Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones that control the secretion of insulin, glucagon, and somatostatin to facilitate glucose disposal. The actions of incretin hormones are terminated via enzymatic cleavage by dipeptidyl peptidase-4 (DPP-4) and through renal clearance. GLP-1 and GIP promote β-cell proliferation and survival in rodents. DPP-4 inhibitors expand β-cell mass, reduce α-cell mass, and inhibit glucagon secretion in preclinical studies; however, whether incretin-based therapies sustain functional β-cell mass in human diabetic subjects remains unclear. GLP-1 and GIP exert their actions predominantly through unique G protein-coupled receptors expressed on β-cells and other pancreatic cell types. Accurate localization of incretin receptor expression in pancreatic ductal or acinar cells in normal or diabetic human pancreas is challenging because antisera used for detection of the GLP-1 receptor often are neither sufficiently sensitive nor specific to yield reliable data. This article reviews recent advances and controversies in incretin hormone action in the pancreas and contrasts established mechanisms with areas of uncertainty. Furthermore, methodological challenges and pitfalls are highlighted and key areas requiring additional scientific investigation are outlined.
Incretins are gut-derived circulating peptide hormones that potentiate glucose-dependent insulin secretion following ingestion of a meal. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones. The insulinotropic actions of endogenously secreted GLP-1 and GIP are transient because both peptides are rapidly cleared by the kidney and inactivated by cleavage at the N-terminus by dipeptidyl peptidase-4 (DPP-4), a ubiquitous exopeptidase. Potentiation of incretin action underlines two therapeutic classes of glucose-lowering agents: GLP-1 receptor (GLP-1R) agonists and DPP-4 inhibitors (1). Original concepts of GIP and GLP-1 biology, which focused primarily on islet β-cells, have been expanded to include actions on other cell types within and outside the pancreas (2,3). There is now considerable interest in understanding how the potentiation of incretin action controls multiple facets of pancreatic biology, encompassing the regulation of glucose sensing, hormone secretion, and cell proliferation, differentiation, and survival. Recent studies have suggested that incretin therapies promote pancreatic inflammation as well as aberrant cell proliferation within the endocrine and exocrine pancreas (4,5); substantial technical and methodological issues, however, limit the generalizability of these findings. This Perspectives in Diabetes evaluates the science supporting existing dogma and discusses new concepts, controversies, and uncertainties in the biology of incretin action in the pancreas.
Several dozen commercial antisera are available for detection of GLP-1R and GIP receptor expression by immunohistochemical techniques and Western blotting, and real-time PCR is widely used to quantify expression of incretin receptor genes in pancreatic exocrine and endocrine compartments. Most antisera used to detect GLP-1R expression (by immunohistochemistry or Western blot analysis) are neither sensitive nor specific (6,7). Important control experiments (absorption of the antibody with a peptide epitope, demonstration that the antibody recognizes only a single protein, and failure to generate a signal in cells that do not express a full-length receptor mRNA transcript or in tissues from Glp1r−/− mice) are usually absent. Furthermore, multiple studies describe GLP-1R protein expression in cells or tissues that do not express full-length Glp1r mRNA. The widespread use of tightly cropped bands in Western blot analysis precludes accurate assessment of whether a putative band/protein detected by Western blotting is the correct size, the only GLP-1R immunoreactive protein visualized, or one of several unrelated immunoreactive proteins detected by the same antisera.
Scientists who are interested in the expression of incretin hormone receptors face the challenging task of assessing how much, if any, of the data published with these antisera is correct. For example, immunoreactive GLP-1R protein expression or Glp1r mRNA transcripts have been detected throughout the heart and ventricle; however, we and others determined that cardiac Glp1r expression was restricted to the atria and absent from the ventricles in mice (8) and rats (9). How do the limitations of available reagents affect our understanding of incretin action in the pancreas? The putative localization of incretin receptor expression in the exocrine pancreas provides an instructive example. Abundant immunohistochemical GLP-1R expression in ductal and acinar cells was reported in rodent and human pancreas, papillary thyroid cancer, and pancreatic adenocarcinoma (10,11). Characterization of multiple GLP-1R antisera, including Abcam39072 (11), one of the reagents used in these studies, revealed major problems with sensitivity and specificity. These antisera detected multiple spurious bands in Western blot analyses of fibroblasts that do not express the GLP-1R and in cellular extracts from Glp1r−/− mice (6). We now extend these analyses to detection of the human GLP-1R. Western blot analysis using fibroblasts transfected with human GLP-1R cDNA shows that Abcam39072 does not detect the human GLP-1R (Fig. 1). A second antiserum, distributed by Novus Biologicals (1940002), recognizes the human GLP-1R protein (Fig. 1) but also detects multiple spurious bands/proteins in control cells that do not express the Glp1r (Fig. 1). Similar problems with the sensitivity and specificity of GLP-1R antisera have been described by others (7). Hence the majority of published studies using multiple GLP-1R antisera must be discounted until the experimental data are independently verified with validated, highly sensitive, and highly specific antisera.
