Revisiting the Complexity of GLP-1 Action from Sites of Synthesis to Receptor Activation Brent A

Endocrine Reviews, 2021, Vol. XX, No. XX, 1–32 doi:10.1210/endrev/bnaa032 Review

Review Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 Revisiting the Complexity of GLP-1 Action from Sites of Synthesis to Receptor Activation Brent A. McLean,1,* Chi Kin Wong,1,* Jonathan E. Campbell,2 David J. Hodson,3 Stefan Trapp,4 and Daniel J. Drucker1

1Department of Medicine, Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, University of Toronto, Ontario, Canada; 2The Department of Medicine, Division of Endocrinology, Department of Pharmacology and Cancer Biology, Duke Molecular Physiology Institute, Duke University, 27701 Durham, NC, USA; 3Institute of Metabolism and Systems Research (IMSR), University of Birmingham, and Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK; 4Centre for Cardiovascular and Metabolic Neuroscience, Department of Neuroscience, Physiology & Pharmacology, UCL, London, UK

*B.A.M. and C.K.W. are joint first authors.

ORCiD numbers: 0000-0002-8641-8568 (D. J. Hodson); 0000-0003-0665-4948 (S. Trapp); 0000-0001-6688-8127 (D. J. Drucker).

Abbreviations: AP, area postrema; ARC, arcuate nucleus; CCK, cholecystokinin; cDNA, complementary deoxyribonucleic acid; cAMP, cyclic adenosine 3′,5′-monophosphate; CNS, central nervous system; DPP-4, dipeptidyl peptidase-4; DREADD, designer receptors activated by designer drugs; EAT, epicardial adipose tissue; EC, endothelial cell; ENS, enteric nervous system; EPAC, exchange protein directly activated by cAMP; ER, endoplasmic reticulum; Ex4, exendin-4; Ex9, exendin(9-39); FACS, fluorescence-activated cell sorting; GABA, γ-aminobutyric acid; GAD, glutamic acid decarboxylase; GCG, glucagon gene or protein; GFP, green fluorescent protein; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; GLP-1RA, glucagon-like peptide-1 receptor agonist; HFD, high-fat diet; ICV, intracerebroventricular; IEL, intestinal intraepithelial lymphocyte; IHC, immunohistochemistry; iNKT cells, invariant natural killer T cells; ISH, in situ hybridization; LA, left atrium; LV, left ventricle; Mab, monoclonal antibody; NASH, nonalcoholic steatohepatitis; NG, nodose ganglion; NG2, neural/glial antigen 2; NTS, nucleus tractus solitarius; PK, protein kinase; PLC, phospholipase C; PPG, preproglucagon; PVN, paraventricular nucleus; qPCR, quantitative polymerase chain reaction; RA, right atrium; RV, right ventricle; RNA-seq, ribonucleic acid sequencing; scRNA-seq, single cell RNA sequencing; RT-PCR, reverse transcription-polymerase chain reaction; SA, sinoatrial; SAT, subcutaneous adipose tissue; TCR, T cell receptor; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; YFP, yellow fluorescent protein; WT, wild type.

Received: 9 October 2020; Editorial Decision: 23 November 2020; First Published Online: 15 December 2020; Corrected and Typeset: 15 January 2021.

Abstract Glucagon-like peptide-1 (GLP-1) is produced in gut endocrine cells and in the brain, and acts through hormonal and neural pathways to regulate islet function, satiety, and gut motility, supporting development of GLP-1 receptor (GLP-1R) agonists for the treatment of diabetes and obesity. Classic notions of GLP-1 acting as a meal-stimulated hormone from the distal gut are challenged by data supporting production of GLP-1 in the endocrine pancreas, and by the importance of brain-derived GLP-1 in the control of neural activity. Moreover, attribution of direct vs indirect actions of GLP-1 is difficult, as many tissue and cellular targets of GLP-1 action do not exhibit robust or detectable GLP-1R expression. Furthermore, reliable detection of the GLP-1R is technically challenging, highly method dependent, and subject to

ISSN Print: 0163-769X ISSN Online: 1945-7189 Printed: in USA © The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: [email protected] https://academic.oup.com/edrv 1 2 Endocrine Reviews, 2021, Vol. XX, No. XX misinterpretation. Here we revisit the actions of GLP-1, scrutinizing key concepts supporting gut vs extra-intestinal GLP-1 synthesis and secretion. We discuss new insights refining cellular localization of GLP-1R expression and integrate recent data to refine our understanding of how and where GLP-1 acts to control inflammation, cardiovascular function, islet hormone secretion, gastric emptying, appetite, and body weight. These findings update our know- ledge of cell types and mechanisms linking endogenous vs pharmacological GLP-1 action to activation of the canonical GLP-1R, and the control of metabolic activity in multiple organs. Key Words: diabetes, obesity, receptor, islets, brain, gastrointestinal tract, cardiovascular Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

Graphical Abstract

Mechanisms of GLP-1 action

GLP1 receptor

GLP-1

ESSENTIAL POINTS • Plasticity of the endocrine pancreas enables the capacity for local GLP-1 production • The β-cell GLP-1R receives signals from both GLP-1 and glucagon to regulate glucose-stimulated insulin secretion • Detection of the GLP-1R in multiple cell types is challenging due to low levels of expression and technical valid- ation of reagents • Brain-derived GLP-1 is synthesized in the brainstem and widely distributed to multiple GLP-1Rs throughout the central nervous system • GLP-1Rs within the central nervous system may be engaged by both CNS-derived and systemically circulating GLP-1 and GLP-1R agonists • The intestinal L cell network is the predominant contributor to circulating GLP-1 • GLP-1 receptor agonists exert nonmetabolic actions in multiple cells and tissues that do not express the canonical GLP-1R Endocrine Reviews, 2021, Vol. XX, No. XX 3

The gut endocrine system contains dozens of specialized and not gut-derived GLP-1, as a key contributor to physio- plurihormonal endocrine cells whose peptide hormone logical GLP-1R-dependent signals regulating appetite and products regulate a wide range of biological functions, body weight. ranging from control of ingestive behavior to the diges- Similarly enigmatic is the challenge of understanding tion, absorption, and assimilation of nutrients (1). Among the actions of GLP-1 and degradation-resistant GLP- the best studied of the gut hormones is glucagon-like 1RA on organs and cell types that do not express the peptide-1 (GLP-1), a product of the proglucagon gene GLP-1R. For example, GLP-1RA reduce the rates and

(GCG). The cloning of the proglucagon cDNAs encoding extent of myocardial infarction in humans and in pre- Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 GLP-1 within the GCG gene in the early 1980s (2, 3), clinical studies (15-17), yet it remains challenging to enabled the study of the physiology of the glucagon-like detect GLP-1R protein expression in the left ventricle peptides. Originally described as an incretin hormone of the heart, the most common site of infarction (18). that potentiates meal-stimulated insulin secretion, sev- Moreover, GLP-1RAs reduce hepatic fat accumulation eral decades of research have broadened the actions of and are under clinical investigation for the treatment of GLP-1 beyond the endocrine pancreas to encompass ac- nonalcoholic steatohepatitis (NASH), yet hepatocytes do tivity within the nervous system, gut, kidney, pancreas, not appear to express a functional canonical GLP-1R heart, and immune system (4-6). Moreover, the clin- (19-21). Moreover, GLP-1RA rapidly reduce the levels ical development of GLP-1R agonists (GLP-1RAs) for of postprandial triglycerides in animals and humans, yet the treatment of type 2 diabetes (T2D) and obesity has enterocytes do not exhibit GLP-1R expression or ac- heightened interest in how physiological and pharmaco- tivity. These and related examples highlight how multiple logical levels of GLP-1 act on key tissues relevant to the pharmacological actions of GLP-1 and GLP-1RA are pathophysiology of diabetes and its complications. highly conserved across species, yet difficult to explain A major advance in understanding GLP-1 action mechanistically based on the known expression patterns stemmed from the cloning and characterization of the of the GLP-1R. GLP-1 receptor (GLP-1R) (7). GLP1R mRNA transcripts Several technological advances have enabled were found to be widely distributed in peripheral tissues re-examination of these questions, refining our under- and the central nervous system (CNS), providing a logical standing of where and how GLP-1 is synthesized and link between the demonstrated actions of GLP-1 and the acts to originate GLP-1R-dependent signals in multiple expression of the canonical GLP-1R in cellular targets organs. Similarly, new tools have enabled more sensitive of GLP-1 action (8-10). Nevertheless, the precise cellular and reliable detection of GLP-1R expression. These in- localization of GLP-1R has proved to be challenging, re- clude single cell RNA sequencing (scRNA-seq), reporter flecting low levels of mRNA expression, and multiple tech- mice illuminating cellular sites of Glp1r transcriptional nical challenges, including the inadvertent application of activity, more sensitive and specific validated GLP-1R incompletely characterized nonspecific antisera for detec- antisera, advanced microscopy, and new chemical probes tion of GLP-1R protein expression (11). for enhanced detection of GLP-1R binding sites at single The concept of GLP-1 as a gut-derived hormone acting cell resolution. on peripheral organs, has been refined through enhanced Here we re-evaluate existing concepts of GLP-1 action, understanding of the importance of pancreatic- and brain- highlighting insights into established and emerging con- derived GLP-1. GLP-1 circulates at very low (picomolar) cepts illuminated by recent progress in the evaluation of levels, is immediately inactivated by local gut and systemic GLP-1 production and in the expression and activity of expression of dipeptidyl peptidase-4 (DPP-4) (12, 13), the canonical GLP-1R. The sites of GLP-1 synthesis within and is rapidly cleared from the circulation by the kidney. the gastrointestinal tract, the brain, and the pancreas are Contemporaneously, evidence supporting the pancreatic described, and linked where possible, to actions of GLP-1 production of GLP-1 has fostered the competing notion within these organs, which are transduced by the canonical that the incretin activity of GLP-1 reflects local GLP-1 GLP-1R. We focus on discussing established and emerging synthesis in α-cells, coupled to paracrine actions on adja- concepts of how circulating GLP-1 produces multiple ac- cent GLP-1R+ β-cells (6). Moreover, notwithstanding the tions, both directly and indirectly, on multiple organs and reduction of food intake and weight loss observed with cell types with variable GLP-1R expression. Collectively, pharmacological administration of GLP-1RAs, inactiva- these findings update established and emerging concepts of tion of gut GLP-1 production does not perturb control of GLP-1 action, and highlight mechanistic controversies sur- food intake or body weight (14), consistent with consider- rounding GLP-1 action on cell types that do not express able evidence implicating local brainstem-derived GLP-1, the GLP-1R. 4 Endocrine Reviews, 2021, Vol. XX, No. XX

