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Which human cell lines do not express the GLP-1 receptor?

Which human cell lines do not express the GLP-1 receptor?



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I need a human cell line that does not express the GLP-1 (glucagon like peptide-1) receptor.

I'm working with HeLa cells, do those express the GLP-1 receptor? Which other cell lines exist that don't express this specific receptor?

Are there any general resources where I could find this kind of information?


AbCam suggests HeLa cells as positive controls for their antibody to GLP1R. They provide the following pictures of HeLa cells labeled with their antibody:

(The image of the right is treated with synthesized peptide.)

According to Wikipedia, GLP1R is also expressed in pancreatic beta cells and the brain.


Cell lines

Cell lines are often used in place of primary cells to study biological processes. However, care must be taken when interpreting the results as cell lines do not always accurately replicate the primary cells. In this article, we will briefly talk about advantages and disadvantages of cell lines and then discuss results using the mouse Sertoli cell line, MSC-1, compared with primary mouse Sertoli cells. MSC-1 cells resemble Sertoli cells morphologically and possess several biochemical markers associated with Sertoli cells. Studies have demonstrated that the function and regulation of retinoic acid receptor α (RARα) is similar between MSC-1 and rat Sertoli cells. However, MSC-1 cells lack some of the immune privilege properties associated with primary Sertoli cells, including survival in animals with a fully functional immune system. Therefore, it has to be kept in mind that cell lines do not behave identically with primary cells and should not be used to replace primary cells. In order to strengthen the findings, key control experiments using primary cells should always be performed.

Immortal cell lines are often used in research in place of primary cells. They offer several advantages, such as they are cost effective, easy to use, provide an unlimited supply of material and bypass ethical concerns associated with the use of animal and human tissue. Cell lines also provide a pure population of cells, which is valuable since it provides a consistent sample and reproducible results. Cell lines have revolutionized scientific research and are being used in vaccine production, testing drug metabolism and cytotoxicity, antibody production, study of gene function, generation of artificial tissues (e.g., artificial skin) and synthesis of biological compounds e.g., therapeutic proteins. 1 - 3 Cell line popularity can be estimated by the numerous publications using cell lines and American Type Culture Collection (ATCC) Cell Biology Collection which consists of over 3,600 cell lines from over 150 different species. However, despite being a powerful tool, one must be careful when using cell lines in place of primary cells. Cell lines should display and maintain functional features as close to primary cells as possible. This may particularly be difficult to determine as often the functions of the primary cells are not entirely understood. Since cell lines are genetically manipulated this may alter their phenotype, native functions and their responsiveness to stimuli. Serial passage of cell lines can further cause genotypic and phenotypic variation over an extended period of time and genetic drift can also cause heterogeneity in cultures at a single point in time. Therefore, cell lines may not adequately represent primary cells and may provide different results. The other major problems associated with cell lines are contamination with other cell lines and mycoplasma. The bitter truth of cross-contamination of cell lines either inter or intraspecies was exposed by Walter Nelson-Rees in the early 1970s. He showed that at that time point the majority of cell lines being used worldwide and distributed by cell banks were contaminated with HeLa cells. 4 This still remains a problem even after 40 y. 5 , 6 When contamination of a cell line occurs whereby a very rapidly proliferating cell line is introduced, it only takes a few passages until the culture is entirely taken over by the contaminating cell line. HeLa cell contamination is well known to cause such problems. Additionally, mycoplasma contamination can persist undetected in cell cultures for a long period of time and cause extensive alterations in gene expression and cell behavior. Based on submissions to cell banks, 15�% of cell lines were estimated to be contaminated with mycoplasma. 7 , 8 Therefore, great care should be taken when using cell lines and experiments where key findings are confirmed in primary cultures should always be included.

