I- Birth and death of the α-cell
1. Structure of the islets of Langerhans
The islets of Langerhans are scattered throughout the pancreatic tissue and make up less than 2% of the organ’s total mass. The islets, first observed by Paul Langerhans in 1869, contain five distinct cell subtypes, each associated with the secretion of an endocrine hormone (Figure 1). In the mouse, insulin-secreting β-cells make up an estimated 60% to 80% of the islet cells and tend to segregate to the islet core whilst the remaining cells are arranged in the so-called mantle region. The next most prominent cell type corresponds to glucagon-secreting α-cells representing 10% to 20% of the islet cells, followed by smaller numbers of the somatostatin-secreting δ-cells, pancreatic polypeptide (PP) PP-cells and ghrelin-secreting ε-cells (Lefebvre, Rorsman,Lectures). In humans, non-β-cells are often observed both at the periphery and also seemingly in clusters within the center of islets. Such a difference in islet structure between humans and rodents suggests species-specific paracrine regulation, which will be discussed in Chapter III (Kulkarni, Lecture).
2. Origin of α-cells and control of differentiation
a. Pancreatic endocrine cells
The pancreas originates from a dorsal and ventral bud of the foregut endoderm, which are later referred to as dorsal and ventral lobe, or the “tail” and “head” of the adult pancreas. In mouse, the first hormone-expressing cells are detected at E9.5, most of which elaborate glucagon; the first insulin-expressing cells are seen at E10.5 and the majority coexpress glucagon. A second wave of hormone-expressing cells is detected from E13.5 onward and includes glucagon-, insulin-, somatostatin-, ghrelin-, and PPexpressing cells.3 Migration of differentiated cells and formation of the islets of Langerhans take place during the tertiary transition (E16.5 to birth). The perinatal period is marked by rapid islet cell proliferation and the coalescence of endocrine cells into their final, compact islet structure (Kaestner, Lecture; Nuria et al, unpublished). Human pancreatic endocrine cells originate from ductal cell precursors in fetal life. Limited studies on post-mortem pancreas in human pregnancy indicate a marginal increase(~1.4-fold) of β-cell mass with no change in islet or β-cell size compared with the larger expansion of islet mass in pregnant rats (2- to 5-fold). The endocrine population expands during early infancy. In adults, subsequent proliferation is thought to involve replication of existing islet cells or a combination of replication and neogenesis4 (Clark, Lecture; Cnop et al, unpublished). However, islet cell proliferation declines dramatically with age, both in rodents and humans.
Figure 1. Anatomical characteristics of pancreatic islets (from Kawamori et al, unpublished) (A) Immunohistochemicalanalysis of mouse pancreatic islets. Insulin-positive β-cells (blue) are located in the core region of the islet. Glucagon-positive α-cells (red) and somatostatin positive δ-cells (green) are located in the mantle area of the islet. A schematic image of the structure is shown on the right. (B) Schematic image of the structure of human islets. α-cells observed in the center of the islets face to intra-islet vascular vessels and are also surrounded by β-cells.
Aristaless related homeobox (Arx) and paired box gene 4 (Pax-4)
Arx and Pax4 have antagonistic functions for proper islet cell specification (Figure 3). The Arx gene is necessary for α-cell developmentand is sufficient to promote the α- and PP-lineages during pancreas morphogenesis. The persistent expression of Arx in early pancreatic and/or endocrine precursor cells during embryonic development results in a dramatic reduction in β- and δ- cell numbers, concurrent with an increase in α- and PP-cell populations. More interestingly, the misexpression of Arx in adult β-cells was found to induce their conversion into cells exhibiting α- or PP-cell characteristics.7 In contrast, the Pax4 gene favors the β- and δ- cell fate and its deletion results in the absence of β- and δ- cells and an increase in α-cell number.8 Pax4 ectopic expression in the developing mouse pancreas resulted in oversized islets mostly composed of cells displaying a β-cell phenotype,9 indicating that Pax4 misexpression is sufficient to promote the β-cell lineage allocation during development. Furthermore, its misexpression in glucagon-expressing cells resulted in a loss of α-cells concurrently with a dramatic increase in the number of insulin-producing cells (5- to 6-fold compared with their wild-type counterparts). Lineage tracing experiments demonstrated a conversion of glucagon-positive cells into insulin-expressing cells that showed most of the features of true β-cells.9 It was shown that the continual conversion of α-cells into β-like cells induced by Pax4 misexpression led to the continual stimulation of α-cell neogenesis, via a continuous activation of progenitor cells located in the lining of the ducts that reactivated Ngn3 expression. This continuous cycle ultimately resulted in the formation of hyperplastic islets. Interestingly, this process was sufficient in young animals to reverse the effects of chemically induced diabetes (Colombat, Lecture; Courtney et al, unpublished).