The Pancreas GLP1R/Glp1r mRNA expression in different islet GLP-1 production, GLP-1R expression, and cell types GLP-1R signaling in the pancreas Prior to the development of validated antibodies for the GLP-1R, the expression patterns of Glp1r/GLP1R in islet Is pancreas-derived GLP-1 relevant for islet function? cells were derived from analysis of ligand binding and RNA An ongoing debate centers on whether proglucagon pro- levels. The GLP-1R cDNAs were originally cloned from cessing in α-cells is capable of producing meaningful rat (7) and human (32) pancreas, and later shown to be amounts of bioactive GLP-1 to influenceβ -cell func- expressed in the mouse pancreas by reverse transcription- Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 tion and glucose tolerance. Early reports demonstrated polymerase chain reaction (RT-PCR) (10). Currently, islets/ increased α-cell expression of Pcsk1, the prohormone pancreas samples are often used as a positive control when processing enzyme required for GLP-1 production, in re- exploring Glp1r/GLP1R expression in other tissues (18). sponse to pancreatic injury or induction of experimental However, it is continuously debated which endocrine cell pancreatic inflammation (22-26), raising the hypothesis population within the islet express the GLP-1R. Multiple that α-cells could adapt to metabolic stress by increasing strategies have been deployed to identify Glp1r/GLP1R+ GLP-1 production. Active GLP-1 was detected, albeit at cell populations, including transcriptional analysis of sorted low levels relative to corresponding levels of glucagon in islet populations. Using genetic reporter lines to enrich for the same extracts, from both mouse and human pancreas populations of -, -, and -cells, Glp1r was shown to be (14). However, induction of metabolic stress in high-fat α β δ robustly expressed in mouse -cells, moderately expressed diet (HFD)–fed mice did not alter the levels of GLP-1 in β in -cells, and expressed at low to undetectable levels in the pancreas (14). Genetic deletion of Pcsk1 to eliminate δ -cells (33). These data align with a study employing bulk prohormone convertase 1 (PC1) reduced islet content and α RNA-seq to analyze enriched cell populations obtained secretion of GLP-1 by ~40%, leading to modest impair- from reporter mice, showing Glp1r to be highly expressed ment of glucose intolerance in obese mice (25), reinforcing in β-cells, less so in δ-cells, but absent in α-cells (34). the key role of PC1 in α-cell GLP-1 production. However, An alternative approach to identifying islet Glp1r lo- analyses using the mouse perfused pancreas and a spe- calization utilized mice expressing Cre recombinase under cific assay to detect active, fully processed GLP-1 reported control of the Glp1r promoter. Using Glp1r-Cre mice mostly undetectable levels of active GLP-1 in the per- crossed with a fluorescent reporter line, cells are indelibly fusate, arguing that α-cell GLP-1 production in the normal marked in response to Glp1r transcription (35). Such ap- murine pancreas is negligible (27). On the other hand, the proaches are of high fidelity, since only a few molecules GLP-1R antagonist exendin(9-39) (Ex9) did not impair –/– of Cre are required to excise the stop cassette and drive glycemic excursion in Gcg mice with preserved intes- reporter protein expression. A downside of this approach tinal GLP-1 production, yet selective rescue of pancreatic –/– is the potential disconnect between transcription and trans- Gcg expression in Gcg mice, together with Ex9 adminis- lation, although this is less likely in light of newer data (see tration, implicated pancreatic GLP-1 in the control of glu- below). Moreover, relative levels of GLP-1R cannot be de- cose homeostasis in this mouse model (28). Complicating termined between and within cell types, since reporter ex- this interpretation are findings that glucagon, produced pression is generally driven using a Rosa26 promoter. at much higher levels in the islet, is also capable of stimu- The first study to utilize this approach to investigate lating insulin secretion and controlling glucose homeo- Glp1r promoter activity in islets reported strong activity stasis through the GLP-1R (27, 29-31). Consequently, in both β- and δ-cells, although Glp1r levels were ~10- paracrine interactions between α- and β-cells mediated fold higher in the β-cell population (35). Subsequent by GCG peptides and the GLP-1R could be completely analysis of α-cell populations with double reporter mice accounted for by glucagon secretion. Interestingly, while (Glp1r-Cre:ROSA26-tdRFP:GLU-Venus) produced an the levels of glucagon secreted from isolated mouse islets estimate that 9.5% of α-cells are Glp1r+. Interestingly, are ~300-fold greater than GLP-1, the potency of GLP-1 the fraction of Glp1r+ α-cells also contained signifi- for insulin secretion is ~300-fold more than glucagon cantly more Ins1 expression than Glp1r– α-cells. Could (31). Finally, if α-cells produce active GLP-1, this source these represent a subpopulation of unique α-cells that does not contribute to the circulating concentrations in are GLP-1 responsive? Do they arise from dedifferenti- the plasma as mice with intestine-specific inactivation of ated β-cells, explaining the presence of both Glp1r and the Gcg gene exhibit almost undetectable levels of active Ins transcripts? Or do these results arise from contamin- circulating GLP-1 (14). Figure 1A summarizes paracrine ation of a non–α-cell population? The importance of this GLP1 signaling in the islet. Endocrine Reviews, 2021, Vol. XX, No. XX 5 Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

Figure 1. GLP-1 source, GLP-1R labeling and GLP-1R signaling in pancreatic islets. (A) Potential sources of GLP1 in the islets, as well as paracrine signaling pathways. Proglucagon products from α-cells that activate islet GLP-1Rs include glucagon and potentially GLP-1. The biological relevance of GLP-1 produced by α-cells remains an unresolved line of investigation. (B) GLP1R labelling is observed at the membrane in β-cells. Predominantly cytoplasmic labelling is nonspecific unless the GLP1R is agonist-bound in which case some surface and punctate intracellular staining is detected due to GLP1R internalization. (C) Major GLP1R signaling pathways reported in primary rodent and human beta cells or islets. It is well established that GLP-1R signals through cAMP pathways, including PKA and EPAC2. β-arrestin 1 has been proposed to regulate GLP-1R signaling; however, the mechanisms of this pathway remain unresolved. (D) Actions mediated by the GLP-1R in pancreatic islets. ADCY, adenylate cyclase, GLP1R, Glucagon- like peptide-1 receptor; PKA, protein kinase A; EPAC2, exchange protein directly activated by cAMP 2; INS, insulin SST, somatostatin. subpopulation of α-cells remains unclear. A second study assess for potential heterogeneity in expression within a examined Glp1r activity in islet cell populations using an particular cell population. For instance, in samples from independent Glp1r-Cre mouse crossed with the mTmG human islets, GLP1R mRNA was robustly detectable reporter mouse line (36). The expression of Ins and Sst in β- and δ-cell populations, with very few GLP1R+ α- were found in the Glp1r+ population (GFP+), while cells (37), largely aligning with the results of cell popula- Gcg expression was exclusive to the Glp1r– population tion analysis from transgenic mice described above (Fig. 2). (TdTomato+). This data set indicates little to no Glp1r However, it was interesting to note the considerable range expression in α-cells, conflicting with previous reports, in expression levels between individual β-cells (and δ- but this approach cannot rule out a small population of cells), particularly since a significant portion of individual α-cells being Glp1r positive. cells in either population were reported to be GLP1R–. scRNA-seq has been utilized for interrogation of the Heterogeneity in β-cell GLP1R/Glp1r expression was Glp1r expressing cell population within islets, and to common among all human and mouse scRNA-seq data 6 Endocrine Reviews, 2021, Vol. XX, No. XX Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

Figure 2. Expression of GLP1R in 9940 human pancreatic islet endocrine cells. The UMAP (Uniform Manifold Approximation and Projection) plots were created to visualize gene expression in each cell. The x-axis and y-axis indicate the overall transcriptional difference of each cells arbitrated as “distance” in a 2-dimensional space; the closer the 2 cells on the plot, the more similar their transcriptomes are, and in turn they are more likely to share a lineage. The expression scales are continual and log2 normalized, ranging from light blue (low expression) to dark red (high expression) in color, Cell types were classified based on markers genesINS and MAFA (mature β cells), GCG (α cells), SST (δ cells), and PPY (PP cells). Count matrices were accessed on September 19, 2020, and aggregated from the scRNA-seq datasets reported by Segerstolpe et al. (37) (GEO accession number GSE73727), Enge et al. (220) (GEO accession number GSE81547), and Camunas-Soler et al. (38) (GEO accession number GSE124742). Human gene nomenclature: INS, insulin; GCG, glucagon; SST, somatostatin; PPY, pancreatic polypeptide Y; MAFA, V-maf musculoaponeurotic fibrosarcoma oncogene homologue A. analyzed (38), indicating this outcome is consistent des- expressed in the β-cell compartment, with no detectable pite the varied platforms used to obtain these data. It is GLP-1R protein or mRNA in α-cells and δ-cells (41, 42). exciting to imagine heterogeneity in the relative expres- Comparable results were obtained by an independent sion of GLP1R/Glp1r within specific cell populations, as group using 2 commercial antibodies (42) although one this could reconcile the reports that only a fraction of of the reagents was subsequently shown to lack specificity α-cells are GLP-1R+ and potentially contribute to the on- for the GLP-1R (43). All of these early studies detected going discussion of β-cell heterogeneity (38, 39). However, GLP-1R in the β-cell membrane as well as in the cyto- the varying levels of GLP1R/Glp1r transcripts in different plasm. While this could reflect the imaging technique used, cells was subsequently proposed to reflect technological cytoplasmic localization could also reflect nonspecific limitations associated with scRNA-seq, due to the signifi- signal, since GLP-1R should be present predominantly at cant drop out of low-expressing genes during the process the plasma membrane in its unstimulated state (Fig. 1B). of generating the cDNA library (39). While a validated monoclonal antibody (Mab 7F38) has been reported which displays a clean membrane signal (44), it has not yet been used to extensively re-examine GLP-1R protein expression and localization in the GLP-1R cellular localization in mouse islets. Recent rodent islet studies using a novel antibody raised against the GLP-1R Multiple approaches have localized GLP-1R in pancreatic N-terminal region, and validated using a new floxed GLP- islets. Early studies using immunohistochemistry (IHC) 1R-null reporter mouse line, detected GLP-1R throughout to examine rat pancreas showed immunoreactivity for the β-cell and α-cell cytoplasm (45). The reason for this GLP-1R in the majority of β-cells and δ-cells, with 1 in discrepancy in the expression of the α-cell GLP-1R across 5 α-cells also expressing the receptor, in line with single studies is unclear, but might reflect the use of antibodies cell RT-PCR findings (40). Later studies using the same raised against different GLP-1R epitopes (eg, N-terminus rat antibody showed that GLP-1R was predominantly or the wider extracellular domain). Endocrine Reviews, 2021, Vol. XX, No. XX 7

A second approach to detect GLP-1R in islets re- in β-cells and δ-cells, but not in α-cells from human lies upon orthosteric agonists or antagonists con- pancreata resected from individuals with insulinomas, jugated to various fluorophores (Cyanine 3 and 5, chronic pancreatitis, or pancreatic cancer (51). Most re- tetramethylrhodamine, silicon rhodamine) (46-48). cently, studies using the same antibody combined with Inherent advantages of this approach include visualiza- confocal imaging showed that GLP-1R is expressed tion of GLP-1R capable of ligand binding and activation in most β-cells and very few α-cells (52). Of interest, and live tissue imaging, which reduces background signal GLP-1R was shown to be moderately expressed in som-

introduced by fixation. However, because peptides and atostatin+ cells (ie, δ-cells) (52). Thus, based upon IHC, Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 some fluorophores are cell impermeable, GLP-1R already GLP-1R expression patterns in human reflect those seen within the cell will not be readily detectable. Studies using in the mouse, but with some immunoreactivity also de- fluorescent agonists reported similar findings to those tected in δ-cells (the majority of mouse studies did not with monoclonal antibody: GLP-1R expression largely quantify somatostatin+ cells). confined to theβ -cell compartment, with expression in a small subpopulation of α-cells (~1%) (48). Along these lines, well characterized fluorescent antagonists revealed predominant GLP-1R expression in β-cells (~85%), with Tying together GLP-1R transcript and protein slightly higher expression in α-cells (~5%) than shown levels in the same cell with fluorescent agonist or antibody (49) but in agree- While the measurement of Glp1r mRNA transcripts is not ment with Glp1r-Cre:ROSA26-tdRFP:GLU-Venus burdened with the limitations present when assessing pro- double reporter mouse approaches (35). Notably, for all tein levels based on current approaches, extending Glp1r fluorescent ligand localization approaches, staining over- transcript abundance to protein is a necessary step to iden- lapped with data obtained using IHC with antibodies tify cells that permit GLP-1 activity. However, there ap- validated in Glp1r–/– mouse tissue (48, 49). pears to be significant discordance between detection of gene and protein expression of the GLP-1R in mouse islets (36). These observations stemmed from sequential efforts GLP-1R protein expression and localization in the to first validate a novel GLP-1R antibody (53) in Glp1r–/– primate and human islet mice to demonstrate specificity for the GLP-1R in dispersed Most of the studies investigating GLP-1R distribution in islets cells during flow cytometry. Next, this antibody was primate and human islets have used IHC. Using a validated applied to Glp1r-Cre:mTmG reporter mice to compare monoclonal antibody (Mab 3F52), GLP-1R colocalized Glp1r promoter activity with GLP-1R protein levels. The with insulin-positive cells in the monkey islet (44). antibody failed to stain any TdTomato+ cells (Glp1r–), Conversely, less colocalization was detected in glucagon- enhancing the validity of the 2 reagents (36). This popu- positive cells, with the observed overlap attributed to cyto- lation of Glp1r– cells were enriched for Gcg, marking this plasmic extensions from β-cells (44). However, given the population as α-cells. Remarkably, 2 populations emerged axial resolutions needed to accurately separate cell mem- with respect to GLP-1R staining when examining the GFP+ branes, further studies will be required to exclude GLP-1R cells (Glp1r+). The population of GFP+ cells that stained expression in monkey α-cells. positive with the antibody were enriched for Ins2 expres- Early studies looking at human islets showed strong sion, while the population of cells that stained significantly colocalization of GLP-1R with β-cells, with no staining lower with the antibody were enriched for Sst (36). This seen in α-cells (41). Similarly, studies using a commer- suggests that β-cells have robust GLP-1R protein levels, cial antibody confirmed these results, showing GLP-1R and that δ-cells have much less GLP-1R. These findings immunopositivity in 0.25% of α-cells (50). It is worth were corroborated in experiments in wild-type (WT) islets, noting however that the antibodies used in both these where the GLP-1R antibody robustly stained β-cells, but studies gave rise to predominantly cytoplasmic staining, failed to detect protein expression in either α- or δ-cells. at odds with the known localization of nonstimulated The potential discrepancy between RNA and protein ex- GLP-1R. Studies using a validated antibody with pression in δ-cells warrants further investigation to elim- membrane-localized staining again showed GLP-1R ex- inate the potential contribution of technical limitations, pression largely confined toβ -cells, but staining was not especially given the relative paucity of this cell type. Finally, performed for glucagon, making α-cell quantification im- the majority of β-cells express the GLP-1R and this per- possible (44). Consistent with these observations, Waser centage was not changed by various stressors reported to and colleagues utilized the Mab 3F52 antibody and de- modulate Glp1r levels, indicating little plasticity in expres- tected membrane-localized GLP-1R immunopositivity sion of the β-cell GLP-1R. 8 Endocrine Reviews, 2021, Vol. XX, No. XX