Herein we share our experience using an immortalized mouse Sertoli cell line (MSC-1), that was developed in 1992 by Peschon et al. 9 This cell line was isolated from transgenic mice containing Sertoli cells transformed by the small and large T-antigens of the SV40 virus, which were targeted to Sertoli cells using the promoter for Mullerian inhibiting substance. MSC-1 cells were similar to primary Sertoli cells morphologically and expressed many of the same genes as primary Sertoli cells. 9 , 10 Although, follicle-stimulating hormone receptor (FSHr) and Mullerian inhibiting substance were not detected in MSC-1 cells. 9 , 10

Previously, MSC-1 cells were used to study the function and regulation of retinoic acid receptor α (RARα). In these studies, retinoic acid, activation of protein kinase C (PKC) and mitogen activated protein kinase (MAPK) were shown to increase the nuclear localization and transcriptional activity of RARα. 11 Additionally, peroxisome proliferators inhibited the retinoic acid-induced nuclear localization and transcriptional activity of RARα, while increasing the nuclear localization and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in MSC-1 cells. 12 Importantly, the results were confirmed in primary Sertoli cells isolated from 20-d old rats, 11 , 12 which verified that RARα nuclear localization and transcription were regulated by retinoic acid, PKC, MAPK and peroxisome proliferators. This demonstrates that RARα regulation and function is similar in MSC-1 and primary Sertoli cells and that MSC-1 cells can be used as a model to study RARα regulation in primary Sertoli cells.

However, not all results using the MSC-1 cell line are consistent with results from primary Sertoli cells as illustrated by studies on immune privilege. 13 , 14 Immune-privileged sites are anatomical sites where foreign tissues survive for extended periods of time because immune surveillance is reduced, and thus foreign antigens can be tolerated without evoking a detrimental immune response. The testis is an immune-privileged site that results in protection of the auto-immunogenic germ cells (when germ cells are removed from the testis and injected at a different site in the same animal, the cells are rejected). 15 Sertoli cells play an important role in creating this immune-privileged environment by expressing several immunoregulatory factors. 16 - 19 Moreover, isolated Sertoli cells survive and prolong the survival of co-transplanted cells when transplanted as allografts 20 , 21 or xenografts. 22 Similarly, Sertoli cells grafted alone across species survive longer than other cell types. 23 , 24

To compare the immunoprotective properties of MSC-1 cells with primary Sertoli cells, MSC-1 cells were co-transplanted with BALB/c pancreatic islets as allografts into diabetic C3H mice. The islets were rejected in 32.8 ± 8.4 d, which was not significantly different from control mice that received allogeneic islets alone (26.9 ± 2.1 d). In contrast, co-transplantation of BALB/c primary Sertoli cells with BALB/c islets as allografts into diabetic C3H mice significantly prolonged islet graft survival (> 61.1 ± 6.9 d), with 59% of the Sertoli cell/islet co-grafts surviving throughout the study period. 14 In addition, 100% graft survival was observed when primary Sertoli cells were transplanted alone as allografts into naïve BALB/c mice for 20 d. 13 In contrast, MSC-1 cells were unable to protect co-grafted cells in diabetic animals and were themselves rejected when transplanted into naïve mice with a fully functional immune system. 13 , 14 This emphasizes the importance of being cautious before assuming results obtained from cell lines are the same as those obtained using primary cells.

Interestingly, MSC-1 cells did survive in 66% of the recipient diabetic mice even though the islet grafts were rejected. 14 This is most likely due to the suppressed immune system associated with diabetes and suggests MSC-1 cells express some immunoprotective factors but lack or have lower expression of the key factors needed for immune protection of co-grafted cells and for fully functional immune privilege. Thus, MSC-1 cells may not mimic the survival and immune privilege properties of primary Sertoli cells but are useful as a control cell line to identify the key mechanisms or factors important for primary Sertoli cell immune privilege. To identify genes and immune-related functional pathways that are differentially regulated in these cells gene expression profiles of primary mouse Sertoli cells and MSC-1 cells were compared by microarray and ontological analyses. 13 We found that 2,369 genes were expressed with a ± 4-fold or higher level in primary Sertoli cells than in MSC-1 cells. Genes involved in immune functions were identified and differentially expressed. 13 While the Sertoli cells and MSC-1 cells express many of the same genes, they were expressed at different levels which appear to result in different immune regulatory functions. This confirms that the MSC-1 cell line is substantially different from primary mouse Sertoli cells and reiterates the importance of being cautious when making conclusions based on the results from cell lines.

