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β-Cell Replication by Loosening the Brakes of Glucagon-Like Peptide-1 Receptor Signaling

¹ Gene Expression Unit, Department of Molecular Cell Biology Katholieke Universiteit Leuven, Belgium
² Banting and Best Diabetes Centre, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, University of Toronto, Canada

Address correspondence and reprint requests to Frans C. Schuit, frans.schuit{at}med.kuleuven.be, or to Daniel J. Drucker, d.drucker{at}utoronto.ca

Abbreviations: GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor

A decrease in β-cell mass is a well-known key pathogenic event in diabetes, not only in human subjects with type 1 patients, where β-cells are destroyed by the immune system, but also in type 2 diabetes where reduced β-cell function results in hyperglycemia and associated metabolic abnormalities (1,2). These concepts have made the search for the set of rules that control pancreatic β-cell mass an important area of islet research. An interesting emerging topic with clinical relevance is the notion that the actions of glucagon-like peptide-1 (GLP-1) on islet β-cells could be harnessed to improve and preserve β-cell function and potentially reverse defects in β-cell mass (3). GLP-1 is a proglucagon-derived peptide secreted from gut endocrine cells that acts on β-cells at multiple levels, acutely stimulating insulin secretion while chronically promoting proinsulin biosynthesis and growth and survival of β-cells. During meals, GLP-1 is secreted and acts immediately as an incretin, acutely potentiating glucose-dependent insulin release. However, GLP-1 also enhances glucose competence of β-cells and restores glucose sensitivity to diabetic β-cells in vivo. These findings, taken together with the clinical development of GLP-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 inhibitors, have focused on attention to the extent to which incretin-based agents may exert long-term beneficial effects on preservation of β-cell function in subjects with type 2 diabetes (4).

In an exciting study published in this issue of Diabetes (5), two mechanisms by which GLP-1 causes β-cell replication have been explored. As the authors state, "the overall effect of GLP-1 on increasing β-cell mass in both in vivo and in vitro conditions is relatively small, and augmenting this effect would be beneficial for the treatment or prevention of both type 1 and type 2 diabetes." The goal of the study by Klinger et al. was to elucidate molecular mechanisms of GLP-1–induced proliferation in order to develop new strategies for enhancement of β-cell replication. GLP-1 acts on a G-protein–coupled receptor (the GLP-1R) that activates both cAMP-dependent (6) and MAP-kinase (7) signaling pathways. A remarkable aspect of the new study was that the approach did not consist of simply enhancing known stimulatory signaling events downstream of GLP-1R activation (e.g., by overexpressing the responsible replication-stimulating proteins) in the β-cell; rather, the strategy was to screen for negative regulators of GLP-1R signaling and then attenuate these negative signals to further enhance the pro-proliferative effect of GLP-1 on β-cells. In other words, GLP-1 action reflects a regulated balance of positive and negative signals, and a novel solution to obtain more β-cell proliferation is to attenuate inhibitory signals restraining GLP-1 action.

The experimental approach of the study by Klinger et al. (5) started with a genome-wide screen for genes whose expression is altered in β-cells cultured in the presence of GLP-1. Using microarrays to measure mRNA transcript abundance in β-cells, the authors identified four negative regulators of signaling whose expression is upregulated by GLP-1. Altered expression was confirmed at the protein level, and the GLP-1 induction of these genes was also noted in primary β-cells. The negative regulators identified in this screen included RGS2 (regulator of G protein signaling 2) (8), Dusp14 (dual-specificity phosphatase 14, also called MAP kinase phosphatase 6), a negative feedback regulator of the mitogen-activated protein kinase signaling cascade (9), and Icer (inducible cAMP early repressor) and Crem- (cAMP responsive element modulator), two related cAMP-induced transcriptional repressors (10,11). The effects of these proteins on GLP-1 induction of β-cell replication was studied next using several experimental approaches, including mRNA inactivation via siRNA. Knockdown of mRNA encoding Crem- and Dusp14 enhanced β-cell replication in vitro, following exendin-4 administration, measured as BrdU or [³H]thymidine incorporation. This stimulation of β-cell proliferation was observed in the presence of low and high glucose and was noted after both transient and stable expression of silencing reagents. Importantly, as β-cell lines are quite different from normal β-cells in terms of replication, the same type of effect was observed in primary cultures of rodent β-cells. Final confirmation of the importance of the feedback role of Dusp14 was obtained by expressing a dominant-negative variant of the protein in β-cells: this protein inhibits the normal action of endogenous Dusp14 and augmented the pro-proliferative effect of GLP-1 on β-cells.