Consensus view of islet GLP-1R expression adenosine 3′,5′-monophosphate (cAMP) accumulation fol- Given the number of studies, what is the consensus view lowing ligand activation, with nanomolar concentrations of GLP-1R expression in the islet? It is clear that β-cells of GLP-1/exendin-4 (Ex4) acting through cAMP and ex- abundantly express GLP-1R (80-95%), while the receptor change protein directly activated by cAMP (EPAC) path- is absent or present in a very small proportion in α-cells ways in β-cells (56). cAMP acts as a potent stimulus of (0-20%) (35, 36, 40, 41, 48, 49). δ-Cells are less well char- insulin secretory granule exocytosis by increasing voltage- acterized and although some analyses reveal expression of dependent Ca2+ channel activity, Ca2+ influx, as well as dir-

the Glp1r transcript in these cells (33, 34), analyses at the ectly sensitizing vesicles themselves for release (57, 58). Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 protein level are less firm showing little to no GLP-1R ex- While cAMP constitutes the major mediator of GLP-1R pression (36, 41, 46, 49). These figures should however be signaling, contributions from β-arrestin are more debated. interpreted with some limitations. Quantification based On one hand, both β-arrestin 1 (59) and β-arrestin 2 (60, upon IHC is notoriously challenging. Indeed, it can be dif- 61) have been shown to be dispensable for acute stimu- ficult to assign GLP-1R expression to adjacent cells whose lation of insulin secretion by GLP-1/GLP-1RA. However, membranes overlap (as is often the case for α-cells, β-cells, other studies have shown that β-cell knockout of Arrb1 and δ-cells) (49). Nonspecific background signal in cells ex- enhances insulin secretion in response to GLP-1, sug- pressing low levels of GLP-1R is problematic, and differ- gesting β-arrestin 1 limits GLP-1R signaling in β-cells ences in sensitivity/settings between different microscopes (62). The differing concentrations of GLP-1 used in these means that results are difficult if not impossible to calibrate experiments could reconcile the discrepant results in the across studies (compared with RNA-seq and fluorescence- β-arrestin 1 knockout models, as high concentrations of activated cell sorting [FACS] which are more standardized). GLP-1 (100 nM) (59) may elicit different signaling path- On the other hand, visualizing GLP-1R expression in the ways compared with GLP-1R activation with lower con- intact tissue avoids artefacts introduced by cell dissociation centrations (0.3 nM) (62). Moreover, the absence of a as well as removal of cells from the tissue setting. Moreover, phenotype in the single β-arrestin knockout models (59-61) utilizing FACS to isolate endocrine cell populations based could equally reflect the presence of redundancy in the on primary hormone expression can mask heterogeneous GLP-1R signaling network and as such studies in double Glp1r expression in subpopulations of cells. For instance, β-arrestin 1/2 knockout mice are needed. The major if less than 10% of α-cells are Glp1r+, the remaining 90+% GLP-1R signaling pathways in rodents and human β-cells of α-cells in the binned sample may dilute this signal to are shown in Fig. 1C. yield the interpretation that α-cells are Glp1r–. Thus, com- Activation of GLP-1R by picomolar concentrations of bining multiple approaches to interrogate GLP-1R expres- GLP-1 has been demonstrated in mouse and human β-cells sion remains necessary to account for the limitations of (63, 64). Rather than engaging cAMP synthesis, low con- a single approach. Table 1 summarizes GLP-1R transcript centrations of GLP-1 instead activate a phospholipase C and protein levels based on the studies outlined above. (PLC)-protein kinase C (PKC)-Transient Receptor Potential Cation Channel Subfamily M Member 4/5-signaling cascade through the Gqα subunit, stimulating insulin se- Where and how does GLP-1 act in the islet? cretion (63). Low concentrations of GLP-1 also increase β-Cells Ca2+ fluxes in humanβ -cells, which fits with a role for The molecular pharmacology of GLP-1R signaling and inositol 1,4,5-trisphosphate 3–dependent mobiliza- trafficking has been extensively studied in heterologous tion of endoplasmic reticulum (ER) Ca2+ stores (inositol expression systems and is reviewed in detail elsewhere 1,4,5-trisphosphate 3 being downstream of PKC) (63, (54, 55). Here we briefly cover facets of GLP-1R signaling 64). Most recently, GLP-1R has been shown to signal pre- pertinent to insulin release from primary β-cells where dominantly via Gqα in mouse and human β-cells subject the investigation of the sites and mechanisms of GLP-1R to chronic depolarizing stimuli (KATP channel mutations, signaling remain less well characterized. Activation of hyperglycemia) (65). Since several of these experiments the GLP-1R strongly induces adenylate cyclase and cyclic used chemical inhibitors with known off-target effects (eg,

Table 1. Relative GLP1R transcript and protein levels in the islet endocrine cell types based upon(33-36, 40, 41, 46, 48, 49)

Cell type Glp1r/GLP1R GLP1R

β-cell +++; robust and ubiquitous expression +++; robust and ubiquitous expression, established direct GLP-1 activity α-cell +/–; little to no expression +/–; little to no expression, direct GLP-1 activity is debated δ-cell ++; ubiquitous, but lower expression relative to β-cells +/–; little or no expression, evidence for direct GLP-1 activity is minimal Endocrine Reviews, 2021, Vol. XX, No. XX 9 on store-operated Ca2+ entry, phosphodiesterases), it will δ-Cells be important to examine whether similar phenotypes exist Relatively few reports demonstrate direct actions of GLP-1 in β-cells specifically deleted or knocked down for Gqα or on somatostatin secretion. Studies using the perfused mouse Gsα. Lastly, GLP-1 also engages a network of β-cells in pancreas preparation showed that GLP-1 lowers glucagon human islets, leading to coordinated Ca2+ rises (64). Such secretion in a paracrine manner via an increase in somato- signaling is dependent on intercellular gap junction commu- statin output (which negatively regulates α-cell function) nication, which Ex4 has been shown to facilitate in a cAMP (68). Notably, the action of GLP-1 to inhibit glucagon se-

protein kinase (PK) A-EPAC–dependent manner (66). cretion was prevented by blocking somatostatin receptor Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 2, which is expressed on human α-cells and β-cells (69). α-Cells Similar results have been reported using the perfused rat The hypothesis that GLP-1 directly affects α-cell func- pancreas (70). Such paracrine regulation of glucagon secretion remains controversial. Despite evidence supporting tion by GLP-1 likely requires an intact microvasculature, GLP-1R expression in only a small subpopulation since the somatostatin receptor antagonist CYN154806 did of α-cells, studies have shown that GLP-1 influences not block the glucagonostatic effects of GLP-1 in isolated α-cell signaling and glucagon secretion by inhibiting N mouse islets (42). It remains unclear how GLP1 increases and P/Q voltage-dependent Ca2+ channels (42, 45, 50). somatostatin secretion in mouse δ-cells given the low levels α-cells could plausibly express low levels of GLP1-R, of GLP-1R protein detected following FACS to isolate islet which would be difficult to detect using immunostaining, cells (see above). The expression and functional importance yet sufficient to evoke a response in a highly ampli- of the GLP-1R in δ-cells in the intact pancreas, and the con- fied signaling cascade. However, low abundance Glp1r sequences arising from conditional deletion of the GLP-1R mRNA transcripts should still lead to recombination in the δ-cell compartment requires further analysis. in the various GLP-1R-reporter mice discussed above, where somewhere between 0% (36) and 10% (35) of α-cells were labelled with reporter. Moreover, the most GLP-1R expression and function in the exocrine recent fluorophore antagonist approaches have single- pancreas molecule resolution and detected GLP-1R in only 1% to Interest in the roles of GLP-1R expression and activity in 5% of α-cells, with either complete absence or strong pancreatic acinar cells was heightened by the hypothesis signal, suggesting that graded expression in the popula- that the clinical use of GLP-1RA might be linked to the de- tion is unlikely (49). velopment of pancreatitis or pancreatic cancer (11). Indeed, Another explanation for the actions of GLP-1 on GLP-1RAs such as exenatide or liraglutide increase pancre- α-cells could be through GLP-1 degradation products. atic mass and protein synthesis independent of changes in Several reports suggest that GLP-1 (7-36) and GLP-1 (9- cell proliferation in mice (71). Moreover, native GLP-1 dir- 36) are glucagonostatic in WT and Glp1r–/– islets (67). ectly increases pancreatic enzyme secretion from and cAMP Effects of GLP-1 (7-36) but not GLP-1 (9-36) were abol- formation in mouse pancreatic acinar cells (72), actions re- ished by pretreatment with a DPP-4 inhibitor, suggesting quiring the canonical GLP-1R. The Glp1r mRNA transcript that GLP-1 (7-36) is a propeptide for GLP-1 (9-36) in is detectable in RNA isolated from mouse pancreatic acini this setting (67). Notably, GLP-1 (9-36) was found to (72), and weak variable GLP-1R immunopositivity was signal at the human glucagon receptor with a median ef- detected in pancreatic acinar cells, but not in ductal cells, fective concentration of 30 nM. While such concentra- with the Mab 3F52 antibody in sections from pancreata tions are pharmacological, GLP-1 (9-36) is the major obtained from monkeys with or without diabetes (44). circulating form of GLP-1 and DPP-4 is present in is- Similar patterns of acinar cell GLP-1R-immunopositivity lets, which could plausibly generate high local levels of were detected in pancreatic specimens obtained at sur- cleavage products. These findings are however difficult gery from 10 nondiabetic individuals, as well as in 12 to reconcile with previous studies showing that the GLP- pancreatic specimens from individuals with T2D. In situ 1R-specific antagonist Ex9 (27) blocks the effects of ligand binding detected GLP-1 binding sites in the pan- GLP-1 on α-cell electrical activity and glucagon secretion creatic acinar cells, with radiolabeled ligand displaced in (42). Although interesting, these data are unlikely to be the presence of excess unlabeled GLP-1 (44). Waser and relevant for degradation-resistant GLP-1RA and DPP-4 colleagues examined pancreatic GLP-R expression in the inhibitors, which suppress glucagon secretion without human pancreas by IHC using the Mab 3F52 antibody, generating GLP-1 (9-36) through mechanisms requiring as well as by in situ ligand binding autoradiography using the known GLP-1R. 125I-GLP1(7-36)-amide (51). A combination of both diffuse 10 Endocrine Reviews, 2021, Vol. XX, No. XX and heterogeneous weak GLP-1R expression was observed observation that GLP-1 immunoreactivity was localized in some but not all acini from pancreatic samples resected to axon terminals in the hypothalamus further solidified from insulinoma tumors, chronic pancreatitis, and a few the view that GLP-1 -producing cells in the brainstem cases of pancreatic cancer. No immunoreactive GLP-1R ex- are neurons and utilize GLP-1 as a neurotransmitter that pression was detected in pancreatic ducts (51). Similar pat- is stored until release in the axon terminals. A first proof terns of acinar cell GLP-1R localization were reported in that this assumption is correct was provided by the obser- the mouse pancreas, using the 125I-labeled GLP-1R antag- vation that injection of a virus encoding shRNA against