As mentioned earlier, FSHr was not detected in MSC-1 cells. 10 FSHr is activated by follicle-stimulating hormone (FSH) and is important for Sertoli cell proliferation, macromolecular synthesis, morphological structure, and ultimately the spermatogenic capability. 25 However, the role of FSH in creation of an immune privileged environment is not clear. In one study there was an increase in testicular graft size/survival after transplanting to oophorectomized rodents that correlated with FSH and luteinizing hormone (LH) levels. 26 Additionally, Selawry et al., demonstrated that media collected from rat Sertoli cells cultured at 37ଌ for 24 h supplemented with FSH significantly inhibited the ConA stimulated proliferation of spleen lymphocytes, suggesting FSH may be important for Sertoli cells immune protection. 27 In contrast, the same group also demonstrated that protection of cellular grafts within the testis was not dependent on FSH or LH as treatment of rats with a gonadotropin-releasing hormone (GnRH) analog or hypophysectomy had no effect on the survival of transplanted intratesticular islet allografts. 28

Since FSHr is known to be important for the function of primary Sertoli cells and MSC-1 cells lack FSHr, the survival of MSC-1 cells stably transfected with FSHr (MSC-1FSHr) was examined after allotransplantation. MSC-1FSHr cells were shown previously to express functional FSHr as demonstrated by northern blot analysis and increased c-fos mRNA after FSH treatment. 29 Prior to transplantation, the expression of FSHr was confirmed by RT-PCR and as expected, FSHr mRNA was not detected in MSC-1 cells ( Fig.ꀚ , lane 3) while MSC-1FSHr cells expressed FSHr mRNA ( Fig.ꀚ , Lane 2). Four million MSC-1 or MSC-1FSHr cells were cultured as aggregates ( Fig.ꀜ and D ) and transplanted into naïve BALB/c mice as allografts. Graft-bearing kidneys were removed 20 d post-transplantation and examined for cell survival by immunohistochemistry for SV-40 large T antigen and RT-PCR for FSHr. Consistent with the previous survival data in naïve mice, MSC-1 cell grafts were rejected (0/6) by 20 d post-transplantation ( Fig.ꀟ ). Similarly, MSC-1FSHr grafts were also rejected in naïve BALB/c animals and no large T antigen positive MSC-1FSHr cells or FSHr mRNA were detected at 20 d post-transplantation (0/7) ( Fig.ꀞ , data not shown). In contrast, both MSC-1 (2/2) and MSC-1FSHr (4/4) cells survived in diabetic mice at 20 d post-transplantation as shown by large T antigen staining ( Fig.ਁG–H ) and RT-PCR for FSHr mRNA (MSC-1FSHr only data not shown). However, the MSC-1FSHr grafts were slightly smaller than the MSC-1 cell grafts ( Fig.ਁ , compare G and H). This indicates that the addition of functional FSHr to MSC-1 cells does not compensate for the loss of immune privilege.

