diabetes progression (Unger & Cherrington 2012). There is also growing evidence of remarkable α cell plasticity that may have a negative role on the ability to maintain optimal α cell identity and function but may have a positive role on their ability to functionally adapt to metabolic changes, proliferate, or transdifferentiate into β cells. In this review, we outline the current status of the regulation of α cell identity and function, how these regulatory processes become disrupted in diabetes, and how α cell plasticity contributes to their robust response to environmental stressors.
Section 1: genetics of α cell specification and development
Development of the murine endocrine pancreas involves three main stages referred to as ‘transitions’ and is orchestrated in large part by the timely expression of transcription factors (Fig. 1). With limited access to human fetal tissue less is known about corresponding human pancreas development; however, several studies have highlighted both similarities and differences (reviewed in Jennings et al. 2015). The most notable known differences are the apparent lack of a primary transition in humans and some temporal differences in transcription factor expression. For the purpose of this review, we are focusing on the development of murine α cells. The description of murine pancreas developmental morphology has been well-characterized and widely supported. There has also been extensive research related to the genetic regulation of pancreas development and endocrine cell differentiation via the function of transcription factors; however, until recently, much of the focus has been on β cells. Less is known about α cell development, but this is a continually expanding area of research and is summarized below. A comprehensive review of pancreas development with a focus on α cell differentiation was published previously (Bramswig & Kaestner 2011).
The stages of murine α cell development and the transcription factors involved. Murine pancreas development is described in three transitions. The primary transition occurs from ~E8.5 to E12.5 and PDX1+ pancreas progenitors develop into NEUROG3+ endocrine progenitors. The secondary transition occurs from ~E12.5 to E16.5 and NEUROG3+ endocrine progenitors specify into α cells and the other endocrine cell types. The tertiary transition occurs from ~E16.5 postnatally and immature endocrine cells differentiate into fully functional and mature cells. The transcription factors with genetic and functional evidence of their involvement in α cell developmental transitions are listed, where red text indicates α cell-restricted expression at that timepoint. A ‘?’ after the factors name indicates that only preliminary evidence exists for their role in α cells. Figure made using https://www.biorender.com/.
Citation: Journal of Endocrinology 258, 2; 10.1530/JOE-22-0297
Primary transition
The primary transition of the murine pancreas begins at approximately E8.5 as PDX1 expression marks the emergence of pancreatic progenitors from pancreatic endoderm. The PDX1-expressing pancreatic progenitor cells give rise to the exocrine/ductal and endocrine progenitor lineages. Null mutations of Pdx1 result in pancreas agenesis; however, pancreatic rudiments do initially form and contain a small number of glucagon-expressing cells (Gannon et al. 2008). In the absence of unique markers, the origin and developmental trajectory of this early α cell population remains poorly defined, although single-cell RNA-seq and pseudo-time analyses are beginning to clarify their potential fate (Bastidas-Ponce et al. 2019). Transcription factors such as GATA4/6, ISL1, and FOXA2 are also critical in the formation of pancreatic cell lineages, and deficiency of these factors also leads to pancreas agenesis (Ahlgren et al. 1997, Gao et al. 2008, Carrasco et al. 2012, Xuan et al. 2012). Through the mutually repressive action of the transcription factors NKX6.1 and PTF1A, the pancreatic progenitor population bifurcates into either a ‘trunk’ endocrine/ductal lineage or a ‘tip’ acinar lineage (Schaffer et al. 2010).
Secondary transition
The majority of murine endocrine differentiation occurs during the secondary transition beginning at approximately E12.5. During the secondary transition, expression of the transcription factor NEUROG3 canalizes cells into the endocrine progenitor lineage that subsequently differentiate into immature α, β, δ, PP, and ε hormone-producing cells (Pictet et al. 1972, Smith et al. 2004, Prasadan et al. 2010, Bishop et al. 2013). Studies show that the transcription factor ARX is necessary for the specification of α cells in the islet; pancreas-specific deletion of Arx leads to the complete lack of α cells and a concomitant increase in β cell mass (Collombat et al. 2003, 2005, Mastracci et al. 2011). Conversely, the overexpression of Arx in the developing pancreas and ectopic expression in β-lineage cells, respectively, causes an increase in α cell differentiation and β-to-α conversion, suggesting that ARX is also sufficient for α cell differentiation (Collombat et al. 2007). During the secondary transition, BRN4 expression is restricted to differentiated α cells and functions as a positive regulator of glucagon expression (Hussain et al. 1997). However, Brn4-deficient mice still possess normal islet morphology and glucagon expression and secretion in α cells, indicating that regulation of glucagon by BRN4 is either redundant and/or can be compensated by alternative factors (Heller et al. 2004).
Transcription factors that have shared expression between α cells and other endocrine lineages are also important to the development of functional α cells. The transcription factors PAX6, NKX2.2, and NEUROD1 are some of the most well-studied factors involved in endocrine cell specification. Global mutations in Pax6 and conditional deletion of Pax6 in the endocrine progenitors caused defects in endocrine differentiation, islet morphology, and hormone expression, predominantly affecting the α cell lineage (Sander et al. 1997, St-Onge et al. 1997, Ashery-Padan et al. 2004). Within the α cells, molecular evidence shows that PAX6 regulates transcription of the Preproglucagon gene, while in β cells PAX6 promotes the expression of insulin, highlighting the potential of PAX6 to have distinct roles when expressed in different cell types (Gosmain et al. 2007, Swisa et al. 2017). Nkx2.2-null mice possess a total loss of β cells, a partial loss of α and PP cells, and a concomitant increase in ghrelin-producing ε cells (Sussel 1998). Furthermore, deletion of Nkx2.2 from the NEUROG3+ endocrine progenitors resulted in a decrease in glucagon+ and insulin+ cells and possessed significant overlap in dysregulated genes when compared to the Nkx2.2-null transcriptome (Churchill et al. 2017). Global knockout of NeuroD1 resulted in the complete loss of cells expressing glucagon, insulin, and somatostatin (Naya et al. 1997). Furthermore, conditional knockouts of NeuroD1 in PDX1+ pancreas progenitors and in NEUROG3+ endocrine progenitors also caused a loss of α cells (Mastracci et al. 2013). Together these temporal genetic analyses suggest that NEUROD1 is critical for the specification of α cells. Additional studies support important functions for the transcription factors GATA6, ISL1, and RFX3/6 in all endocrine cell types, including the specification of α cells (Ahlgren et al. 1997, Ait-Lounis et al. 2007, Soyer et al. 2010, Carrasco et al. 2012, Xuan et al. 2012, Tiyaboonchai et al. 2017).