onist Ex9 to detect in situ binding, as well as the Mab 7F38 preproglucagon (PPG) into the nucleus tractus solitarius Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 antibody to localize GLP-1R immunopositivity in the exo- (NTS) causes a decrease in GLP-1 immunoreactivity crine pancreas of WT but not Glp1r–/– mice (73). Hence, the in the paraventricular nucleus (PVN) of the hypothal- available data support low level GLP-1R expression in the amus (82). Functional studies in the rat combining pancreatic acinar but not ductal cells in mice and humans. immunohistochemical identification of these neurons with c-Fos immunodetection revealed their activation by malaise, stress, and gastric distension and linked them to the Concluding Remarks for GLP-1R Expression suppression of food intake (83-86). and Function in the Pancreas The GLP-1-producing neurons are also called PPG Actions mediated by GLP-1 in the islet are shown in neurons or GCG neurons. For the purpose of this review, Fig. 1D. The major source of GLP-1 that binds GLP-1R the term PPG neurons will be used, independent of spe- in the healthy islet is still unresolved, although GLP-1 pro- cies, to describe the neurons that produce GLP-1 within the CNS. These are largely equivalent cell populations in duced in α-cells may play a relatively more important role in the injured pancreas. Insulinotropic effects of GLP-1 higher mammals (87, 88). CNS GLP-1 expression has also and GLP1-RA are mediated by GLP-1Rs expressed pre- been described in other vertebrates, such as fish and bird, but specific accounts of PPG neurons in their brainstem are dominantly (if not completely) in the β-cell compartment in both mouse and human islets, assessed using validated lacking (89, 90). antibodies, probes and reporter mice across a range of The ability to interrogate the function of the CNS GLP-1 system was enabled by development of transgenic techniques. Direct effects of GLP-1 on α-cell function and glucagon secretion remain debated and are unlikely to be mice targeting the GLP-1-producing neurons using en- mediated by the GLP-1R. Instead, degradation of GLP-1 by dogenous Gcg promoter sequences (91). The first mouse DPP-4 may yield truncation products that act upon GCGR strain allowed fluorescent visualization of these cells in vitro and thus electrophysiological studies of these expressed in α-cells. These findings are however unlikely to be pertinent for the glucagonostatic actions of most GLP- neurons in brain slices (92). Additionally, the strong ex- 1RA, which are largely completely DPP-4-resistant. While pression of yellow fluorescent protein (YFP) in the cytoplasm of PPG neurons allowed visualization not only of GLP-1 increases somatostatin secretion, δ-cells likely express GLP-1R at low abundance and indirect effects cannot the cell body and terminals of cells enabling Gcg tran- be excluded. Finally, GLP-1R is present in the exocrine scriptional activity, but of the entire dendritic and axonal pancreas, localized to the acinar cells, where GLP-1 acts structure of these cells. These studies provided further to increase digestive enzyme release. Table 2 lists validated characterization of the main population of PPG neurons antibodies and probes for specific GLP1R detection. in the NTS and in the intermediate reticular nucleus (IRT), and the population in the olfactory bulb that was previously detected by in situ hybridization (ISH) (93- The Brain GLP-1 System 95) (Fig. 2). Additional PPG neurons were located in the piriform cortex and in the lumbar–sacral spinal cord Where does brain GLP-1 originate from? (93, 96). Widespread projections throughout the brain Proglucagon (GCG) mRNA transcripts are detected pre- were confirmed in unprecedented detail by revealing not dominantly in the brainstem (74, 75), whereas the glucagon- only terminal fields, that can be visualized with GLP-1 like peptides (including GLP-1), and the GLP-1R are more immunoreactivity, but also axons in passage. Importantly, widely distributed throughout the CNS (10, 75-80). Further these transgenic mice established that hippocampus and characterization of the GLP-1 immunoreactive cells in the most areas of the cortex do not receive GLP-1 projections, lower brainstem solidified the view that these cells are a findings with implications for understanding the mech- specific population of neurons, rather than glial cells or anisms of neuroprotection that have been postulated for endothelial cells (ECs), which are not catecholaminergic physiological GLP-1 action (97). Subsequent studies util- but primarily glutamatergic (79, 81). Additionally, the ized mice with Cre-recombinase under the control of the Endocrine Reviews, 2021, Vol. XX, No. XX 11

Gcg promoter thereby facilitating functional manipula- of brainstem Gcg mRNA affected glucose tolerance in tion of these neurons in vivo (98-102). Importantly, these regular chow–fed mice or rats (82, 100, 102). Interestingly, mice allowed the selective ablation of NTS-PPG neurons shRNA knockdown of GLP-1 expression from PPG and thereby the demonstration that loss of these neurons neurons in HFD-fed rats resulted in moderately impaired substantially decreased GLP-1 content in both hypothal- glucose tolerance, whereas chemogenetic activation of PPG amus and spinal cord (100) and thus corroborated the neurons in HFD-fed mice did not impact glucose tolerance. findings from rat, that interference with GLP-1 translation Intriguingly, correlation of NTS levels of Gcg mRNA with

in the brainstem affects GLP-1 content in the forebrain fat mass was observed in rats, implying signals linking the Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 (82). These Gcg-Cre transgenic mice enabled the use of extent of adiposity to central PPG activity. Cre-dependent viral expression of Channelrhodopsin2- Intracerebroventricular (ICV) injection of the GLP-1R Green Fluorescent Protein (ChR2-GFP) to demonstrate antagonist Ex9 in chow-fed rats and mice impaired glu- that NTS-PPG neurons project to most (if not all) fore- cose tolerance (82, 105), but ablation of PPG neurons in brain targets (103). These findings confirmed results of chow-fed mice did not impair glucose tolerance (100). The prior studies utilizing injection of retrograde tracers into simplest explanation for this discrepancy would be that various forebrain areas and colocalizing the tracer with PPG neurons are not involved in the regulation of glucose GLP-1 immunoreactivity in the NTS and IRT (79, 104). tolerance, but that gut-derived GLP-1 is able to reach (parts of) the brain to affect glucose tolerance, or that ICV Ex9 actually accesses the general circulation and exerts direct Manipulation of PPG neurons to interrogate their effects, such as in the pancreas. Alternatively, basal consti- function in vivo tutive GLP-1R signaling may exert tonic control of these Studies in mouse and rat established that GLP-1 is pro- processes, which may be disrupted by pharmacological duced by PPG neurons and is found in their cell bodies administration of GLP-1R antagonists independent of the and axon terminals. Functionally, experiments using source of the GLP-1 ligand (106-108). Finally, chemogenetic shRNA to knockdown GLP-1 expression produced a mild activation of PPG neurons increases heart rate, but their in- hyperphagic effect, mainly limited to rats on a HFD (82). hibition or ablation has no effect on resting or active heart Subsequent studies making use of transgenic mice with vir- rate or blood pressure (109). All these results point at the ally mediated Designer Receptors Exclusively Activated by central GLP-1 system as being capable of exerting signifi- Designer Drugs (DREADD) expression in the NTS PPG cant effects on food intake and on autonomic nerve ac- neurons, demonstrated that selective activation of these tivity, but not producing these effects tonically in daily life. neurons produces a strong hypophagic effect (99-101). This was followed by a study employing both inhibitory DREADD receptor expression as well as selective What is the physiological role of ablation of NTS PPG neurons by the expression of diph- brain-derived GLP-1? theria toxin A subunit to examine effects on food intake These findings raise multiple questions, namely how im- and brain GLP-1 content (100). Both hypothalamic and portant is brain-produced GLP-1 for physiology, how is spinal cord GLP-1 content was severely reduced upon ab- it regulated and how and where does it act in the CNS? Is lation of these neurons, thus demonstrating that brainstem its regulation linked to circulating GLP-1? Interpretation GLP-1 neurons are the principal source of GLP-1 within of these findings requires careful scrutiny of the specific the CNS and that GLP-1 is stored in the axon terminals diets, age and sex of the animal species, housing condi- of the PPG neurons, all of which have their cell bodies in tions, and experimental manipulations utilized to infer the lower brainstem. Interestingly, as observed before with conclusions about CNS GLP-1 action. For example, the shRNA knockdown in rat, there was little effect on basal actions of Ex9 to increase food intake were observed in food intake, but reduction of brainstem Gcg expression or satiated but not in hungry rats (110). Similarly, multiple ablation of PPG neurons increased post fast refeeding and studies have employed scheduled meals to observe CNS impaired stress-induced feeding reduction (82, 100). effects of GLP-1 on food intake (107, 111, 112). GLP-1 These findings, together with the phenotype of normal and GLP-1Rs within the brain may not be critical for food intake and body weight observed in GcgGut–/– mice feeding under ad libitum access to food, but only under with intestine-specific deletion of the proglucagon-derived conditions where a large meal is consumed, such as after peptides (14), clarify the modest physiological importance a prolonged fast, or in the case of sporadic availability of either GLP-1 derived from PPG neurons or the gut for of highly palatable food to these rodents. A likely con- control of food intake. Moreover, neither chemogenetic ac- tributing signal here is the extent of gastric distension. tivation nor ablation of CNS PPG neurons or knockdown Notably, c-Fos activation in NTS PPG neurons requires 12 Endocrine Reviews, 2021, Vol. XX, No. XX a higher degree of gastric distension, then for instance GLP-1Rs are expressed throughout the CNS, from c-Fos activation in neighboring catecholaminergic the olfactory bulb down to the spinal cord (35, 75, 128, neurons (83, 85). 129) (Fig. 3). The use of genetic targeting approaches has PPG neurons may also have developed as a risk as- revealed specific cell types producing various GLP-1 re- sessment system to balance predatory risk (manifested as sponses. Adams et al. deleted the GLP-1R from either stress leading to meal termination) with the need for food/ vesicular glutamate transporter 2 or vesicular GABA trans- energy intake. Progressive gastric distension may shift ac- porter–expressing cells, thus removing the receptor from

tivity away from intake towards the avoidance of preda- glutamatergic and GABAergic neurons, respectively. Only Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 tion as the stomach becomes full and a higher percentage those mice lacking GLP-1R in glutamatergic cells lost the of PPG neurons becomes activated (84). Simultaneously, hypophagic and long-term body weight–reducing effect of PPG neurons are also activated in situations of stress, be liraglutide, suggesting that liraglutide affects food intake this psychological or restraint stress or consumption of a and body weight via GLP-1Rs located on glutamatergic, toxic substance, as mimicked experimentally by injection but not GABAergic cells (125). In contrast, Fortin et al. of lithium chloride LiCl or lipopolysaccharide (104, 113). used Glutamic Acid Decarboxylase (GAD)-Cre rats to Additionally, PPG neurons are activated by local (NTS) target GABAergic neurons (130). Their strategy was to availability of leptin or cholecystokinin (CCK), as well as virally express an inhibitory DREADD receptor in GAD- electrical stimulation of vagal terminals in the NTS (92, expressing cells in the NTS and to activate this receptor in 114). Thus, PPG neurons are activated by signals linked to the presence of liraglutide. Inhibition of NTS GABAergic short- and long-term energy balance and can reduce food neurons prevented the anorectic effect of liraglutide. They intake, but do not seem to play a major role in determining concluded that GABAergic neurons in the NTS mediate the food intake and body weight in ad libitum fed rodents. hypophagic effects of liraglutide. Reconciling these studies Rather, their physiological role seems to be tied to the re- in mice and rats, Adams et al. demonstrated key roles for sponse to various forms to stress, but might lend them- GLP-1Rs on glutamatergic but not GABAergic neurons yet selves to be exploited clinically to achieve hypophagia and they did not show that GABAergic neurons per se are not weight loss. A role for PPG neurons beyond the monitoring required for the feeding response (125). Similarly, Fortin of short- and long-term energy balance was also suggested et al. demonstrated that the activity of GABAergic neurons by a comprehensive study of their mono- and polysynaptic in the NTS is required for the hypophagic response to inputs within the central and peripheral nervous system liraglutide, but they did not prove that it is actually the (115). The localization of GLP-1Rs that serve to mediate GLP-1R expressed on these neurons that is required for the the actions of GLP-1 released from PPG neurons is dis- response (130). Thus, one might hypothesize that liraglutide cussed below. binds to GLP-1Rs on glutamatergic neurons, and these in turn signal to GABAergic neurons and their release of inhibitory transmitter is required for the suppression of food GLP-1 receptors within the nervous system intake. Hence, caution is needed when using chemogenetic Various techniques, employing radiolabeled or fluorescent approaches to extrapolate the precise physiological path- GLP-1R agonists or antagonists, or specific GLP-1R anti- ways employed by GLP-1 within the CNS. sera, together with ISH, have localized GLP-1R expression within the nervous system (49, 116-120). Complementary studies have used mice with germline or conditional tissue- Neuronal GLP-1Rs Outside the Blood–Brain specific deletion of the GLP-1R to examine the importance Barrier of different GLP-1R populations in the CNS and periphery (121-126). Notably, the hypophagia caused by ICV injection GLP-1Rs on vagal afferent neurons of GLP-1 was fully abrogated in Glp1r–/– mice, consistent GLP-1Rs are expressed by sets of vagal afferent neurons with the loss of binding sites for Ex4 in the Glp1r–/– mouse (35, 131, 132), which are mechanoreceptors (133). It has brain (121). These findings align with the demonstration been tempting to assume that these GLP-1R expressing that PPG neuron activation produces hypophagia, but that neurons are those stretch-activated vagal afferents that ac- the loss or attenuation of PPG neuronal activity or genetic tivate PPG neurons (85). However, a study examining this deletion of gut GLP-1 does not cause overeating or obesity hypothesis found that GLP-1R expressing vagal afferent (14, 100-102, 127). Thus, although GLP-1 has an essential neurons represent only a very small fraction of the vagal role in the control of blood glucose, it is dispensable for the afferent inputs to PPG neurons (135). GLP-1R-expressing suppression of food intake at least under standard animal vagal afferent neurons have their sensory inputs within house holding conditions. the gastrointestinal tract, their cell bodies in the nodose Endocrine Reviews, 2021, Vol. XX, No. XX 13

GLP-1RA actions Food intake Leaky blood-brain barrier Aversive response PPG+ cell bodies Gut motility PPG+ axon GLP-1R Neuroprotection Brain GLP-1 GrO GLP-1RA PPG neuron actions Food intake Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 Heart rate