Figureਁ. FSHr mRNA expression and survival of MSC-1 and MSC-1FSHr cells as allografts. MSC-1 cells stably transfected with rat FSHr cDNA were obtained from Dr. Griswold (Washington State University, Pullman, WA). 29 MSC-1FSHr cells were maintained and cultured essentially the same as MSC-1 cells with the exception of the addition of 250 mg/ml G418 (Invitrogen, Carlsbad, CA). A and B) RT-PCR was performed for FSHr (A), Lanes 2 and 3 Primers-For 5� TTG TGT CCT CAT CAA GC, Rev 5�T GGA AGT TGT GGG TAG CG) or cyclophilin (B), Lanes 2 and 3 Primers-For 5� ACC GTG TTC TTC GAC, Rev 5𠌪TC TTC TTG CTG GTC TTG CC) with RNA isolated from MSC-1FSHr (A and B, Lane 2) or MSC-1 cells (A and B, Lane 3). Lane 1 (A and B) is 1 kb Plus DNA Ladder (Invitrogen). (C and D) MSC-1 (D) or MSC-1FSHr (C) cells were cultured as aggregated for 48 h. Aggregates were fixed, dispersed in agar, embedded in paraffin, sectioned and immunostained for large T antigen (brown color) and hematoxylin (blue color). (E-H) Four million of these aggregated cells were transplanted under the kidney capsule of naïve (E and F) and diabetic (G and H) BALB/c mice. The grafts were collected at day 20 post-transplantation, and tissue sections were immunostained for MSC-1 cell marker, large T antigen (brown color, E-H). All sections were counterstained with hematoxylin (blue color). A dotted line separates the kidney from the graft. K, kidney Arrow, large T antigen positive cells. Care and maintenance of animals described in (E-H) was performed in accordance with the Institute for Laboratory Animal Research Care and Use of Laboratory Animals, and Texas Tech University Institutional Animal Care and Use Committee-approved protocols.

Immune privilege involves a complex interplay between immunoregulatory factors, the transplant environment and the host’s immune system. Thus, addition of just one factor e.g., FSHr to a cell line does not make it immune-privileged. Other studies have identified several potential pathways or factors that may contribute to Sertoli cell immune privilege. 13 For example, Sertoli cells express or secrete complement inhibitors, apoptosis inhibitors and factors that modulate the immune response. Thus, it seems likely that a combination of several factors is required to make Sertoli cells immune-privileged. Overall, the MSC-1 cell line may serve as a good comparison cell line to study key factors/mechanisms required for primary Sertoli cell immune privilege but they should not be used in place of primary Sertoli cells to study survival mechanisms.

A different MSC-1FSHr cell line was created by Eskola et al. 30 In this cell line, intact FSHR signaling and function, similar to Sertoli cells was verified by cAMP response to FSH and PKC. Antiproliferative effects of FSH on MSC-1FSHr further demonstrated that these cells resemble adult Sertoli cells and thus can be used a model to study posttranscriptional regulation of FSHR and its signal transduction. 30 However, regulation of inhibin-α expression in response to FSH was different from primary Sertoli cells. In a separate study, the basal and cAMP regulated expression of PKA subunits was compared in MSC-1 cells to rat Sertoli cells. 31 This study demonstrates that the RIIβ mRNA basal levels, magnitude of induction of RIIβ mRNA by cAMP, half-life after cAMP removal and mRNA induction independent of protein synthesis is different from primary rat Sertoli cells. 31 These results further demonstrate that even though a Sertoli cell line retains major characteristics of primary Sertoli cells they do not completely replicate primary Sertoli cells.

In conclusion, cell lines are a powerful tool and offer several advantages over primary cells. However, it must be understood that cell lines do not completely mimic primary cells. Therefore, great caution should be taken when designing experiments to assure that the conclusions drawn from cell line are sound. Key experiments should also be replicated in primary cells.

Finally, it should be recognized that a weakness of in vitro cell cultures, both primary cells and cell lines, is that they are being studied in the absence of their local environment that often includes interactions with other cell types that may be critical to the hypothesis being tested. Sertoli cells are well known to interact with other cell types in the local environment and therefore these cells are particularly vulnerable to deficiencies of the isolated or enriched culture environment.