Epistasis analyses have also provided evidence that genetic interactions between NeuroD1 or Arx and Nkx2.2 regulate the development of α cells. While mice carrying individual Nkx2.2-null and NeuroD1-null mice resulted in a major loss of α cells, the Nkx2.2-null/NeuroD1-null double mutant partially rescued the α cell lineage (Chao et al. 2007, Anderson et al. 2009). This suggests Nkx2.2 and Neurod1 genetically interact to regulate α cell development. Although the full molecular mechanism has not yet been determined, authors of the double Nkx2.2/Neurod1 double mutant study hypothesize that the counterintuitive rescue of α cell mass in the double mutant mice may be due to the repression of Neurod1 by NKX2.2. Two studies investigating Nkx2.2 and Arx double mutant mice found that simultaneous removal of Nkx2.2 and Arx from the endocrine progenitors caused an additive reduction in glucagon expression and major alterations in the expression of other endocrine hormones (Mastracci et al. 2011). These results support the model that ARX and NKX2.2 are both critical for promoting α cell differentiation into immature α cells and also that Nkx2.2 and Arx may interact genetically to establish monohormonal identity and regulate appropriate hormone transcription in the developing islet.
Tertiary transition
The tertiary transition begins at ~E16.5 when the maturing endocrine cells begin to form proto-islet clusters (Pictet 1972). Postnatally, endocrine cells continue to mature into fully functional cells and proliferate to establish the mature Islets of Langerhans. Transcription factors MAFB and FOXA2 have been implicated in the maturation of α cell identity and function. In mice, MAFB is strongly expressed in both α and β cells but becomes extinguished in β cells after birth (Nishimura et al. 2006, Artner et al. 2010). MafB-null mice show a substantial decrease in glucagon+ and insulin+ cell numbers and the respective hormone mRNA expression at E15.5 and E18.5; however, there was no difference in the total endocrine cell numbers (Artner et al. 2007, 2010). Furthermore, mRNA expression of cell identity factors Arx, Brn4, and Nkx2.2 were not significantly altered when MafB was depleted (Artner et al. 2007). These data suggest that MAFB acts as a maturation factor for α cells rather than functioning in their specification. FOXA2 is expressed in the pancreatic endoderm as well as in all major mature endocrine lineages (Lee et al. 2005, Gao et al. 2008). Despite the broad expression of FOXA2 in the developing pancreas, deletion of Foxa2 in the pancreatic endoderm did not affect early pancreas development but instead caused a decrease in glucagon+ cells and severe hypoglycemia. The authors concluded FOXA2 was essential for the differentiation and maturation of the α cell lineage.
Transcriptomics of α cell development
Although there has been a minimal functional assessment of transcription factors regulating α cell development compared to the regulation of β cells, the advancement of transcriptomic interrogation is uncovering novel regulators of the islet cell lineages. In particular, single-cell analysis of pseudo-time clusters of cells that obtain an α cell-like fate has revealed several insights into potential pre-α markers and novel α cell lineage markers. For example, in a cell population on an α cell trajectory – prior to glucagon expression, there was an enrichment of Peg10 and Auts2, two factors that have established roles in differentiation, and Smarca1, a mature α cell marker and epigenetic regulator (Byrnes et al. 2018). The α cell-enriched expression of Peg10 in both endocrine progenitors and mature α cells was validated by RNA in situ hybridization, supporting the hypothesis that Peg10 was a marker of pre-hormonal α cells. Another study using murine single-cell transcriptomics and pseudo-time analysis showed that the expression dynamics of the differentiation factor Prom1 is restricted to the α cell branch, peaking in expression prior to the initiation of glucagon expression, but after NEUROG3, suggesting it could be a candidate for α cell specification (van Gurp et al. 2019). Lastly, several bulk- and single-cell RNA-seq datasets have identified the expression of Irx1, Irx2, and Etv1 as α cell-enriched transcription factors, although genetic studies to validate their roles in α cell development have yet to be performed (Benitez et al. 2014, Byrnes et al. 2018, Krentz et al. 2018, van Gurp et al. 2019).
Future directions
There are still many gaps in our knowledge of α cell development. Many of the initial genetic studies in mice were performed before the availability of advanced RNA-, ChIP- and ATAC-seq techniques that can be applied to small numbers of cells. Thus, we have sparse knowledge of the molecular mechanisms of factors such as NKX2.2, ARX, MAFB, NEUROD1, and PAX6 in developing α cells. In the future, these advanced sequencing techniques will provide more comprehensive insights into the molecular roles of these transcription factors in α cell development. Since the combinatorial function of transcriptional regulators is also important for cell lineage specification, the genetic interplay between well-established transcription factors in α cell development could also provide important information. Mice containing Neurog3-driven double knockouts of Nkx2.2/Mafb, Mafb/Arx, Pax6/Mafb, and Pax6/Nkx2.2 are only a few high-priority experiments that could further elucidate the transcription factor regulatory networks at play in α-lineage cells. In addition, RNA-seq technology has identified many novel candidates as potential markers for pre-α lineage cells (Peg10, Prom1) and differentiating α cells (Etv1, Irx1, Irx2). Further validation using functional assays in mice and assessing corresponding expression analysis in human islets will be critical for understanding their relevance to α cell development and function.
Section 2: genetics of α cell function
Impaired glucagon secretion in individuals with diabetes has long been appreciated in the field but is especially important to those managing their diabetes, due to the health challenges associated with α cell dysfunction. Early reports found that after insulin-induced hypoglycemia, individuals with mid- and long-term T1D were slower to normalize to euglycemia and displayed significantly reduced plasma glucagon, suggesting a dysfunction in hypoglycemia-induced glucagon secretion (Special collection article by Robertson et al., Gerich et al. 1973, Bolli et al. 1983, Unger & Cherrington 2012, Fig. 2). With the increase of studies exploring the of glucagon secretion in individuals with T1D, evidence for hyper-secretion of glucagon, lack of high glucose suppression have also been reported (Müller et al. 1970, Marchetti et al. 2000). These dysfunctions are potentially partially driven by non-cell-autonomous mechanisms, where insulin-stimulated suppression of glucagon secretion is decreased due to the decline in β-cell numbers and function (Unger & Cherrington 2012). However, a study using human α cells that were positive for the T1D-correlated glutamic acid decarboxylase autoantibody (GADA+) found that these cells were already dysfunctional – displaying an increased response to glucagon secretagogues and impaired high glucose suppression of secretion prior to T1D onset (Doliba et al. 2022). Furthermore, the major dysregulation of glycolytic pathway genes in GADA+ α cells suggests that the functional impairment in these cells could be due to the loss of proper α cell gene expression. In comparison, GADA+ β cells showed no functional impairment, supporting the notion that α cells have a unique early response to pre-T1D environments. People with T2D can also experience dysfunctional glucagon secretion, occasionally resulting in insensitive or inappropriate responses to glucose levels (Müller et al. 1970, Unger & Cherrington 2012, Zhang et al. 2013, Dai et al. 2022). Similar to T1D, dysfunction of α cells in T2D has been partially linked to the breakdown of insulin’s repressive effect on α cells, but research suggests that α cells in T2D are also becoming insulin resistant (Lee et al. 2014). Human T2D α cells have also been shown to possess dysregulated expression of key α cell genes but paradoxically, T2D α cells with high expression of the transcription factors ARX, NKX2.2, NEUROD1, and ISL1 correlated to a decrease in total glucagon exocytosis (Segerstolpe et al. 2016, Xin et al. 2016, Dai et al. 2022). Although the precise mechanisms have yet to be defined, there is mounting evidence to suggest that a common characteristic of diabetes includes α cell dysfunction.