Pir NAc Ventricle

Sep OVLT BNST SFO

ARC VMPO PVN

AH Caudal DMH hippocampus PVT

CeA

PAG

Barr PBN

LC VLM DMNX AP T IR

RPa NTS GLP-1RAs IML CAA in circulation

Figure 3. GLP-1 receptors (GLP-1Rs) and brain-derived GLP-1 in the central nervous system. Within the CNS GLP-1 is produced by PPG neurons (light yellow) and distributed to their axon terminals ready for synaptic release (dark green). Names of brain areas that receive PPG innervation and express GLP-1 receptors are given as abbreviations in blue. Systemically distributed GLP-1 receptor agonists (GLP-1RAs) are depicted as red circles and access the circumventricular organs with a leaky blood–brain barrier, and the ventricles, but not the brain parenchyma. The olfactory bulb and the piriform cortex (Pir) form areas that express GLP-1Rs and harbor PPG neurons that project locally only. Abbreviations: AH, anterior hypothalamus; AP, area postrema; ARC, arcuate nucleus; Barr, Barrington’s nucleus; BNST, bed nucleus of the stria terminalis; CAA, central autonomic area (lamina X); caud Hipp, caudal ventral hippocampus; CeA, central nucleus of the amygdala; DMH, dorsomedial hypothalamus; DMNX, dorsal vagal motornucleus; GrO, granule cell layer of the olfactory bulb; IML, intermediolateral nucleus; IRT, intermediate reticular nucleus; LC; locus coeruleus; NAc, nucleus accumbens; NTS, nucleus tractus solitarius; OVLT, organum vasculosum of the lamina terminalis; PAG, periaqueductal grey; PBN, parabrachial nucleus; Pir, piriform cortex; PVN, paraventricular nucleus; PVT, paraventricular thalamus; Sep, lateral septum; SFO, subfornical organ; RPa, raphe pallidus; VLM, ventrolateral medulla; VMPO, ventromedial posterior nucleus. ganglion, and their axon terminals in the NTS; where delivery of shRNA targeting the nodose ganglion (NG) exactly their GLP-1Rs are located, remains unclear. Thus, GLP-1R in male Sprague Dawley rats (134). GLP-1R the source of GLP-1 activating these neurons might be the knockdown in vagal afferent neurons did not affect long gut-derived GLP-1 for the peripheral nerve endings, or term energy balance but did accelerate gastric emptying, the PPG neuron-derived GLP-1 if their central terminals findings consistent with the phenotype of mice with gen- express GLP-1R. The physiological role of GLP-1Rs ex- etic reduction of Glp1r expression using Phox2b-Cre pressed in vagal afferent neurons was examined by viral to target autonomic neurons including the NG (126). 14 Endocrine Reviews, 2021, Vol. XX, No. XX

Reduction of NG Glp1r expression also blocked the ef- supported by studies dissociating the hypophagic effects of fects of intraperitoneal, but not intraventricular, Ex4 on PPG neuron activation from the food intake suppressing food intake and gastric emptying in rats (134), whereas actions of subcutaneous semaglutide (135). It seems pos- the sustained actions of large GLP-1RA such as dulaglutide sible that GLP-1Rs expressed in the ARC may be activated to generate weight loss were also modestly attenuated in by high circulating levels of GLP-1 under conditions of se- mice with genetic reduction of NG Glp1r expression (126). vere intestinal malaise or following bariatric surgery. This Collectively, these observations imply that the GLP-1Rs distinction may explain why GLP-1RA therapy is associ-

expressed by vagal afferent neurons are located in the per- ated with an increased sensation of nausea (136), while Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 iphery, outside the blood-brain barrier, rather than on the chemogenetic activation of brainstem PPG neurons does nerve terminals inside the NTS. Thus, GLP-1Rs expressed not elicit adverse responses in mice (102, 135). by vagal neurons seem to be a physiological target for gut- derived or pharmacologically administered GLP-1. GLP-1Rs inside the blood–brain barrier Most other GLP-1R-expressing brain regions receive GLP-1Rs in circumventricular organs axonal inputs from PPG neurons, and are likely activated Similar uncertainty arises in the context of interrogating by GLP-1 originating from those cells. However, brain- the importance of GLP-1Rs expressed by neurons in the stem PPG neurons do not project to the hippocampus or circumventricular organs that lack a functional blood brain to the cerebral cortex (93). Thus, it is currently unclear barrier. These regions, such as the area postrema (AP), me- how GLP-1Rs located in these areas are activated. A small dian eminence and part of the neighboring arcuate nu- population of PPG neurons has been reported in the piri- cleus (ARC), subfornical organ, and organum vasculosum form cortex (93), but these do not give rise to axons in of the lamina terminalis, are accessible from the circula- other parts of the cortex or the hippocampus. Activation tion, and thus are potential targets for postprandially re- of GLP-1Rs in the ventral hippocampus also results in leased gut-derived GLP-1 as well as confirmed targets for hypophagia, and it has been suggested that volume trans- systemically administered GLP-1RAs (Fig. 3). However, mission from the lateral ventricles, which had detectable some of these regions are also innervated by PPG neurons levels of GLP-1, might account for their physiological (93). The two circumventricular regions that have been activation (137). These observations are consistent with investigated in some detail are the median eminence/ARC the possibility that PPG neurons might be the source of and the AP. While systemic administration of GLP-1RA GLP-1 within the ventricle, as PPG axons are found along leads to activation visualized by c-Fos immunoreactivity, the ependymal cell layer covering the surface of the vent- in many areas of the brain, it is particularly these areas ricles and intraventricular injection of a retrograde tracer where clear binding of fluorescent GLP-1RA is observed labelled a fraction of NTS PPG neurons (137). While (116-118). this is plausible, it should be noted, that both GLP-1RA Most likely, engagement of GLP-1Rs in these areas, as and GLP-1R antagonists given subcutaneously access indirectly visualized by c-Fos staining in GLP-1R+ cells the ventricular space (49, 116, 118). Hence, there is no (125), leads to additional secondary c-Fos activation along discernable barrier for pharmacological or gut-derived downstream pathways in the CNS (116-118). Experiments GLP-1 to reach the brain ventricles, but it does not provide employing physical lesions to destroy the AP, the ARC, or evidence that post-prandially released GLP-1 would mean- the PVN, as well as subdiaphragmatic vagal afferent de- ingfully access the ventricles. Additionally, the observation afferentation indicated that the majority of the hypophagic that there is very little entry of systemically administered effect of the GLP-1RA liraglutide resides within the ARC GLP-1R agonists into the brain parenchyma beyond the (118). Chronic infusion of Ex9 into the PVN, but not the circumventricular organs, and particularly into the ven- ARC, increased body weight (118); however, Ex9 reduced tral hippocampus, suggests that there is no free diffusion the body weight–lowering effect of liraglutide when ad- between the ventricular space and the brain parenchyma ministered into the ARC, but not the PVN. These results (116, 118). This would then argue against PPG neuron- indicate that while the ARC may be a key target for the derived GLP-1 entering the ventricles and exiting at the anorectic actions of liraglutide there is no tonic activation level of the ventral hippocampus. A final area that is not of the ARC GLP-1Rs by endogenous GLP-1 released either innervated by brainstem PPG neurons is the olfactory by the gut or the PPG neurons. bulb. However, the olfactory bulb contains both GLP-1- The available evidence distinguishes between the weight and GLP-1R-expressing cells (75, 95, 138), and could thus lowering effects of systemically administered GLP-1RA constitute an independent local GLP-1 system. Whether from those of PPG neuronal activation. This notion is or not the olfactory bulb is accessible to GLP-1 from the Endocrine Reviews, 2021, Vol. XX, No. XX 15 general circulation is not clear; no studies utilizing GLP-1R reduce the rates of stroke in humans with risk factors for agonists or antagonists to date have visualized binding in or established cardiovascular disease (149). Furthermore, the region of the olfactory bulb. GLP-1RA also reduce albumin excretion, and trials are When interrogating the function of GLP-1R populations underway to determine whether these agents may reduce within the brain, modulation of food intake is often the pri- rates of dialysis, decline of eGFR, and renal replacement mary measured output, but the physiological mechanisms in people with diabetic kidney disease. Collectively, these underlying modulation of this output varied substantially observations have created great interest in understanding

between studies and brain regions. For example, food in- how GLP-1RA exert their mechanisms of action in the Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 take might also be reduced due to a reduction in the reward cardiorenal system. Nevertheless, there is still relative un- value of the specific substance tested, findings potentially certainty and some controversy about the precise cell types relevant for the actions of substance abuse, the sensation of and tissues that express the GLP-1R in the heart, peripheral nausea or other malaise, or the presence of acute or chronic vasculature, and kidney (Fig. 4). stress (139-142). Additionally, neural GLP-1R activation has been linked to an increased cardiovascular output, or to changes in body temperature, or increased peripheral Localization of GLP-1R in the heart brown fat activity, all likely consequences of activation of Despite considerable effort, the precise identity of GLP-1R+ the sympathetic nervous system (143-146) and therefore in cells in the heart remains elusive (44). Analysis of hearts line with the proposed functional role of PPG neurons in from 3 cynomolgus monkeys, including sections from stress responses as discussed above. males and females, revealed GLP-1R-immunopositive cells using Mab 3F52 only in SA myocytes, which were identified by costaining sections for hyperpolarization activated Concluding Remarks about GLP-1R Expression cyclic nucleotide gated potassium channel 4. In a single and GLP-1 Action in the Nervous System frozen sample taken adjacent to the SA node confirmed to The emergence of fluorescently labeled GLP-1RA and be GLP-1R immunopositive by IHC, 125I-GLP-1 binding transgenic mouse models combined with the use of viral was detected and diminished following addition of ex- vectors to dissect the brain GLP-1 system over the past cess unlabeled ligand. GLP-1R-immunopositive cells were decade has highlighted our lack of deep understanding of also visualized in sections from the SA node from a single GLP-1 action in the brain. It is now emerging that there normal human heart (44). is no direct link between the gut and brain GLP-1 system, Wallner et al. reported GLP1R expression in right and that GLP-1RAs exert their appetite-suppressing effects atrium (RA) tissue and right ventricle (RV) tissue from primarily, if not exclusively, by interacting with GLP-1Rs 7 human heart samples that were not suitable for trans- on cells in the circumventricular organs outside the blood– plantation. Relative GLP1R expression by qPCR was brain barrier, that then signal deeper into the brain but do 3.4-fold higher in the RA than in the RV (150). GLP1R not involve activation of PPG neurons, the brain’s own mRNA transcripts were also amplified by conventional source of GLP-1. Understanding the physiology of brain- PCR generating a 103 base pair cDNA product from RNA derived GLP-1 is even more nascent and unravelling the isolated from the RA and RV, and from 3 samples of iso- details of its regulation and various actions including, but lated cardiomyocytes from the left ventricle (LV). Clarke not limited to, the reduction in food intake, promises to be and colleagues analyzed GLP-1R protein expression in an exciting journey of discovery. the human heart by IHC using Mab 3F52 to analyze cardiac tissue obtained from anonymous donors from 2 tissue biobanks (151). Extensive GLP-1R-immunopositivity was GLP-1R expression in the cardiovascular system reported in atrial and ventricular cardiomyocytes, but not Cardiovascular safety trials with GLP-1RA have demon- within ECs or vascular smooth muscle cells (VSMCs). The strated reduction in all-cause and cardiovascular mortality, proportion of GLP-1R+ cells, and the number of hearts and lower rates of major cardiovascular events including or sections examined was not described (151). Giblett stroke and myocardial infarction (15, 16). GLP-1RA also and colleagues from the same research group also ana- increase heart rate in humans and animals, findings at- lyzed GLP-1R protein expression in human heart tissue tributed in part to activation of the sympathetic nervous obtained from nondiabetic human subjects with ischemic system, inhibition of the parasympathetic nervous system, heart disease using Mab 3F52 (152). Patchy GLP-1R and activation of the sinoatrial (SA) GLP-1R (143, 144, 147, immunoreactivity was observed in cardiac myocytes from 148). GLP-1R signaling also attenuates the extent of ex- the RV and LV; however, the precise location of the GLP- perimental stroke in preclinical studies (97), and GLP-1RA 1R-immunopositive cardiomyocytes was not described, 16 Endocrine Reviews, 2021, Vol. XX, No. XX

GLP1R expression Heart rate SA node myocytes Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

Myocardial infarction

Heart atria, ventricles

cell types)

Endothelial function

Select vascular beds Atherosclerosis

Aorta intima, media

cell types)