Figure 3. Schematic model of endocrine subtype specification during pancreatic development (from Courtney et al, unpublished). A: Following redirection towards the pancreatic lineage (Pdx1-expressing cells), the endocrine program is initiated as outlined by the expression of Ngn3 in all endocrine progenitors. These cells have the potential to become either an α-cell or a second progenitor with β- and δ-cell potentials and able to express both Arx and Pax4. Mutual inhibitory interactions between Arx and Pax4 lead to “competition” between the two transcription factors resulting in subtype allocation. If Arx prevails, Pax4 expression is extinguished and an α-cell fate is chosen. If Pax4 predominates and Arx expression is extinguished, the resulting progenitor cell (β/δ) is poised to undergo a second round of competitive fate allocation. In this second event, a hypothetical “factor X” is envisioned to promote the δ-cell fate and to display a mutual inhibitory interaction with Pax4 similar to the interaction between Arx and Pax4 in the first round of cell fate allocation. B-D: Fate changes in the case of Pax4- (B), Arx- (C), or combined Arx-/Pax4-(D) deficiency.
α-cells are one of the four epithelial cell types of the intestine—with enterocytes, goblet cells, and Paneth cells—arising from common multipotent cells in the intestinal crypts, deriving from the endoderm. All these enteroendocrine cells differentiate as cells migrate up the crypt-villus axis turning over every 3 to 4 days; they actively self-renew and differentiate throughout the life of an animal from a large reservoir of stem cells (Leiter,Lecture; Li et al, unpublished). The differentiation of α-cells is governed by numerous transcription factors (Figure 2). The Pdx1 gene is activated in the progenitor cells of the foregut endoderm as early as E8.5 in the mouse. During early development, Pdx1 is expressed in the entire epithelium. In later fetal stages and in the adult pancreas, lowlevels of Pdx1 expression are found in the entire pancreas, including α-cells, while highexpression is restricted to β-cells, a subset of δ-cells, and occasional ductal epithelialcells. In the various models of Pdx1-deficiency, an increase in α-cell number is observed. Whether this is due to loss of inhibition of α-cell development by β-cells—whose number is greatly reduced—or to transdifferentiation of β-cells into α-cells is not known.5 Pdx1+ cells differentiate into Neurogenin3+ (Ngn3 orNeurog3) endocrine progenitor cells. Cells expressing Ngn3 at high levels are observed only during embryonic development. These cells do not co-express glucagon or insulin, but give rise to all pancreatic endocrine cells (Kaestner, Lecture). Recent evidence has shown that, in fact, Ngn3-expressing progenitor cells pass through different phases of competence where early Ngn3-expressing cells give rise exclusively to α-cells, whereas expression of Ngn3 in later pancreatic developmental stages gives rise successively to β-, PP-, and finally δ-cells.6 Following the initiation of the endocrine program, a set of transcription factors are required to direct Ngn3-positive cells towards the four mature endocrine cell fates. These factors can be subdivided into early-acting factors (such as Nkx2.2, Nkx6.1, Pax4 or Arx) that are coexpressed with Ngn3 inendocrine precursors and late factors (including Pax6, Isl1, MafA or Pdx1) that are detected in more mature cells (Collombat, Lecture).
Figure 2. Transcription factors involved in α-cell differentiation and activation of preproglucagon transcription(from Kaestner et al, unpublished). Pdx1+ cells differentiate into Ngn3+ endocrine progenitor cells, which give riseto α-, β-, δ-, ε-, and PP-cells. Arx and Foxa2 play crucial roles in initial and terminal differentiation of α-cells, respectively.Preproglucagon transcription is regulated by Foxa1, Pax6, Brn4, Isl-1, and MafB.
3. β- and α-cell life: mass plasticity and cell lifespan and longevity
In animal models, β-cell mass flexibly adapts to insulin requirement (plasticity) which is thought to be regulated by an equilibrium of programmed cell death and neogenesis and/or replication of existing β-cells. Conditions associated with increased demand for insulin as insulin resistance and obesity result in larger β-cell mass in rodents (30-fold) compared with the very modest increases with BMI in humans (1.2-fold) (Clark, Lecture).
Excessive nutrient intake is reported to induce hyperplasia and/or hypertrophy of α-cells. α-cell mass is increased in diabetes. Interestingly, α-cell−specific insulin recepto rknockout (αIRKO) mice10 exhibited a progressive increase in β-cell area, while α-cell area was unchanged by aging, indicating a relative decrease in α-cells in the islets. Thus insulin signaling is likely involved in the proliferation of α-cells (Kulkarni,Lecture).
The half-life of β-cells in young rodents (<1 year old) has been estimated to be 30 to 60 days; cell divisions in mouse islets decrease with age and are very low in animals at age one year, suggesting that mouse islet cells are largely post-mitotic by 12 months. In humans the “birthdate” of β-cells was shown to be in the first three decades of life and no substantial proportion of cell division occurred subsequently. Replication of human adult islet β-cells has been estimated to be 10-fold less than that in adult mouse and to be highest in children under the age of 5 years. Interestingly, using mathematical modeling of lipofuscin body accumulation—a feature of aging in post-mitoticcells—Cnop et al demonstrated that both α- and β-cell populations are largely established by the age of 20 in humans with little evidence for continuous turnover or replication after this age11 (Clark, Lecture; Cnop et al, unpublished).
“Pancreatic α-cells and glucagon—neglected metabolic actors”
I- Birth and death of the α-cell
II- Regulation of glucagon expression
III- Regulation of glucagon secretion
IV- Role of glucagon in metabolism
V- Therapeutic perspectives
Lectures during IGIS meeting and unpublished reviews