Diagram of α cell functional components and the genes related to glucagon secretion. At low blood glucose levels, ATP-gated K+ channels are open. This allows for Na+ and Ca2+-based action potentials to occur causing an influx of Ca+ into the cell. The increase in cytosolic Ca+ stimulates the activation of Ca2+-dependent exocytosis machinery, leading to glucagon secretion. Other stimuli of secretion regulation can act through G-protein-coupled receptors. Hormones such as somatostatin and GIP can directly suppress or promote glucagon secretion, respectively. Juxtacrine regulation via Ephrin signaling between α and β cells can suppress glucagon secretion. Genes with published correlations, dysregulation in diabetes, or direct evidence in α cells are included for ion channel subunits, G-protein-coupled receptors, and juxtacrine regulation. Figure made using https://www.biorender.com/.
Citation: Journal of Endocrinology 258, 2; 10.1530/JOE-22-0297
Ion channels involved in glucagon secretion
The regulation of glucagon secretion by ion channels is reviewed extensively in the article by Gao et al. in this special collection. Interestingly, changes in ion channel gene expression and function during diabetes also suggest there is a disruption in α cell identity and function (Fig. 2). Although murine α and β cells express similar voltage-gated Na+ channel subunit genes, they differ in the relative expression and role of these subunits in relation to the Na+ current. The Na+ channel subunit genes Scn3a and Scn3b are more highly expressed in α cells than in β cells where Scn9a is the predominant subunit (Zhang et al. 2014). Furthermore, SCN3A and SCN3B were highly correlated to Na+ current in human α cells (Dai et al. 2022). In a high-fat diet (HFD) model of T2D in mice, α cell expression of Scn9a was elevated and the α cells appeared to shift toward a more β cell-like Na+ current. The authors suggested that the α cells were transitioning into a ‘β-like’ electrophysiological state. Furthermore, blocking L-type Ca2+ channels caused impaired glucagon secretion in HFD-fed mice while blocking P/Q-type channels had no effect, the exact opposite of the non-diabetic α cell response and more typical of a β cell response (Dai et al. 2022), again suggesting a transition of these channels from an α cell to β cell like identity. Interestingly, several of the α cell ion channel subunits are downregulated in human T1D α cells, including SCN3B, CACNA1A, CACNA1C, CACNA1D, CACNA1H, CACNA2D1, and CACNA2D2 (Brissova et al. 2018, Dai et al. 2022).
ATP-gated potassium (K-ATP) channels have also been implicated in the regulation of glucagon secretion by sensing levels of cytosolic ATP generated from the uptake of blood glucose (Reviewed in Rorsman et al. 2014). Studies that increase the K-ATP channel activity in non-diabetic human islets recapitulated the lack of glucagon secretion suppression found in T2D islets (Zhang et al. 2013). This supports the importance of K-ATP channels in the suppression of glucagon secretion but also their role in T2D. The K-ATP channel subunit KCNJ11 has several human variants associated with increased diabetes risk (Gloyn et al. 2003, 2004). Furthermore, the ATP-sensitive K+ channel subunit ABCC8 (SUR1) as well as other K+ channel subunits KCNJ8, KCNJ6, and KCNK3 have significantly downregulated mRNA expression in T1D α cells (Gromada et al. 2004, Brissova et al. 2018).
Paracrine and endocrine regulation of glucagon secretion
Glucagon secretion is also regulated via paracrine and endocrine signals, including somatostatin, insulin, gut-derived incretin hormones, and amino acids (reviewed in Muller et al. 2017). These different mechanisms of regulating glucagon secretion are often mediated through the binding of extracellular ligands to G-protein-coupled receptors in the plasma membrane of α cells. Receptor binding can cause either activation or inhibition of an adenylate cyclase/cAMP cascade that regulates the activity of ion channels thus contributing to additional modulation of exocytosis.
There is good evidence for hormone-triggered paracrine regulation of glucagon secretion. Insulin secreted from β cells has been shown to suppress glucagon secretion via interaction with the INSR receptor expressed on α cells (Banarer et al. 2002, Franklin et al. 2005, Kawamori et al. 2009). INSR transcription has been shown to be slightly upregulated in α cells in individuals with T1D (Brissova et al. 2018). Somatostatin secreted from δ cells regulates both insulin and glucagon secretion through SSTR receptors (Koerker et al. 1974a,b, Strowski et al. 2000, Hauge-Evans et al. 2010). Although the evidence is limited, human α cells appear to primarily express SSTR2, while β cells appear to express both SSTR1 and SSTR5 (Kumar et al. 1999). In mice, Sstr3 was also found to be expressed in the primary cilia of β cells (Iwanaga et al. 2011). Importantly, glucagon secretion in mice fed a HFD was insensitive to endogenous and exogenous somatostatin, suggesting a breakdown of normal paracrine regulation of glucagon secretion by somatostatin during T2D (Kellard et al. 2020). Lastly, although SSTR1 appears to be primarily expressed in human β cells, it has been implicated in human α cell function as well as being slightly downregulated in T1D α cells (Brissova et al. 2018, Dai et al. 2022).
Incretin peptides are secreted by enteroendocrine cells in the intestine and peptides such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) have also been implicated in the regulation of hormone secretion in the islet (reviewed in Muller et al. 2017). GLP-1 and GIP effectively signal to α and β cells via the specific receptors GLP1R and GIPR, respectively. There has been much attention toward using GLP1R inhibitors to ameliorate hyperglucagonemia in diabetes, but recent evidence on GIP and GIPR in α cells supports a novel role for GIP in the pathogenesis of diabetes (Pederson & Brown 1978, Ding et al. 1997, Chia et al. 2009, DiGruccio et al. 2016). A study looking at the effects of an enzyme-resistant form of GIP on α cells in mice with induced major β cell loss found that the modified GIP peptide caused an increase in α lineage cells not expressing glucagon and used genetic lineage tracing with the GluCreERT2; ROSA26-eYPF alleles to demonstrate the occurrence of α-to-β cell interconversion (Sarnobat et al. 2020). In human studies, GIPR was found to be highly correlated with the peak Na+ current of human α cells and was significantly downregulated in human T1D α cells (Dai et al. 2022). Further review of these topics can be found in the Gao et al. and Armour et al. articles in this special collection. These findings in β cell-deficient mice and α cells from T1D individuals could support the role of GIPR/GIP in the loss of α cell identity and/or function in diabetes (Brissova et al. 2018). Novel research into the communication between α cells and other cells of the islet has also found evidence of cell–cell regulation of glucagon secretion via juxtacrine signaling mediated through an interaction between the EPHRIN-A5 ligand on the surface of β cells and the α cell surface receptor EPHA4 (Hutchens & Piston 2015, Ng et al. 2022). This topic is reviewed extensively in the article by Gao et al. in this special collection.