Blood pressure Vascular smooth muscle cells

GLP1R

Figure 4. GLP-1 receptor expression in heart and blood vessels within select organs and actions of GLP-1 associated with these organ and cell types. The arrow depicts the relative levels of GLP-1R expression deduced from human gene expression databases such as the Genotype Tissue Expression portal. and the proportion of GLP-1R-immunopositive cells de- by Western blot analysis using Mab 3F52, although this tected within each analyzed heart was not reported (152). antibody did recognize the GLP-1R protein in extracts sub- GLP1R expression was examined in RNA samples from jected to immunoprecipitation and immunoblotting from all 4 chambers of 15 healthy and diseased human hearts by cells transfected with a cDNA encoding the GLP1R. The qPCR, and RT-PCR for the full-length mRNA transcript discrepant results for GLP-1R localization in the human (18). GLP1R mRNA transcripts were detected in all of heart using the same antibody in various reports likely re- the hearts examined, with similar levels in RA vs RV but flect different technical conditions and controls employed lower levels in the LV vs the LA. The relative abundance for IHC in various laboratories. of the GLP1R in heart tissue was similar to that detected Analysis of Glp1r expression in the mouse heart reveals in human islet RNA samples. Nevertheless, the cellular levels of Glp1r mRNA transcripts are higher in the atria localization of GLP-1R expression in the heart, assessed than ventricles. PCR amplification of Glp1r transcript in using both ISH and IHC with Mab 3F52, was not identi- 3 samples from normotensive or hypertensive angiotensin fied in the majority of sections analyzed, except in the RA, II-infused mice detected Glp1r mRNA transcripts in atria where GLP1R RNA was detected by ISH (18). Moreover, however much lower levels were detected in ventricular GLP-1R protein was not detectable in human cardiac tissue tissue (153). Whether this discrepancy reflects true-species Endocrine Reviews, 2021, Vol. XX, No. XX 17 differences or region-specific differences in RNA sampling cell types in vivo. Indeed GLP1R mRNA transcripts were of human ventricular biopsies is unclear as comparable not detected by qPCR in RNA isolated from human cor- levels of GLP1R mRNA transcripts were detected in RNA onary artery VSMCs or ECs (18). However, Richards et al. extracted from biopsies obtained from all 4 chambers of reported Glp1r promoter-directed fluorescent cells in the the human heart (18). Consistent with Glp1r expression aorta, and arteries and arterioles of multiple tissues, and in an atrial myocyte-like cell type, Glp1r expression was Glp1r mRNA transcripts were detected by qPCR in mouse reduced in mouse atria using the cardiomyocyte specific aorta, albeit at levels 90% lower than corresponding ex-

αMyosin Heavy Chain promoter to drive tamoxifen indu- pression of Glp1r in RNA isolated from mouse atrial Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 cible Cre-ER recombinase in cardiomyocytes (147). To fur- tissue (35). Fluorescent cells within the intestinal vascu- ther determine the identity of mouse atrial GLP-1R+ cells, lature costained with α-smooth muscle actin and neural/ Baggio et al. used the Hcn4 promoter, classically expressed glial antigen 2, a marker for pericytes, and were thought in SA node cells, to drive Cre recombinase expression and to represent arterial, rather than venous blood vessels. specifically target SA gene expression (148). Although Nevertheless, Glp1r expression was not examined in the Hcn4-Cre reduced levels of Gcgr mRNA transcripts in majority of cells+ for the fluorescent reporter. control experiments using floxed Gcgr mice, no reduction GLP1R mRNA transcripts were detected by qPCR in of atrial Glp1r levels was achieved using Hcn4-Cre and the aortic intima and media obtained from subjects, with floxedGlp1r mice. Hence, the precise atrial cell type(s) that and without obesity, scheduled for aortic surgery (154). express the murine cardiac GLP-1R remain uncertain. Extensive diffuse GLP-1R-immunopositivity was detected in the aorta using Mab 3F52. Notably, GLP1R/GLP-1R expression was not quantified relative to other GLP1R+ Assessment of GLP-1R expression in tissues, nor assessed using the same reagents and conditions blood vessels for comparative purposes of specificity in organs known Despite the multiplicity of studies describing actions of not to express the GLP-1R. GLP-1RA on blood vessels, there is surprisingly little Single cell analysis of murine endothelial cells from 20 rigorous information that describes GLP-1R expression organs showed Glp1r+ cells most prominently in the lung in normal or diseased VSMCs, ECs, or other vascular cell (Fig. 5), with some Glp1r+ positive ECs also detected in types (Fig. 4). Much of the data in this area rely on ECs cells from the liver and heart (155). Given the limitations and VSMCs analyzed after cell culture ex vivo, which may of read depth needed to identify low abundance transcripts not be representative of GLP-1R expression in the same with scRNA-seq, this is likely a conservative snapshot of

Figure 5. Expression of mouse Glp1r in 40 449 murine endothelial cells. Count matrices were accessed on September 19, 2020, and reported by Kalucka et al. (221) (ArrayExpress ID E-MTAB-8077). 18 Endocrine Reviews, 2021, Vol. XX, No. XX

EC Glp1r expression. Consistent with Glp1r expression in GLP-1R, and studies using validated reagents have not de- ECs, mice expressing EC-specific Cdh5-dependent Cre re- tected the GLP-1R in glomerular epithelial or tubular cells combinase exhibited reduced Glp1r expression in a lung (44, 157, 158). Hence, the mechanisms by which GLP-1 EC preparation isolated using CD31 and CD102 coated modulates renal function, including control of albumin, beads (156). However, no markers were used to assess the water and salt excretion, either directly through control of extent of EC enrichment, and knockdown of Glp1r was the renal vasculature, or via indirect mechanisms, remain not examined in resistance arteries, kidneys, heart, aorta, uncertain.

or other vascular sites that might contain EC GLP-1R ex- Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 pression. Using Glp1r-Cre mice crossed with a ROSA26- tdRFP reporter, Richards and colleagues also detected GLP-1 production in enteroendocrine L cells Glp1r-directed reporter gene expression in a few blood along the gut vessels within the cardiac ventricles, with a morphology Enteroendocrine L cells have classically been defined as the consistent with VSMCs (35). Overall, the data suggest GCG+ cell type giving rise to GLP-1 in the gut (1). In hu- that Glp1r/GLP1R expression in the vascular system is mans and mice, the majority of L cells reside in the ileum highly heterogeneous, with high expression in mouse lung and colon, with fewer cells located in the duodenum and ECs and restricted expression in subsets of VSMCs in the jejunum. L cells sense various nutrients from the luminal kidney (discussed next), rather than broadly expressed surface and in turn secrete GLP-1 into the local circula- throughout the vasculature. tion (160). Numerous cognate receptors for non-nutrient GLP-1 secretagogues are expressed on the basolateral side of L cells, thus enabling L cells to simultaneously sense mol- GLP-1R expression in renal blood vessels ecules in the circulation (161-163). L cells in the proximal The location of GLP-1R expression within the kidneys gut are capable of responding briskly to nutritive stimuli has been controversial, at times localized to glomerular (14, 164); the available data suggest that distal gut L cells or tubular epithelium in studies using GLP-1R antisera exhibit secretory responses biased towards non-nutrient se- that do not detect the GLP-1R or lack specificity (11, 20, cretagogues (164). Analysis of mice with marked reduction 43). In vivo autoradiography using 125I-labeled GLP-1 of Gcg expression within the entire, or selectively in the (7-36) amide, IHC using well-characterized GLP-1R anti- distal gut, reveals essential contributions of intestinal Gcg bodies, and ISH have detected GLP-1R in the rat, mouse expression to the pool of circulating GLP-1, and in the con- and monkey renal vasculature, particularly in arterial trol of gastric emptying and glucose homeostasis (14). VSMCs (44, 157-159). Pyke et al. (44) analyzed kidney scRNA-seq analyses have illuminated the transcriptomic from 7 normal rhesus monkeys using Mab 3F52. GLP- heterogeneity evident across subsets of L cells (Fig. 6). One 1R-immunopositivity was limited to a small number of of the first scRNA-seq studies on the mouse small intes- VSMCs within the preglomerular vascular compartment. tine recovered 310 out of 7216 (4.3%; which is higher than These findings were corroborated by analysis of matched often achieved) quality control–passed Enteroendocrine sections using in situ radiolabeled ligand binding, which cells (EECs) (165), and 10% of these were identified as identified binding in a vessel that also stained positive with Gcg+. Subsequently, several reports characterized indi- Mab 3F52 (44). vidual L cells isolated from mouse reporter lines. Sequencing RT-PCR of RNA from isolated mouse kidney fractions, of 259 individual YFP+ cells isolated from the small intes- and ISH analysis of mouse kidney detected Glp1r mRNA tine of GLU-Venus mice identified 3 main subgroups of L transcripts in blood vessels and not in the tubular fraction cells. These corresponded to (1) Gcg/Pyy double-positive (157). A few GLP-1R+ renin+ juxtaglomerular cells were cells, which account for 50% of L cells; (2) Gcg/Tph1/Pzp reported in the afferent arterioles of the monkey and mouse triple-positive cells, which account for 35% L cells; and (3) kidney (44, 158). Detection of GLP-1R immunopositivity Gcg/Gip double-positive cells, which account for 15% L in several VSMCs within the preglomerular vascular com- cells (166). Similarly, a comprehensive survey of 6906 small partment was corroborated by analysis of matched sections intestine EECs from Neurog3Chrono reporter mice iden- using in situ radiolabeled ligand binding, which identified tified more than 90% of L cells as multihormonal (167). binding in a blood vessel that also stained positive with Mab The detection of Gcg in EECs correlated strongly with 3F52 (44). Moreover, the same antisera failed to identify Cck, followed by Pyy, Ghrl, and Sct coexpression (Fig. 6). GLP-1R immunopositive cells in the kidney from Glp1r–/– A scRNA-seq study on 1560 intestinal YFP+ cells isolated mice. Despite detection of GLP-1R+ cells within a subset from Neurod1-Cre; Rosa26-YFP mice confirmed that co- of the renal vasculature, the majority of renal blood vessels lonic L cells are also mostly multihormonal (168). Although and VSMCs examined by ICC or ISH do not express the both small and large bowl L cells can produce neurotensin, Endocrine Reviews, 2021, Vol. XX, No. XX 19 Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

Figure 6. Coexpression of the proglucagon gene (Gcg) and enteroendocrine genes in 411 murine small intestine L cells. Count matrices were accessed on September 19, 2020, and retrieved from the scRNA-seq datasets reported by Gehart et al. (167) (GEO accession number GSE113561). Mouse gene nomenclature: Pyy, peptide YY; Gip, glucose-dependent insulinotropic polypeptide; Nts, neurotensin; Tph1, tryptophan hydroxylase 1; Sst, somatostatin; Ghrl, ghrelin; Sct, secretin; Cck, cholecystokinin.

L cells in the distal colon preferentially expressed Pyy and organoid-derived L cells sequenced in the study was not Insl5. Studies of human EECs have relied primarily on bulk high (<100), and the L cell subtypes in the organoid might RNA-seq for transcriptome profiling (169-171). To circum- not faithfully recapitulate the ones in the human intestine in vent the difficulty of isolating L cells from human intes- situ. Nevertheless, the analyses and corroboration of mouse tine with high purity, a recent study sequenced 2255 cells and human L cell scRNA-seq datasets support a con- single cells from human small intestinal organoids trans- served transcriptional program in L cells across the species. duced with doxycycline-inducible NEUROG3 transgenes Analysis of human jejunal L cell transcriptomics, and L cell to drive differentiation towards endocrine lineages (172). density identified dysregulation of enteroendocrine differen- Three L cell subtypes among the 2255 EECs sequenced tiation signatures associated with reduced L cell density in were PYYHigh, PYYLow, and NTSHigh cells, all of which were obese people with T2D (173), providing a possible explan- also detected in the mouse L cell scRNA-seq datasets. A dis- ation for reduced meal-stimulated levels of GLP-1 in some crepancy between the mouse and human data was the ab- of these individuals. Collectively, these findings raise several sence of TPH1+ and GIP+ L cells, although the number of important questions that warrant further interrogation; do 20 Endocrine Reviews, 2021, Vol. XX, No. XX different L cell subtypes produce and secrete GLP-1 in a Stomach and Brunner’s glands express secretagogue-selective manner with different secretory cap- the GLP1R acity? Can we attribute the functional heterogeneity of L Human gastric mucosa and parietal cells express GLP1R cells predominantly to their transcriptional signatures or mRNA transcripts as detected by RT-PCR (176) and mul- does their location within the gut, or along the crypt–villus tiple GLP-1R-immunopositive cells were detected within axis, cellular neighborhood and coexisting disease states the gastric epithelium using a partially characterized mono- modify their phenotype? Are there new molecular targets clonal antibody Mab 28141, findings consistent with the that could be identified from these datasets for augmenting detection of Glp1r transcripts in rat parietal cells (177). Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 endogenous GLP-1 secretion? GLP-1 may act on the parietal cell GLP-1R to block gastric acid secretion (178, 179); however, few studies have GLP-1R expression in the gastrointestinal tract explored the function of GLP-1R signaling within parietal cells in vivo. GLP-1R expression is detected across the proximal and Brunner’s glands, found within the proximal duo- distal regions of gastrointestinal tract. The close proximity denum, are a set of submucosal GLP-1R+ exocrine glands of intestinal GLP-1R-expressing immune cells and enteric specialized in secreting mucus. The glands were highly GLP- neurons to L cells within the intestine may facilitate im- 1R-immunoreactive using validated GLP-1R antibodies in mediate paracrine actions of GLP-1. Remarkably, despite both mice (using Mab 7F38) and in humans (using Mab robust actions of GLP-1RA to reduce enterocyte chylo- 3F52) (44, 119). CD-1 mice injected with a single dose of micron synthesis and secretion, the canonical GLP-1R is Ex4 (1 mg/kg) showed upregulation of Il33 and Ccl20 in not detectable within the small or large bowel enterocytes. RNA isolated from Brunner’s glands (180); however, the Below we highlight regional considerations and GLP-1R+ physiological importance of the Brunner’s gland GLP-1R cell types within the gut. system remains poorly defined.