The exciting aspect of this study is that it furthers our understanding of the interplay between feed-forward and feedback signals for the control of β-cell mass. Given the importance of cyclic AMP for control of differentiated cell function and survival, it seems likely that the actions of Dusp14 and Crem- may reflect integration of information from a diverse number of receptors that converge on cAMP-dependent signal transduction in the β-cell. Considerable recent progress has elucidated a multiplicity of signals that function to restrain β-cell proliferation. For example, inhibition of cyclin-dependent kinase inhibitors (CKIs), proteins that function as negative regulators of cell cycle progression, results in expansion of β-cell mass and amelioration of experimental murine diabetes (rev. in 12). Moreover, menin, the tumor suppressor implicated in the pathogenesis of multiple endocrine neoplasia, has recently been identified as a prolactin-dependent negative regulator of β-cell proliferation, and modulation of menin expression in β-cells plays a critical role in the capacity for adaptive β-cell growth and replication (13). Similarly, Wnt signaling promotes β-cell proliferation, which is in turn repressed by Axin, a negative regulator of Wnt signaling (14). Furthermore, a panoply of fibroblast growth factor receptors (FGFRs) are expressed in the exocrine and endocrine pancreas, and reduction of FGFR3 expression results in enhanced epithelial proliferation and larger islets, possibly through expansion of islet cell precursors (15). Hence, these observations establish the biological importance of a new class of negative regulators of β-cell growth that may be key components of pathways regulating β-cell growth.

Despite the identification of new therapeutic targets that control β-cell replication, challenges remain for translation of these findings into clinical applications. Indeed, an important unanswered question is whether the proteins identified as critical for growth of rodent β-cells also subserve identical roles in human islets. It has long been appreciated that rodent β-cells exhibit a greater capacity for replication relative to human islets. These concepts have been reinforced by recent studies demonstrating that partial pancreatectomy, a potent stimulus for rodent β-cell replication, has only limited effects on β-cell replication in human subjects (16). Similarly, the extent of ongoing β-cell replication appears quite modest in the pancreas of human subjects with newly diagnosed type 1 diabetes (17). Hence, the remarkable capacity for adaptive replication demonstrated for mouse β-cells remains to be demonstrated for human β-cells, underscoring the importance of testing the biology of positive and negative regulators of β-cell growth in studies with human islets.

A second obvious question to be further studied is whether loosening the biological brakes of β-cell signal transduction is acceptable in terms of safety. Unrestrained β-cell proliferation may be undesirable as it would predispose to tumor formation. Second, some of the proliferation-stimulating effects observed after mRNA knockdown in β-cells in the experiments of Klinger et al. (5) were also seen in the absence of exendin-4, implying that these brakes are not specific for the GLP-1 receptor but used as well for other (still unidentified) stimulatory signals. Third, the same type of molecular brakes seem to operate in other cell types. For instance, Dusp14 dephosphorylates ERK, JNK, and p38 MAPKs in T-cells of the immune system; hence, this enzyme also opposes the action of stimulated T-cell receptors (9). In fact, the Dusp14 phosphatase was discovered in T-cells, where it could effectively suppress T-cell receptor/CD28 costimulation-mediated IL-2 secretion (9). More disturbing in terms of loosening this important general brake, Dusp14 was identified recently as the nonspecific suppressor factor that suppressed delayed-type hypersensitivity induced by intravenous injection of hapten-conjugated syngeneic spleen cells (18). Consistent with the importance of achieving increased numbers of normal functional β-cells capable of navigating in safe waters between diabetes and repeated episodes of hypoglycemia, a balance of signaling events in the immune system is similarly responsible for guarding against infections while preventing autoimmunity. It is clear that these immunomodulatory aspects of Dusp14 need further clarification, particularly with the knowledge that T-cells contribute to the pathophysiology of autoimmune diabetes. Moreover, some of the negative regulators, as appears to be the case for the menin and the CKIs, may exhibit dual roles, as both tumor suppressors and modulators of normal β-cell replication. Hence, understanding the extent to which negative regulators can be safely downregulated is an important area for further study.

In summary, Klinger et al. (5) have opened a new window that illuminates another side of the sophisticated machinery controlling β-cell signaling. The excitement resides in the knowledge that it may bring new perspectives on details of β-cell signaling that are important for the control of β-cell mass. If aspects of safety and specificity can be solved, this could facilitate efforts to expand β-cell replication via the GLP-1 axis. Hence, understanding the exquisite balance between the positive and negative regulators of β-cell growth has important implications for clinical efforts directed at expansion of β-cell mass for the treatment of both type 1 and type 2 diabetes.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

See accompanying Original Article, p. 584.

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This Article Extract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schuit, F. C. Articles by Drucker, D. J. Search for Related Content PubMed PubMed Citation Articles by Schuit, F. C. Articles by Drucker, D. J. Related Collections Related Article Social Bookmarking                What's this?