Future directions
It is currently unknown to what extent alterations in factors regulating glucagon secretion are primary or secondary events in diabetes. In particular, in-depth genetic analyses of the α cell ion channels remain incomplete, leaving the field with knowledge gaps about their exact roles in α cell function and how they are regulated and maintained during physiological and pathophysiological conditions. In adult β cells, the transcription factor RFX6 directly regulates the transcription of several Ca2+ channel subunits as well as K+ channel ABCC8, but whether RFX6 has similar regulatory roles in α cells has not been explored (Chandra et al. 2014, Piccand et al. 2014).
Section 3: α cells have inherent proliferative potential
Expanded α cell mass has been well documented in rodent models that have a breakdown of glucagon-liver signaling and in individuals with diabetes. Morphometric analyses of islets from humans with diabetes have reported significant increases in the numbers of α cells compared to control islets (Maclean & Ogilvie 1959, Orci et al. 1976, Brissova et al. 2018). T1D-related α cell hyperplasia has also been recently recapitulated in mouse models of β cell loss (Sarnobat et al. 2020). Lastly, the species specificity of α-to-β cell ratios seems to be associated with the wide variation of animal glucose homeostasis and supports the idea that there is inherent plasticity in α cell population regulation (Steiner et al. 2010). This is especially true when considering the dysregulation of α cell mass in diabetes.
Liver and α cell axis
A feedback loop between α cells and the liver has been linked to non-diabetes-related α cell hyperplasia (Fig. 3). Normally, once α cells secrete glucagon into the blood, glucagon binds to GCGR receptors in the liver (Mayo et al. 2003). This stimulates hepatic cells to uptake circulating amino acids, leading to the production of glucose from amino acids and subsequent release into the blood to restore euglycemia (Boden et al. 1984, Wakelam et al. 1986). However, interference of glucagon/hepatic-GCGR signaling stimulates α cell mass hyperplasia. Manipulation of this pathway in several mouse models, including the Gcgr-KO (Longuet et al. 2008), hepatic-Gcgr-KO (Longuet et al. 2013), Gcg-KO (Hayashi et al. 2009), Pcsk2-KO (Webb et al. 2002), and pharmacological blockade of GCGR in the liver (Gu et al. 2009), have all resulted in the expansion of α cell mass. Studies have found that prolonged disruption of GCGR stimulation in hepatocytes causes an increase in circulating amino acids (Solloway et al. 2015, Dean et al. 2017, Kim et al. 2017, 2019). Aminoacidemia in Gcgr-deficient mice and increases in circulating L-glutamine were shown to be causative to α cell hyperplasia and were linked to an upregulation of the mTOR pathway in α cells (Solloway et al. 2015, Dean et al. 2017). mTOR is known to be a critical regulator of proliferation and Rapamycin inhibition in Gcgr-KO mice significantly decreased α cell proliferation, supporting the model that the Gcgr-KO-induced aminoacidemia stimulates mTOR-dependent α cell proliferation (Solloway et al. 2015, Saxton & Sabatini 2017). Surprisingly, a recent report shows evidence that blockade of GCGR via monoclonal antibodies in T2D mice causes α cells to dedifferentiate to a Neurogenin3+ state, α-to-β conversion, and increased insulin secretion in α-lineage cells (Cui et al. 2022). This suggests a greater role for a liver-α cell feedback loop in T2D but also a potential influence on the loop’s effects on transcription during diabetes.
Breakdown of the liver-α cell feedback loop causes α cell proliferation. Glucagon receptor inhibition in the liver causes increased hepatic amino acid output into the blood. Amino acids are taken up by α cells via transporters SLC38A5 (in mice), SLC38A4 (in human), and SLC7A2. Increased amino acids in the cytosol of α cells stimulate mTOR signaling, which triggers α cell proliferation and further activates Slc38a5/SLC38A4 expression. Increased amino acids in the α cells also stimulate the activity of FOXP1/2/4 transcription factors, which activate α cell proliferation through cell-cycle regulators. Figure made using https://www.biorender.com/.
Citation: Journal of Endocrinology 258, 2; 10.1530/JOE-22-0297
Two independent reports on the liver-α cell feedback loop mechanism have revealed that the amino acid transporter SLC38A5 is critical for the amino acid-induced response of α cells (Dean et al. 2017, Kim et al. 2017). These studies found that Slc38a5 was upregulated by GCGR inhibition and that disruption of mTOR signaling via rapamycin in GCGR-blocked mice ablated the upregulation of Slc38a5 (Kim et al. 2017). Importantly, Slc38a5/Gcgr compound mouse mutants and compound slca38a5a/slc38a5b/gcgra/gcgrb mutants in zebrafish displayed reduced α cell hyperplasia. These data led the authors to conclude that SLC38A5 is necessary for glucagon pathway dependent α cell hyperplasia and that an mTOR-SLC38A5 positive feedback loop in α cells amplifies α cell proliferation during aminoacidemia.
Interestingly, human α cells do not express SLC38A5 and when transplanted into Gcgr-deficient mice, α cells still proliferate despite lacking human SLC38A5 (Kim et al. 2017). Recent investigations using human donor islets grafted into the kidney capsule of SCID mice found that human amino acid transporter SLC38A4 was upregulated in α cells after GCGR-blockade (Kim et al. 2019). This group also demonstrated that the increase in SLC38A4 expression was mTOR-dependent and that this increase in expression was higher in proliferating α cells. In addition, islets from individuals experiencing ‘glucagon cell hyperplasia and neoplasia’ (GCHN) due to GCGR loss-of-function mutations were found to have a significant increase of SLC38A4 expression in their α cells. Taken together, these authors concluded that SLC38A4 has a role in human amino acid-induced α cell hyperplasia and that it could be a likely human analog for the mouse SLC38A5’s role in the liver-α cell axis.
Lastly, recent work has revealed that the amino acid transporter SLC7A2 is also important to amino acid-induced α cell hyperplasia (Spears et al. 2019). Using full-body mouse knockouts of Slc7a2 and siRNA knockdown of Slc7a2 in immortalized αTC cells, researchers found decreased amino acid-dependent α cell hyperplasia as well as reduced glucagon and insulin secretion. These studies into α cell amino acid transporters highlight how sensitive α cells are to circulating amino acid levels and their adaptability to metabolic changes.
Genetic insights into α cell proliferation
It is still unknown if the liver-α cell axis and/or additional mechanisms are at play in diabetic α cell proliferation. Evidence in non-diabetic human donor islets suggests that α cells are more proliferative in early life but proliferative potential wanes over time (Lam et al. 2018), although α cells in humans seem to maintain proliferation longer than human β cells.