GLP-1R expression and biology in gut intraepithelial lymphocytes GLP-1R expression in the enteric nervous system Intestinal intraepithelial lymphocytes (IELs) are a unique The enteric nervous system (ENS) is unique in its cap- population of T cells with innate-like immune function. ability to transduce signals somewhat independently from Percoll-purified IELs express high levels ofGlp1r relative the brain or the spinal cord (181). It comprises 3 main cell to the thymus, spleen, and lymph nodes as shown by qPCR types: enteric neurons, enteric glial cells, and interstitial (174). Furthermore, IELs from the small bowel express cells of Cajal. These cells cluster in the myenteric plexuses, higher levels of Glp1r than colonic IELs. Whole body loss found in the muscle layer of the gut, and in the submucosal of the Glp1r does not perturb the number and composition plexuses, found in the submucosal layer of the gut, along of IELs (174). The IEL GLP-1R is functional, as Ex-4 dir- the entire gastrointestinal tract (181). ectly augments cyclic adenosine monophosphate (cAMP) Multiple lines of evidence demonstrate GLP-1R expres- levels in murine gut IELs ex vivo (174). Within the intestinal sion in subsets of enteric neurons. A study using mouse epithelium, both TCRγδ + and TCRαβ + IELs express com- reporter lines expressing Rosa26-tdRFP or Rosa26-eYFP parable levels of Glp1r as shown by qPCR on sorted IELs under the control of Glp1r-Cre found a small number (175). The importance of gut IEL GLP-1Rs for regulating of fluorescent-positive enteric neurons (35); treatment incretin biology and metabolism has been examined in the with 10 nmol/L GLP-1 increased their action potential context of β7–/– mice, which lack gut IELs due to defects in firing frequency ex vivo. About 60% and 19% of these lymphocyte gut homing (175). Reconstitution of irradiated fluorescent-positive neurons from the small and large in- Ldlr–/– mice with a chimera of β7–/–/Glp1r–/– bone marrow testine, respectively, were positive for neuronal nitric oxide raised plasma GLP-1, improved intraperitoneal glucose synthase, implying that these neurons are likely inhibitory tolerance, lowered plasma cholesterol, and reduced aortic motor neurons (35). Complementary analyses employed plaque size after 14 weeks of high-fat high-cholesterol diet Wnt1-Cre to knock down Glp1r in the mouse central and feeding, relative to irradiated WT mice with a chimera of enteric nervous system (126). Glp1r mRNA transcripts β7–/–/WT bone marrow. How gut GLP-1R-expressing IELs were more abundant in the muscle layer (including the control the bioavailability of local and systemic GLP-1 re- myenteric plexus) than in the submucosal layer (including mains uncertain (175). The physiological importance of the the submucosal plexus). Wnt1-Cre enabled knockdown enteroendocrine–IEL axis for local and systemic immunity of more than 95% of Glp1r mRNA in the ileal muscle remains incompletely defined. layer, but only 80% in the jejunal and as low as 60% in Endocrine Reviews, 2021, Vol. XX, No. XX 21 the duodenal muscle layer (126). Human myenteric plex- detection (133). Consistent with these findings, mice uses were reported to contain GLP-1R immunoreactivity; lacking Glp1r in their enteric neurons exhibited normal however, these experiments utilized an antibody, Ab39072 food intake, gastric emptying, body weight, and glucose subsequently shown to lack sensitivity and specificity (20, control (126), suggesting that GLP-1R expression in en- 182). A single cell atlas of the murine (and human) ENS teric neurons is physiologically dispensable for the classical affirmed the presence of Glp1r/GLP1R mRNA transcripts metabolic actions of GLP-1 and some aspects of nutrient in various neuronal subsets within the ENS (Fig. 7) (183). sensing. Notably, adenosine triphosphate coreleased with

Beyond enteric neurons, it remains unclear whether enteric GLP-1 from L cells, might also trigger vagal activation Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 glial cells and interstitial cells of Cajal express the Glp1r. through P2Y2/3, adding further complexity to L cell–ENS The role(s) of GLP-1R signaling in enteric neurons communication (184) have not been fully elucidated. Inference from the scRNA- seq data suggest that the GLP-1R-expressing enteric neurons are secretomotor/vasodilator neurons. Analysis Potential GLP1R expression in intestinal of mice expressing a novel Glp1r-Cre; lox-tdTomato re- smooth muscle porter demonstrated tdTomato-positive neurons lining the Whether GLP-1R agonism inhibits gut motility indirectly stomach and the intestine—these neurons were identified through neural mechanisms, or in part through direct as mechanosensitive and were not involved in nutrient interaction with a smooth muscle GLP-1R remains unclear

Figure 7. Expression of Glp1r in 4974 murine ileal and colonic enteric neurons. Enteric neuron subtypes types were classified based on neuropep- tide genes Adcyap1 (PACAP neurons and Gal (galanin neurons). Count matrices were accessed on September 19, 2020, and aggregated from the scRNA-seq datasets reported by Drokhlyansky et al (183) (Broad Institute Single Cell Portal SCP1038). Mouse gene nomenclature: Adcyap1, pituitary adenylate cyclase activating polypeptide or PACAP; Gal, galanin. 22 Endocrine Reviews, 2021, Vol. XX, No. XX

(185-187). GLP-1 may also reduce smooth muscle cell in- antigen-presenting CD1d that binds a variety of foreign flammation in the context of lipopolysaccharide-induced lipids (194). A case report examined the putative direct ef- Tnf and Il1a expression in colonic smooth muscle cells iso- fects of GLP-1 on human iNKT cells in the context of psor- lated from BALB/c mice (188). Notably, ascertainment of iasis. Two individuals with T2D and psoriasis treated with definitive nonvascular smooth muscle cell GLP-1R expres- liraglutide exhibited reduced number of circulating iNKT sion remains challenging to date, as contamination from cells after 6 weeks of treatment (195). Flow cytometric ana- rare cell types within ex vivo preparations, including en- lyses revealed that 55% of CD3+ T cells and 96% of TCR- + teric neurons, cannot be excluded. Vα24 iNKT cells showed positive intracellular staining Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 Thus, the local gut GLP-1 system is comprised of 3 main using an unspecified PE-GLP-1R antibody, accompanied by components: L cells as the main GLP-1 producer, and en- increased levels of cAMP levels in these cells following in- teric neurons and IELs as the primary responders to GLP-1. cubation with GLP-1. Exogenous liraglutide treatment of Although GLP-1 secretion by L cells contributes to a ma- HFD-fed mice for 5 days reduced the number of iNKT cells jority of active GLP-1 levels in the circulation, it is worth in the blood and adipose tissues (196). Moreover, HFD-fed mentioning that the local gut environment is frequently ex- Cd1d–/– mice, which lack iNKT cells in the body, lost less posed to very high levels of GLP-1 (likely higher than the body weight when treated with liraglutide. Nevertheless, circulatory levels), especially when L cells are stimulated the expression of the GLP-1R in mouse iNKT cells was not after meal ingestion or under various stress conditions. One definitively established and there is little rigorous evidence can hypothesize that the presence of GLP-1R-expressing that B cells, NK, NKT, or innate lymphoid cells express the enteric neurons and IELs may present a feedback mech- canonical GLP-1R. anism to modulate GLP-1 levels, as recently suggested (175). Moreover, local GLP-1 bioactivity is further modi- fied by vascular and potentially enterocyte DPP-4 activity GLP-1R expression in myeloid lineages (189, 190). As enteric neurons and IEL subpopulations are Immunoreactive GLP-1R protein has been detected in difficult to isolate and study, the actions of GLP-1 on these mouse peritoneal macrophages by Western blotting using cell types remains incompletely understood. incompletely characterized antisera (197, 198), and in macrophage-like cell lines. However, full-length Glp1r transcripts have not been consistently detected in macro- GLP-1 action in the immune system phages from mouse tissues or peritoneum, in the basal state GLP-1R agonists reduce systemic inflammation in preclin- or after induction of inflammation (20). Flow cytometry ical and clinical studies; however, the mechanisms linking was used to investigate GLP-1R expression in peripheral GLP-1R signaling to modulation of immune cell activity blood from individuals with allergic asthma (199). About remain uncertain (191). Low levels of Glp1r transcripts 10% and 5% of blood neutrophils and eosinophils, re- have been detected in isolated thymocytes, splenocytes, and spectively, from healthy control subjects stained positive lymph node cells from C57BL/6J mice by RT-PCR (192). with an Alexa Fluor 700-conjugated GLP-1R antibody. Glp1r expression was also detected by RT-PCR in The proportion of GLP-1R-immuno-positive eosinophils murine T cells, both in the basal state and after stimulation was reduced to 2% in subjects with asthma. The specificity with aCD3/CD28, as well as in TH1 and TH17 polarized of the GLP-1R antibody used in this study, AF700-GLP-1R, cells by RT-PCR; however, levels were not compared rela- remains uncertain. Overall, the expression of GLP-1R has tive to Glp1r expression in other cell types or tissues (193). not yet been robustly confirmed in myeloid cells. Whole body Glp1r–/– mice display normal cellularity and immunophenotypes in the thymus, spleen, lymph nodes, and bone marrow, suggesting that expression of Glp1r is GLP-1R transcripts in miscellaneous tissues dispensable for the normal development of these immune Adipose tissue organs (192). Recent studies have begun to explore the GLP1R transcripts are reported in adipocyte cell lines ex biology of GLP-1R expression in tissue-resident immune vivo and in human visceral and subcutaneous adipose tis- cells. sues by Taqman-based qPCR (200). Nevertheless, the precise GLP-1R+ cell type(s) within rodent or human adipose tissue, ranging from vascular ECs or VSMCs, immune Invariant natural killer T cells as targets of cells, or adipocytes themselves, has not been defined with GLP-1 action certainty. Invariant natural killer T (iNKT) cells exhibit proper- Iacobellis et al. detected GLP1R and GLP2R RNA by ties of T cells and NKT cells; iNKT cells recognize the RNA-seq and qPCR in 8 human epicardial adipose tissue Endocrine Reviews, 2021, Vol. XX, No. XX 23

(EAT) samples from individuals with coronary artery Eyes disease with and without T2D (201). GLP2R mRNA Diabetic complications such as retinopathy, neuropathy, and transcripts were ~5-fold more abundant than GLP1R nephropathy heighten the interest in the presence of GLP-1R mRNA; GLP1R expression was described as “relatively in the eyes, peripheral nerves, and kidney. Although GLP-1R low” however precise quantification or comparison with was reported in human retinal pigment epithelia by both GLP1R expression in other tissues was not provided. qPCR and by IHC, these analyses used the subsequently GLP-1R-immunopositivity was detected in EAT and discontinued ab39072 antisera (208). Later studies using