Similar concerns relate to the interpretation of some experiments using regular PCR or real-time PCR to detect expression of the incretin receptor gene. Real-time PCR detects Glp1r mRNA transcripts by generating an amplicon of less than 100 base pairs, whereas regular PCR frequently uses primer pairs that generate Glp1r PCR products that are several hundred base pairs in length; both are far smaller than the entire full-length GLP-1R open reading frame. However, cells may generate noncoding mRNA transcripts detectable by regular or real-time PCR. Analysis of Gipr expression revealed ∼64 possible Gipr mRNA splice variants in RNA from human adipose tissue, only two of which were predicted to contain an open reading frame sufficient to give rise to a fully functional, membrane-spanning GIP receptor protein (12). Whether one or more of these variant Gipr RNA transcripts encodes a truncated GIP receptor protein that might exhibit dominant negative signaling activity, as described in mouse β-cells (13), requires further investigation. Furthermore, using a polyclonal antiserum, an immunoreactive GIP receptor protein was detected in human skeletal muscle (12), a tissue not previously reported to express full-length Gipr mRNA transcripts (14). Despite reports describing the detection of 1) partial Glp1r mRNA transcripts by PCR or 2) immunoreactive GLP-1R proteins by Western blotting or immunohistochemistry in murine liver, macrophages, or ventricular cardiomyocytes (2), we could not detect full-length Glp1r mRNA transcripts in the same cells and tissues (6,8).
Given the considerable limitations of commonly used reagents and techniques, how should we interpret available data reporting localization of GLP-1R expression in the endocrine and exocrine pancreas? The difficulty in isolating pure ductal, acinar, or islet cell RNA that is free from contamination by other cell types renders use of such cell fractions suboptimal for the analysis of cell-specific gene expression. Some groups have localized GLP-1R expression in islet α-cells (15); however, analysis of Glp1r mRNA transcripts in RNA from purified murine α-cells and β-cells that were sorted using a fluorescence-activated cell sorter failed to detect Glp1r mRNA transcripts in α-cells (K. Furuyama, P. Herrera, personal communication). Similarly, Glp1r and Gcgr mRNA transcripts were not detected by in situ hybridization in rat or mouse α-cells, respectively (16,17). Although Gipr mRNA transcripts were detected in rodent α-cells (18), less information is available regarding Glp1r or Gipr expression in human α-cells. GLP-1R activation stimulates secretion of islet somatostatin, but whether some, most, or few somatostatin-producing δ-cells express the GLP-1R has not been established. DPP-4 expression at the cell surface has been identified on murine α-cells and β-cells and even more strongly on ductal cells (19); however, whether DPP-4 activity locally regulates bioactive incretin activity within these pancreatic cell types has not been determined.
Glp1r mRNA transcripts have been detected in pancreatic ductal human pancreatic adenocarcinoma cell lines (20). However the GLP-1R agonist exendin-4 failed to stimulate growth or enhance cell survival in five different human pancreatic cancer cell lines that express an endogenous Glp1r mRNA transcript. Whether Glp1r mRNA transcripts are expressed in non-immortalized pancreatic ductal or acinar cells remains uncertain. Tornehave et al. (16) were unable to demonstrate Glp1r mRNA transcripts in pancreatic duct cells from mice and rats by in situ hybridization, despite detection of an immunoreactive protein in ducts using a GLP-1R antibody that was subsequently shown to exhibit suboptimal specificity (6). Transcriptome analysis of human pancreatic endocrine and exocrine cells detected glucagon receptor (Gcgr) expression in ductal cells, but Glp1r expression was not reported (21). Despite immunohistochemical depiction of robust GLP-1R immunopositivity in human pancreatic cancer cells (22), using transcriptome analysis of publicly available databases (oncomine.com, version 4.4.3, and Genome Expression Omnibus, https://www.frankenthalerfoundation.org we have been unable to find evidence that Glp1r mRNA transcripts are overexpressed in these tumors. Similarly, using in situ ligand binding and autoradiography, Körner et al. (23) were unable to detect GLP-1 binding sites in pancreatic adenocarcinomas. New studies using individual endocrine or acinar cells purified by fluorescence-activated cell sorter analysis or isolation of single pancreatic cells using laser-capture microdissection followed by the use of validated antisera and/or PCR analysis using primers that span the full-length Glp1r open reading frame should refine our understanding of the direct cellular targets of GLP-1 action in the pancreas.
The increasing realization that β-cells exhibit considerable functional heterogeneity begs the question of whether there is a gradient of incretin receptor express