Emerging reports are beginning to reveal the identity of transcription factors that are important for the regulation of α cell proliferation and the corresponding increases in α cell mass. Several members of the forkhead box (FOX) family of transcription factors are important to the development of islet endocrine cells. FOXA2 is important during early endocrine specification and evidence shows FOXM1 as a regulator of β cell mass during periods of metabolic stress (Sund et al. 2001, Ackermann Misfeldt et al. 2008, Gao et al. 2008, Zhang et al. 2010). Research within the past decade has revealed that the FOXP1, FOXP2, and FOXP4 members of the FOXP subfamily are important to glucagon secretion and α cell mass. Single mutants of the Foxp1/2/4 group caused no major effects, but the endocrine-specific Foxp1/2/4 triple mutant found that fasted mice were hypoglycemic due to defective glucagon secretion and that α cell mass was drastically reduced compared to controls (Spaeth et al. 2015). Expression analysis of triple knockout islets found that mRNAs of cyclins Ccna2, Ccnb1 and Ccnd2 were downregulated and the cyclin-dependent kinase inhibitor Cdkn1a was upregulated. Together this evidence supports the model that FOXP1/2/4 regulates α cell proliferation via transcriptional regulation of cell-cycle factors. A more recent report demonstrated that aminoacidemia induced by GCGR blockade in Foxp1/2/4 global knockout mice did not stimulate α cell hyperplasia but also did not affect the mTOR pathway (Dean et al. 2017). This suggests that amino acid-based α cell hyperplasia relies on FOXP transcription factors, but that FOXP activity is either downstream of mTOR or is part of an mTOR-independent pathway.
Another study points to the existence of a distinct highly proliferative α-like population in human islets (Lam et al. 2018). These cells, which persist through adulthood, are ARX+, cytoplasmic-SOX9+, hormone negative, and show high expression of Ki67 proliferation staining. This α cell-related population also express endocrine markers such as NKX2.2, INSM1, and ISL1/2 as well as exocytotic genes SNAP25 and SYT1. Although they do not express glucagon, these proliferative cells express ARX and lack expression of pancreatic polypeptide and ghrelin, suggesting they are potentially α-like.
Future directions
Further work to understand how the α cell transcriptomes change during diabetic conditions could reveal more insights into the inherent adaptive proliferative mechanisms in α cells. More research will be necessary to uncover whether the loss of hormone expression and/or cytoplasmic expression of SOX9 contributes to islet cell proliferation in humans or to ascertain whether this population is connected to amino acid-induced α cell hyperplasia. Lastly, since this SOX9 population is highly proliferative, they could represent a robust source of islet cells for the generation of neogenic β cell populations.
Section 4: regulation of α cell identity
With the advent of single-cell RNA-Seq and the identification of markers and tools with which to purify the α cell population, there are now several studies demonstrating that diabetes affects α cell identity. Recent advances using next-generation sequencing and human islet gene expression analyses have revealed the transcriptomic alterations occurring in T1D and T2D α cells. Using islets donated from individuals with recent-onset T1D, studies found that the key α cell identity factors ARX and MAFB had significantly downregulated mRNA and protein expression, with a corresponding increase in the β cell-specific factor NKX6.1 (Brissova et al. 2018). These studies also identified differential expression of many downstream α cell-enriched factors, including ion channel genes and vesicle trafficking genes. The β cells that remained in these islets were found to retain expression of the key β cell transcription factors NKX6.1 and PDX1, suggesting that although functional β cells were still present in early onset T1D, the α cell transcriptional and functional programs were already altered. Furthermore, a recent study has identified transcriptomic changes in human α cells that were also positive for GADA (Doliba et al. 2022). This study showed that not only were transcription factors such as PDX1 and MAFA dysregulated in GADA+ α cells, but many genes from the glycolysis and gluconeogenesis pathway were also downregulated, including GCK and LDHA. Another study that focused on the transcriptomes of α cells in human T2D also demonstrated that α cells showed a de-repression of genes that are associated with immature α cells in the context of T2D (Avrahami et al. 2020). These findings were supported by studies focusing on α cells with exocytotic dysfunction in T2D that identified expression profiles containing upregulation of progenitor factors and transcripts relating to immature α cells. Overall, these studies demonstrate that α cells in diabetes are undergoing transcriptional changes that resemble loss of maturation or dedifferentiation. Although the occurrence of shifts in α cell identity is difficult to assess in human studies of T1D, mouse models of drastic β cell loss have resulted in the stimulation of α lineage cells to become bihormonal or undergo a full α-to-β conversion (Thorel et al. 2010, Sarnobat et al. 2020). Altogether, these data suggest that in the context of both T1D and T2D, α cells are susceptible to transcriptomic shifts that can cause changes in α cell identity.
Clearly, gene expression in α cells is being substantially altered in both T1D and T2D. This may reflect adaptive functional changes in the cell, a shift in their identity, or a combination of the two events. An obvious factor in the maintenance of α cell function and identity is the role of transcription factors. Transcription factors with cell-specific expression or cell-specific function can define a cell’s distinct gene expression profile, potentiating cellular identity and function as a whole. This is also true in the α cell, and many groups have been working to characterize the molecular mechanisms of α cell transcription factors.
ARX
The Arista-less homeobox-containing transcription factor ARX is restricted to α cells, pancreatic polypeptide-producing cells (PP cells), and ghrelin-producing ε cells in the islet (Mastracci et al. 2011, Wilcox et al. 2013, DiGruccio et al. 2016). Considered the ‘master regulator’ of α cell identity, the ARX transcription factor has been shown to be necessary for the development and maintenance of α cells in mice and humans as well as being sufficient to convert β cells into α cells (Collombat et al. 2003, 2007, Itoh et al. 2010, Mastracci et al. 2011, Courtney et al. 2013, Wilcox et al. 2013, Xu & Xu 2019). The loss of Arx in α cells simultaneously causes decreased glucagon expression and increased insulin and somatostatin expression (Collombat et al. 2007, Mastracci et al. 2011, Wilcox et al. 2013, Chakravarthy et al. 2017; Fig. 4A). Importantly, α cells deficient in ARX erroneously express the β cell transcription factors NKX6.1, MAFA, PDX1, and GLUT2 (Fig. 4A), suggesting a shift in cell identity, rather than merely hormone dysregulation. These identity shifts resulted in both polyhormonal cells and cells that were glucagon−/insulin+ or glucagon−/somatostatin+. Interestingly, when the endocrine-enriched DNA methyltransferase DNMT1 is depleted from α cells simultaneously with ARX, α-lineage cells convert to β-like cells at a higher frequency than the single Arx α cell knockout (Chakravarthy et al. 2017). This group also demonstrated that α cells deficient in both ARX and DNMT1 are more canalized toward the β cell fate than either the α or δ cell fates. Since α cell identity was unperturbed in the single Dnmt1 conditional knockout, the epistasis of Dnmt1 to Arx and the synergy of the double knockout suggests they could be cooperating on an α-to-β axis by maintaining α enriched gene expression and repressing β genes. Using single-cell transcriptomic analysis and electrophysiological assays, they also uncovered that the ARX/DNMT1 depleted α cells that had converted to β-like cells possessed transcriptional signatures and electrophysiological characteristics that were more similar to native β cells than to native α cells (Fig. 4A). The authors of this study speculated that DNMT1’s role in this mechanism was to ‘prime’ the epigenetic state of the α cells creating a chromatin environment that is poised for transcriptional change. Although much of the molecular mechanism of ARX in α cells has yet to be uncovered, the evidence shown here supports its crucial role in the maintenance of α cell identity. The evidence that Arx is commonly dysregulated in α cells from T1D and T2D individuals, further emphasizes its importance in islet biology.