subcutaneous adipose tissue (SAT) from a diabetic and ISH and IHC with the GLP-1R antibody Mab 3F52 did not Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 nondiabetic subject, analyzed by fluorescent IHC (Mab detect GLP-1R in the majority of cells within the human 3F52). Remarkably, in contrast to relatively compar- retina; however, <1% of the cells in the ganglion cell layer able levels of GLP1R mRNA in EAT vs SAT, GLP-1R were found to be GLP-1R+ (209). Using the same antibody, immunopositive cells were much more abundant in EAT, and protocols, GLP-1R-immunopositive cells were detected compared to SAT (201). Tyramide signal amplification in GLP-1R+ tissues including the pancreas, Brunner’s glands, (which also has the potential to amplify signal from and kidney. nonspecific primary antibody binding) was used to detect GLP-1R immunopositivity, due to the very low levels of Liver GLP-1R expression, The precise identity of the GLP-1R+ There is considerable interest in the investigational assess- cells in EAT was not determined. A follow-up study from ment of GLP-1RA for the treatment of NASH, based on the same group reported GLP1R expression in EAT by preclinical and human studies demonstrating these agents quantitative microarray analysis from 17 subjects under- reduce hepatic fat, inflammation and progression of fibrosis going coronary artery bypass grafting. Remarkably (210, 211). Nevertheless, the mechanisms linking GLP-1R GLP2R mRNA transcripts were reported to be ~57-fold signaling to control of NASH remain elusive, as levels of more abundant in EAT, relative to levels quantified for hepatic Glp1r/GLP1R expression are relatively low and GLP1R (202). sometimes undetectable, relative to expression in pancreas, kidney, gut, lung, and heart (10, 212). Indeed, Boland and Thyroid colleagues did not detect GLP1R mRNA transcripts in GLP-1R agonists produce thyroid C-cell hyperplasia human liver, Kupffer or stellate cell fractions, prepared from in rats and mice (203, 204), consistent with expression 3 human liver biopsy samples analyzed by bulk RNA-seq of the GLP-1R in calcitonin-producing C cells within (213). Although a substantial number of studies report hep- the rat and mouse thyroid (203). The development of atocyte GLP-1R expression in mouse and human liver by C cell proliferation and calcitonin secretion in response immunocytochemistry or Western blotting, the great ma- to GLP-1R agonism in mice and rats is mediated by jority of these analyses use antisera that have not been val- the canonical GLP-1R (204, 205) prompting consider- idated for sensitive and specific detection of the GLP-1R able regulatory concern, and more detailed investigation (191). Moreover, isolated murine hepatocytes do not exhibit of GLP-1R expression in the human thyroid. Out of 59 canonical cAMP responses to GLP-1R agonists, fail to bind normal, hyperplastic and neoplastic human thyroid glands fluorescent GLP-1 in vitro, and do not express the Glp1r analyzed by IHC with the Mab 3F52 antibody, no GLP- (19-21, 214). 1R-immunopositive follicular or C cells were detected in GLP1R mRNA transcripts have been detected by normal or hyperplastic thyroid tissue. Two of 10 cases with RT-PCR in RNA isolated from human liver, and levels sporadic medullary thyroid carcinoma exhibited scattered were reported to be reduced in livers from individuals GLP-1R-immunopositive C cells (51). Receptor autoradi- with NASH (215). However, assignment of GLP-1R to ography using 125I-labeled GLP-1 (7-36) amide detected hepatocytes was carried out using an antibody with un- the presence of GLP-1Rs in a human case of medullary known sensitivity and specificity. It seems likely that low thyroid cancer. Several studies have reported more wide- level GLP-1R expression in liver reflects the presence of spread GLP-1R expression in the normal and neoplastic the GLP-1R in nonhepatocyte cells, such as immune cells thyroid (206, 207). However, these analyses used GLP-1R or blood vessels. Indeed a fraction of ECs from mouse antibodies lacking specificity (18, 20, 43). The available liver contained Glp1r mRNA transcripts detected by data suggest that GLP-1R+ cells are generally rare or ab- scRNA-seq (Fig. 5) (155). Hence, it seems reasonable to sent in the normal human thyroid, yet may be expressed in conclude that the actions of GLP-1R agonists on hepato- a subset of C cells in the context of C cell hyperplasia or cytes are likely indirect, through incompletely identified medullary thyroid cancer. pathways. 24 Endocrine Reviews, 2021, Vol. XX, No. XX

Concluding Remarks for GLP-1 Action Outside the expression of the GLP-1R in specific cell types and tis- the Pancreas and Nervous System sues (18, 44, 49, 116, 118) including the use of negative controls and GLP-1R knockout tissues where applicable. The available data highlight multiple actions of GLP-1 Many of the studies described herein utilize mice and and GLP-1R agonists, deduced from both preclinical and rats as models for GLP-1 action and GLP-1R activation. human studies, on key target cell types and tissues that The experimental conclusions may reflect the age, sex, spe- do not exhibit robust GLP-1R expression. For example, cies, housing conditions, diet, and health of the animals, as GLP-1R agonists reduce the rates of myocardial infarction,

well as the specific experimental and technical approaches Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 decrease hepatic lipid accumulation, and attenuate renal al- utilized (216). For example, the use of chemogenetic and bumin excretion, yet the GLP-1R is not expressed within optogenetic studies to interrogate PPG neurons informs most cardiomyocytes, hepatocytes, or renal glomerular and the potential of broadly activating these neurons, but tubular epithelium. Similarly, GLP-1R agonism leads to may not necessarily reflect the selective actions of GLP- reduced atherosclerosis, attenuated chylomicron secretion 1, acting through the GLP-1R, in the nervous system. and decreased inflammation, yet the majority of VSMCs, Moreover, careful controls are required when using both ECs, enterocytes, and immune cells do not express the ca- these techniques, since optogenetic channels alter neuronal nonical GLP-1R. Collectively, these findings highlight the properties (217), and DREADD ligands such as clozapine- importance of indirect mediators of GLP-1 action as key N-oxide can be converted to functional metabolites in the contributors to mechanisms ensuing from activation of the brain (218). Similarly, activation or deletion of the Gcg canonical GLP-1R. gene perturbs the expression of not only GLP-1, but multiple structurally related proglucagon-derived peptides (PGDPS), including oxyntomodulin and glucagon which Caveats and limitations may also signal through the GLP-1R. Hence, attribution Although considerable progress has been made in detecting of the results of these studies to the GLP-1/GLP-1R system GLP-1R expression, including characterization of 2 mono- should be done with caution. Furthermore, most studies clonal antibodies suitable for detection of the primate and localizing GLP-1 or the GLP-1R utilize postmortem tissue mouse/rat GLP-1R (3F52 and 7F38, respectively), it is still and do not reveal information on GLP-1/GLP-1R expres- possible to incorrectly assign GLP-1R expression using these sion in the live animal, or in live tissue. 2 antibodies. Importantly, the presence or absence of GLP- This review did not examine the potential actions of 1R-immunopositivity remains highly technique and tissue GLP-1 degradation products, generated by enzymatic dependent. Ideally, GLP-1R antisera should be assessed for cleavage of GLP-1, which may produce multiple biological specificity simultaneously under the conditions deployed actions independent of the known GLP-1R (219). This for histochemistry or Western blotting, using tissues from biology may be relevant to circumstances characterized Glp1r–/– mice, or with human cells or organoids engineered by pharmacological infusion of GLP-1, bariatric surgery, to delete the GLP-1R. Although a predominantly membrane or GLP-1-producing tumors, and has been discussed else- pattern of cellular staining is ideally observed when using where (6, 191). Moreover, although human islet GLP-1 chemical probes or validated antisera to detect the GLP-1R action and GLP-1R expression has been extensively studied (18, 44, 49), several reports of diffuse cytoplasmic staining in the pancreas, much less is known about GLP-1R local- raise questions about whether the antisera were used under ization in other human organs and their cell types (Fig. 8). conditions optimized for specificity. For example, the 7F38 As noted herein, the very low expression of the GLP-1R antisera has detected diffuse cytoplasmic and some mem- may preclude its reliable detection by RNA-seq, and more brane GLP-1R-immunopositivity throughout the entire particularly in scRNA-seq, even in islet cells known to ex- islet (158), a pattern of staining which can be more difficult press the canonical GLP-1R and often used as a positive to interpret, relative to the more restrictive cellular localiza- control (36). Hence, the old adage “absence of evidence is tion of islet and membrane-localized GLP-1R expression. not evidence for absence” remains relevant for interpret- Indeed, in the context of establishing optimal technical con- ation of these single cell or bulk data sets in tissues known ditions for the sensitive and specific detection of the human to express low levels of the full length GLP1R mRNA. GLP-1R in human heart using IHC and the 3F52 antibody Along these lines, sensitive in situ transcript visualization (18), we detected extensive cytoplasmic staining in tissues techniques such as RNAScope and single molecular fluor- or cell types not known to express the GLP-1R in some of escence ISH might prove transformative for understating our pilot experiments. Hence, wherever possible, multiple GLP-1R mRNA localization and abundance in complex complementary techniques should be used to interrogate tissues. Endocrine Reviews, 2021, Vol. XX, No. XX 25

Glucose Body weight Cardioprotection

GLP1R GLP1R

GLP-1

Neurons Blood Astrocytes

vessels Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021

SA node GLP-1 Acinar cells Blood vessels Endocrine cells

Parietal cells Smooth muscle VSMCs

Brunner’s glands VSMCs Enteric neurons ECs IELs

Figure 8. Schematic depiction of major targets of GLP-1 action, and the cell types within key organs that express the GLP-1 Receptor (GLP1R). The GLP-1 peptide is produced in and shown adjacent to the intestine, pancreas, and brain, the 3 major sites of GCG expression. EC, endothelial cells; IEL, intestinal intraepithelial lymphocytes; SA node, sinoatrial node; VSMC, vascular smooth muscle cell.

Table 2. List of antibodies and fluorescent ligands validated for GLP1R detection specificity using GLP1R-/- tissue or transfected cells (with and without human GLP1R).

Reagent type Catalogue number/name Source Reported cross-reactivity Reference

Antibody Mab 3F52 Iowa DSHB Human, primate (44) Antibody Mab 7F38 Iowa DSHB Human, Mouse, Rabbit, Rat (119) Antibody Glp1R0017/ GLP1R-APC Cambridge University Mouse (36, 53) GLP1R agonist Ex4-Cy3 and Ex4-Cy5 Novo Nordisk Mouse (48) GLP1R agonist Liraglutide750 Novo Nordisk Mouse, rat (118) GLP1R antagonist Ex9-39750 Novo Nordisk Mouse, rat (118) GLP1R antagonist LUXendin555, LUXendin645, and LUXendin651 University of Birmingham Mouse/hESC (49)

Recent technological advances coupled with reagent the expanding direct vs indirect cellular targets of GLP-1 development have fostered a more accurate and com- action. plete understanding of how and where endogenous and pharmacologically administered GLP-1 and GLP-1RA Acknowledgments acts to control metabolism. Rapid ongoing develop- Financial Support: D.J.D. is supported by CIHR grant 154321, ments in this field are likely to further refine existing in- investigator-initiated studies of peptide hormone action sponsored by sights, answering important mechanistic questions, while Novo Nordisk, a Banting and Best Diabetes Centre-Novo Nordisk raising new testable hypotheses designed to illuminate Chair in Incretin Biology and the Novo Nordisk Foundation-Mt. 26 Endocrine Reviews, 2021, Vol. XX, No. XX

Sinai Hospital fund for the study of gut peptides. B.A.M. is supported 11. Drucker DJ. Incretin action in the pancreas: potential by a fellowship from the CIHR and C.K.W. is supported by a Banting promise, possible perils, and pathological pitfalls. Diabetes. and Best Diabetes Centre fellowship. D.J.H. was supported by Med- 2013;62(10):3316-3323. ical Research Council (MRC) (MR/N00275X/1 and MR/S025618/1) 12. Hansen L, Deacon CF, Orskov C, Holst JJ. Glucagon-like and Diabetes UK (17/0005681) Project Grants. This project has re- peptide-1-(7-36)amide is transformed to glucagon-like pep- ceived funding from the European Research Council (ERC) under the tide-1-(9-36)amide by dipeptidyl peptidase IV in the capillaries European Union’s Horizon 2020 research and innovation program supplying the L cells of the porcine intestine. Endocrinology. (Starting Grant 715884 to D.J.H.). J.E.C. is supported by funding 1999;140(11):5356-5363. from the American Diabetes Association (1-18-JDF-017) and the Na-

13. Mulvihill EE, Drucker DJ. Pharmacology, physiology, and mech- Downloaded from https://academic.oup.com/edrv/advance-article/doi/10.1210/endrev/bnaa032/6035170 by guest on 09 February 2021 tional Institutes of Health (DK123075, DK125353). S.T. was sup- anisms of action of dipeptidyl peptidase-4 inhibitors. Endocr ported by the Medical Research Council (MR/N02589X/1), British Rev. 2014;35(6):992-1019. Heart Foundation (FS/14/43/30960), and NIH (R01 DK095757). 14. Song Y, Koehler JA, Baggio LL, Powers AC, Sandoval DA, Drucker DJ. Gut-proglucagon-derived peptides are essential for regulating glucose homeostasis in mice. Cell Metab. Additional Information 2019;30(5):976-986.e3. Correspondence: Dr. Daniel J. Drucker, LTRI, Mt. Sinai Hos- 15. Nauck MA, Meier JJ, Cavender MA, Abd El Aziz M, Drucker DJ. pital, 600 University Ave Mailbox 39 TCP5-1004, Toronto, Ontario Cardiovascular actions and clinical outcomes with glucagon- M5G1X5, Canada. Email: [email protected] like Peptide-1 receptor agonists and dipeptidyl Peptidase-4 in- Disclosures: D.J.D. has been a consultant or speaker for Forkhead hibitors. Circulation. 2017;136(9):849-870. Therapeutics, Intarcia, Kallyope, Merck, and Novo Nordisk Inc. in the 16. Kristensen SL, Rørth R, Jhund PS, et al. Cardiovascular, mor- last 12 months. Mt. Sinai Hospital has received grants from Merck, tality, and kidney outcomes with GLP-1 receptor agonists in Novo Nordisk, and Takeda Inc. for investigator-initiated preclinical patients with type 2 diabetes: a systematic review and meta- studies of glucagon-like peptide action in the Drucker laboratory. analysis of cardiovascular outcome trials. Lancet Diabetes D.J.D. does not hold stock or equity in any of these companies. Endocrinol. 2019;7(10):776-785. 17. Noyan-Ashraf MH, Momen MA, Ban K, et al. 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