Transcription factors with known roles in maintaining α cell identity. Evidence shows that ARX, MAFB, and PAX6 maintain the transcriptional identity of α cells. Left of the vertical dashed line is the known regulatory targets of each transcription factor. Right of the dashed line is insights into the mechanism of each factor. (A) Several studies suggest ARX promotes α cell hormonal identity because without ARX α cells can shift identity toward β- and δ-like cells. Arx also represses the expression of β cell-enriched factors NKX6.1, PDX1, MAFA, and GLUT2. The double knockout (KO) of Arx and Dnmt1 causes a greater frequency of α-to-β conversion than the Arx single knockout and results in β-like transcriptomic signatures and electrophysiology. This suggests a cooperative mechanism between ARX and DNMT1 in maintaining α cell identity. (B) MAFB directly activates the transcription of preproglucagon in α cells. MAFB also has distinct mechanistic roles in development, maturation, and maintenance and acts to maintain the α cell population size. (C) In α cells, PAX6 directly activates the transcription of preproglucagon, MafB, cMaf, and NeuroD1. PAX6 is expressed in both α and β cells but binds at α cell-specific sites due to the α-enriched lncRNA Paupar. Alternate splicing of Pax6 pre-mRNA that includes the 5a exon is promoted by Paupar, causing the α-specific enrichment of a PAX6 5a protein with different DNA binding affinities. Figure made using https://www.biorender.com/.
Citation: Journal of Endocrinology 258, 2; 10.1530/JOE-22-0297
MAFB
MAFB is a leucine-zipper-containing transcription factor that is expressed in murine α and β cells during development but gradually becomes restricted to α cells once mature single hormonal identity is established postnatally (Artner 2006, Nishimura et al. 2006, Artner et al. 2010, van der Meulen et al. 2017). Importantly, MAFB has been shown to bind to the preproglucagon G1 promoter element and activate its transcription (Planque et al. 2001, Artner 2006, Gosmain et al. 2007, Katoh et al. 2018; Fig. 4B). In the βTC cell culture model of murine β cells, misexpression of MAFB caused the ectopic expression of glucagon (Artner 2006). MafB knockouts using pan-pancreatic or pancreatic endocrine Cre systems found gene expression dysregulation of key endocrine markers such as Gcg, Arx, Nkx6.1, Pdx1, Ghrl, Ins2, and Ppy (Conrad et al. 2016, Katoh et al. 2018). However, tamoxifen-induced inactivation of MafB in adult mice found only glucagon was downregulated, suggesting unique transcriptional regulatory roles of MAFB in the different stages of endocrine development, α cell maturation and α cell maintenance (Chang et al. 2020; Fig. 4B). Although there is no evidence that loss of MafB in adult α cells causes any hormonal identity shifts, there is some evidence to show that loss of MafB in adult α cells causes decreased α cell mass and significantly impaired glucagon secretion when exposed to arginine stimulation (Conrad et al. 2016, Katoh et al. 2018, Chang et al. 2020; Fig. 4B). Lastly, even though MAFB expression in mice is restricted to maturing and mature α cells, in humans, MAFB is expressed in both adult α and β cells (Conrad et al. 2016). This suggests potential species-specific roles for MAFB that have yet to be uncovered. Altogether, evidence supports a role for MAFB in the maintenance of the functional and proliferative characteristics of adult α cells.
PAX6
The paired and homeobox domain-containing transcription factor PAX6 is important in the development of pancreatic endocrine cells and is expressed in the majority of mature islet cells (Sander et al. 1997, St-Onge et al. 1997). Initial experiments found that PAX6 works in concert with the MAF transcription factors to directly regulate the transcription of the preproglucagon gene by binding to its G1 promoter element (Gosmain et al. 2007; Fig. 4C). Pax6-knockdown experiments in primary rat α cells and mouse αTC lines have revealed that PAX6 regulates the transcription of many important α cell differentiation and functional genes, including Preproglucagon, Pcsk2, MafB, cMaf, and NeuroD1 (Gosmain et al. 2010). Luciferase promoter activity assays confirmed that MafB, cMaf, and NeuroD1 are regulated by direct PAX6 binding (Fig. 4C). Immunofluorescence expression analysis in a conditional α cell knockout of Pax6 supported the findings that both MafB and glucagon expression are positively regulated by PAX6 in α cells (Ahmad et al. 2015).
Since PAX6 is expressed in both α and β cells understanding the cell-specific role and mechanism of PAX6 in α cells is important to fully understand the distinct identity and function of α cells. Immunofluorescence and transcriptomic analysis done in the β cell-specific knockout of Pax6 shows that PAX6 positively regulates many β-specific transcription factors such as Nkx6.1, Pdx1, and MafA (Ahmad et al. 2015, Swisa et al. 2017). Importantly, even though glucagon is activated by PAX6 in α cells, in β cells PAX6 represses glucagon and activates the insulin genes (Swisa et al. 2017). These studies highlight the often-opposing cell-specific regulatory functions of PAX6 in α versus β cells. A recent report has identified a unique mechanism through which PAX6 can exert cell-specific target selection. This study revealed that an α cell-enriched long-noncoding-RNA (lncRNA) Paupar (Pax6 upstream antisense RNA) facilitates the α cell-specific function of PAX6 (Singer et al. 2019). This report demonstrated that the lncRNA Paupar interacts with SRSF alternative splicing proteins to enable the enrichment of a Pax6-5a splice isoform that inserts an additional 14 amino acids into the paired DNA binding domain to alter its binding specificity (Fig. 4C). PAX6 chromatin immunoprecipitation sequencing (ChIP-seq) in αTC cells depleted of Paupar revealed more than 2000 differentially bound sites, many of which overlapped with regulatory regions of α enriched genes. Importantly, downregulation or deletion of Paupar did not alter overall Pax6 expression levels, but reduced expression specifically of the Pax6-5a isoform to cause downregulation of the α cell genes Gcg, MafB, and Arx and dysregulation of many genes related to α cell ion channels, vesicle trafficking, and cell signaling. These data led the group to conclude that Paupar-induced enrichment of the Pax6-5a isoform in α cells is a large component of the α cell-specific Pax6 mechanism that promotes α cell identity and function.
Future directions
Although foundational research into a small subset of α cell transcription factors has been published, there is still a paucity of information about many others, including the functions of α-enriched IRX1 (Petri et al. 2006), IRX2 (Petri et al. 2006), KLF4, BRN4 (Hussain et al. 1997), and ETV1. Furthermore, emerging sequencing studies have revealed many apparently α cell-specific genes, including transcription factors that have yet to be studied in the pancreas (Van Gurp et al. 2022). Understanding the repressive and stimulatory mechanisms through which these α cell-specific transcription factors function could enrich our understanding of α cell-specific function and identity. There are also a large number of factors that – similar to PAX6 – function in multiple endocrine cell types, including factors such as NEUROD1 (Naya et al. 1997, Anderson et al. 2009), NKX2.2 (Gutierrez et al. 2017), ISL1 (Dassaye et al. 2016), GATA6 (Lorberbaum & Sussel 2017, Tiyaboonchai et al. 2017, Chiou et al. 2021), RFX6 (Chandra et al. 2014, Piccand et al. 2014), and FOXA2 (Sund et al. 2001, Gao et al. 2008). Understanding the cell-specific roles of these dual α/β-expressed transcription factors could illuminate more about the regulation of α cell transcriptional programs and how they become disrupted in diabetes. Currently, there is much known about the functional targets of these factors in β cells and with the growing interest in α cell biology and the availability of novel molecular technologies, these should soon be complemented by their role in α cell development and function.
Section 5: epigenetic plasticity of α cells
As we have demonstrated thus far in this review, α cells lose either part or the whole of their identity during diabetes due to the dysregulation of the expression of genes regulating and related to α cell functional and proliferative identities. The base dynamics of gene transcription in the mature cell can be regulated by inherent chromatin accessibility that is established during differentiation by covalent histone modification and chromatin remodeling. With the expansion of technologies that allow the discovery of the precise placement of histone marks as well as the accessibility of chromatin, novel research has found evidence that the α cell epigenetic landscape supports a distinct plasticity that is different from other islet cells, which could potentially enable α cell pathogenesis in diabetes and allow α cells to more easily transdifferentiate into β cells.
Poised chromatin modification
Using ChIP-seq and antibodies for H3K4me3 that marks active transcription or H3K27me3 that denotes repression, scientists have produced maps of chromatin modification in human donor α and β cells (Bramswig et al. 2013). They reported that α cells have the highest number of locations marked with both H3K4me3 and H3K27me3. Dual marking, or bivalency, is thought to represent a poised state, especially if located at an enhancer or promoter (Bernstein et al. 2006). These bivalent chromatin locations in α cells that were monovalently marked in β cells were nearly 3-fold more abundant than chromatin that was bivalent in β cells but monovalent in α cells (Bramswig et al. 2013). Furthermore, a large number of β cell-enriched genes that are important to transcription regulation and membrane-bound functions have poised chromatin in α cells (Fig. 5A). Specifically, this group found that β-enriched genes MAFA, PSCK1, HDAC9, KCNQ2, and GLP1R all had activating marks in β cells but bivalent marks in α cells. In addition, while the α-enriched genes GCG and IRX2 had H3K4me3 marks in α cells and H3K27me3 marks in β cells, critical α cell factor ARX and α enriched transcription factor IRX1 were bivalently marked in α cells. Interestingly, the β cell gene NKX6.1 and α cell genes ALDH1A1 and PSCK2 have activating H3K4me3 marks in both α cells and β cells. Together, this suggests a plasticity model that α cell chromatin is in a state that may not fully protect from ectopic expression of β genes and may not efficiently preserve α gene expression, especially during times of extreme metabolic stress.
The α cell epigenetic landscape is more accessible and poised than in β cells. Evidence comparing the chromatin accessibility and histone modifications of human donor α and β cells suggests that α cells possess greater epigenetic plasticity than β cells. (A) In α cells, many promoters of repressed β-specific genes possess accessible chromatin and poised histones with both active H3K4me3 and repressive H3K27me3 marks. However, in β cells, these same genes are activated and have open chromatin but only possess the active H3K4me3 mark. (B) ATAC-seq data reveals that α cells have much more unique open chromatin sites than β cells. Figure made using https://www.biorender.com/.
Citation: Journal of Endocrinology 258, 2; 10.1530/JOE-22-0297
Chromatin accessibility
The openness of chromatin can be observed through the use of an Assay for Transposase-Accessible Chromatin paired with next-generation sequencing (ATAC-seq). Researchers have recently conducted ATAC-seq on human donor islets to produce maps of chromatin accessibility in human α and β cells (Ackermann et al. 2016). This data revealed that α cells have a higher proportion of cell-unique open chromatin while β cells have more open chromatin that is shared with α cells. Remarkably, α cells had 27,000 unique ATAC peaks while β cells only have 1850, showing that on a genomic scale, α cells have a huge amount of cell-specific chromatin accessibility (Fig. 5B). Furthermore, α cell-specific open chromatin in humans was found at several T2D risk loci in the KCNQ1 and CDKN2A-CDKN2B genes. When accessible chromatin was paired with cell-specific gene expression data and the chromatin modification maps (Bramswig et al. 2013), this group reported that most α cell-specific genes have unique open chromatin and H3K4me3 active marks at promoters in α cells, while more β cell-specific gene promoters had open chromatin and poised bivalent chromatin in α cells (Fig. 5A). This again illustrates that chromatin at β cell-specific genes is not completely repressed in α cells and suggests unique epigenetic plasticity in α cells, especially at β specific genes.
A recent manuscript assessing chromatin accessibility in mouse α and β cells has revealed that α-enriched accessible chromatin was found at proximal promoters at a two-fold greater frequency than β-enriched accessible chromatin, which was found mostly in distal intergenic regions (Mawla et al. 2023). This report also found that 33% of differentially α/β enriched chromatin at promoters were genes that are normally repressed in α cells but have α-enriched accessible chromatin – an ‘incongruent’ expression/chromatin accessibility signature. Furthermore, the majority of these α-repressed genes with ‘incongruent’ chromatin accessibility were de-repressed in cells that had undergone α-to-β transdifferentiation. This evidence in mice provides more support for α cell epigenetic plasticity but also that this plasticity correlates to changes in α-to-β cell conversion.
α-to-β Transdifferentiation
While not widely observed in human islets, α-to-β cell transdifferentiation has been well documented in multiple mouse contexts. As discussed earlier, the depletion of Arx from mouse α cells can cause α cells to convert into β cells, but researchers have also reported α-to-β cell conversion in healthy mouse islets and islets with β cell ablation (Thorel et al. 2010, van der Meulen et al. 2017, Oropeza et al. 2021). These reports highlight the unique plasticity of the α cell to change their identity in contexts of health and disease.
While the mechanistic basis of this α cell phenomenon remains poorly understood, the search for reliable and high-efficiency means of α-to-β conversion as a means of creating populations of transplantable β cells for diabetic treatment options has become a driving force for α cell study. Key insights from the group that initially observed α-to-β transdifferentiation in β cell-ablated mice recently reported that α cells in this β-deficient environment undergo significant transcriptional changes (Oropeza et al. 2021). This set of ‘transitional genes’ shows a temporally dynamic expression that can be further broken down into expression subsets with distinct and sometimes transient timing from the point of β cell ablation until 30 days post β-cell ablation. Gene expression changes in these α cells consist of genes related to proliferation, mTOR signaling, cell cycle regulators, and interferon signaling but also many transcription factors and genes related to β cell identity and function. Importantly, nearly 70% of the 1386 differentially expressed genes in these α cells possessed accessible chromatin at their human homologous promoters and 40% of these ‘transitional genes’ had 3D chromatin contacts between a putative enhancer and their promoters. Furthermore, islet DNA-binding sites of transcription factors such as NKX2.2, MAFB, and FOXA2 were found to be highly present at promoters and putative enhancers of ‘transitional genes’. Together these findings suggest that the plastic epigenetic environment of α cells could be enabling their response to drastic changes in the islet environment.
A recent discovery in healthy mouse islets suggests that a population of immature β-like cells exists during postnatal life (van der Meulen et al. 2017). These ‘virgin’ β-like cells were primarily found toward the edge of the islets, adjacent to α cells. The virgin cells do not produce insulin, fail to express the β cell maturity marker UNC3 or the functional protein GLUT2, and do not show mature electrophysiological activity. Lineage tracing experiments demonstrated that many of these cells were derived from the α-lineage and expressed moderate levels of both α and β cell identity genes. The authors originally suggested that these cells represented an intermediate stage in α-to-β transdifferentiation. Furthermore, many α-lineage cells were found to have fully transdifferentiated into β cells and showed highly similar gene transcription and functional characteristics to that of ‘conventional β cells’ in young mice. However, a more recent study from the group indicated that the virgin β-like cells were long-lived with low turnover into mature β cells in healthy conditions (Lee et al. 2021). Overall, these observations provide evidence for limited α cell plasticity and the ability of α cells to change their functional state and/or identity early in life and potentially during disease conditions. Although the mechanistic means of stimulating and potentiating functional or identity change in α cells has not been identified, many of the gene expression changes found in the altered α-lineage cells were shown to have the poised and accessible epigenetics described in the sections above, suggesting a role in α cell epigenetic plasticity in the generation of neogenic β cells (Bramswig et al. 2013, Ackermann et al. 2016).
Future directions
Chromatin modifications and accessibility reveal that human α cells possess poised and open chromatin at β cell-specific genes but also poised chromatin at enriched α factor genes ARX and IRX1. These data motivate the hypothesis that α cells possess more plastic epigenetic regulation, which could lead to regulatory shifts of the α cell transcriptome, potentially during metabolic stress and this could be due to a change in transcription factor function. To test this hypothesis, more experiments on how transcription factor gene regulation and DNA occupancy change during metabolic stress could be conducted. Using conditional transcription factor knockouts in HFD-fed mice to recapitulate T2D conditions and STZ-induced β cell loss or non-obese diabetic mice as a model of T1D, RNA-seq and ChIP-seq assays could determine how direct targets of transcription factors change during diabetes. Prioritizing the study of transcription factors that are dysregulated in diabetes, such as ARX, FOXA2, IRX2, ISL1, MAFB, NKX2.2, RFX6, and PAX6, could determine early responders to metabolic stress.
Discussion
In diabetes, α cells have dysregulated gene expression, cellular dysfunction, and proliferative abnormalities. These fundamental changes in α cell identity likely start with the breakdown of transcriptional regulation and could be due to epigenetic plasticity that is unique to the endocrine α cell lineage due to its α cell-specific ‘chromatin meta-states’.
In recent years, many data sets have been generated to deepen our understanding of α cell specificity and plasticity. This includes data sets that (i) compare α, β, δ, and/or exocrine cells in mouse islets (DiGruccio et al. 2016, Schaum et al. 2018); (ii) healthy human islets (Ackermann et al. 2016, Avrahami et al. 2020); (iii) T1D islets (Brissova et al. 2018) and T2D islets (Segerstolpe et al. 2016, Xin et al. 2016, Avrahami et al. 2020); (iv) patch-seq data between healthy human and T2D islets (Camunas-Soler et al. 2020, Dai et al. 2022); (v) human α and β chromatin modification ChIP-seq (Bramswig et al. 2013); and (vi) human α and β chromatin accessibility ATAC-seq (Ackermann et al. 2016, Chiou et al. 2021). While the availability of these datasets has provided exciting new information about α cell biology, they are also accompanied by the challenge of data integration and massive post-hoc multi-omic analyses. Future analyses have the potential to continue to delineate key differences in how the α cell transcriptome changes in T1D and T2D, while also helping pinpoint priority gene candidates for future mechanistic studies.
α and β cells share expression of several transcription factors that have been reported to be critical for β cell function. Although strong evidence shows that α/β enriched PAX6 has important cell-specific roles in α and β cells, many of the other α/β expressed transcription factors have not been studied in α cells. Evidence shows that PAX6 oppositely regulates cell-specific genes such as glucagon and insulin in α and β cells, respectively, and that this cell-specific function is at least partially controlled by cell-specific PAX6 isoforms. Could other factors expressed in both α and β cells that have cell-specific functions and oppositely regulate genes in a cell-specific way underlie a mechanism for how α cells experience transcriptomic dysregulation in diabetes? Characterizing the α cell functions of transcription factors expressed in both α and β cells that are affected by diabetes, such as NKX2.2, PAX6, ISL1, and FOXA2, could not only help us understand α cell identity maintenance but could also lead to the understanding of how the α cell transcriptome becomes dysregulated in diabetes.
In conclusion, many recent studies in the α cell field have revealed that transcriptomic, functional, and proliferative dysregulation of α cells contributes to islet dysfunction in diabetes. It is also evident from these studies that α cells lose many aspects of their cellular identity prior to and during the disease process. Research characterizing the human α cell epigenome suggests that high levels of α cell epigenetic plasticity is unique among the islet endocrine cell populations, especially at β cell-specific gene loci. Considering this plasticity and the poised nature of the α cell epigenome, we suggest that the pathology of α cells in diabetes is at least partially due to transcriptional dysregulation of key factors via their inherent epigenetic plasticity. To that end, more attention and effort toward understanding the maintenance of α cell identity will greatly inform future studies on the pathogenesis of diabetes and could further their potential as a source of neogenic β cells.
Declaration of interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by NIH NIDDK R01 DK082590, R01 DK1181155, R01 DK125360, U01 DK127505 and P30 DK116073 (LS) and NIH F31 DK130281 (E.P.B.).
Acknowledgements
We thank members of the Sussel Lab for discussions about the content of the review. We also thank Dylan Sarbaugh and Cell, Stem Cell and Development Program graduate students for their critical reading of the manuscript.
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