Abstract

This review summarizes current knowledge about glucagon-like peptide 1 receptor agonists (GLP-1 RA) and their effects on bone metabolism and fracture risk. Recent in vivo and in vitro experiments indicated that GLP-1 RA could improve bone metabolism. GLP-1 could affect the fat-bone axis by promoting osteogenic differentiation and inhibiting adipogenic differentiation of bone mesenchymal precursor cells (BMSCs), which express the GLP-1 receptor. GLP-1 RA may also influence the balance between osteoclasts and osteoblasts, thus leading to more bone formation and less bone resorption. Wnt/β-catenin signalling is involved in this process. Mature osteocytes, which also express the GLP-1 receptor, produce sclerostin which inhibits Wnt/β-catenin signalling by binding to low density lipoprotein receptor-related protein (LRP) 5 and preventing the binding of Wnt. GLP-1 RA also decreases the expression of sclerostin (SOST) and circulating levels of SOST. In addition, GLP-1 receptors are expressed in thyroid C cells, where GLP-1 induces calcitonin release and thus indirectly inhibits bone resorption. Furthermore, GLP-1 RA influences the osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK) system by increasing OPG gene expression, and thus reverses the decreased bone mass in rats models. However, a recent meta-analysis and a cohort study did not show a significant relationship between GLP-RA use and fracture risk. Future clinical trials will be necessary to investigate thoroughly the relationship between GLP-1 RA use and fracture risk in diabetic patients.


1 INTRODUCTION

Glucagon-like peptide-1 (GLP-1) is a gut hormone secreted from enteroendocrine cells in the intestine. It derives from posttranslational processing of proglucagon and exerts various metabolic actions on multiple organs and tissues through a family B G-protein-coupled receptor (GPCR), the GLP-1 receptor. In 1987, Jens Holst (Denmark) and Joel Habener (USA) first described and identified the native peptides GLP-1 (7–36, amide extended and 7–37, glycine extended, collectively referred as GLP-1) as secretory products from enteroendocrine cells in the gut of mammals (Holst et al., 1987 Mojsov et al., 1987 ). Further preclinical and clinical studies demonstrated that GLP-1 is a potent insulinotropic agent in healthy rodents and humans with type 2 diabetes mellitus (T2DM). Due to the fact that GLP-1 lowered plasma blood glucose in subjects with persistent hyperglycaemia, it has become the most extensively studied gut-derived hormone (Nauck, Kleine, et al., 1993 ). GLP-1 stimulates glucose-dependent insulin secretion from beta cells in the pancreas, reduces glucagon secretion from alpha cells and slows down gastric emptying, which prohibits postprandial hyperglycaemia (Nauck, Heimesaat, et al., 1993 Wettergren et al., 1993 ). However, a pharmaceutical use of GLP-1 as a new treatment option in T2DM has been complicated by the fact that it is rapidly proteolytically degraded by the ubiquitous protease dipeptidyl peptidase-4 (DPP-4) (see Figure 1). Therefore, GLP-1 receptor agonists resistant to the proteolytic inactivation by DPP-4 (e.g. exendin-4, liraglutide and semaglutide) or inhibitors of DPP-4 (e.g. sitagliptin and linagliptin) were developed and are nowadays established guideline therapy of T2DM.

The treatment of T2DM is of utmost importance since diabetic health complications are associated with 1.5 million global deaths per year plus an additional 2.2 million deaths by an indirect increase of risk factors that mostly comprise cardiovascular (CV) events such as myocardial infarction (MI) or stroke (Disease et al., 2018 ). Accordingly, negative CV side effects are a major risk for diabetic individuals, which is supported by a meta-analysis reporting a hazard ratio of 3.42 (95% confidence interval [CI]: 2.23–5.23) for CV mortality of diabetic individuals (Nakagami et al., 2006 ). In order to obtain approval of GLP-1 receptor agonists as a new treatment option of T2DM from the US Food and Drug Administration (FDA), clinical studies had to be conducted to test the CV safety compared with standard care and placebo in the target population resulting in a number of prominent cardiovascular outcome trials. The typical primary endpoint was the occurrence of a major adverse cardiovascular event (MACE: time to the first event of either CV death or non-fatal MI or stroke). Among all GLP-1receptor agonists that underwent the approval process, four (albiglutide, dulaglutide, semaglutide and liraglutide) overachieved the study goal. They were not only safe, but they significantly reduced MACE in diabetic patients at risk or with established cardiovascular disease (CVD) (Table 1). These promising results from the cardiovascular outcome trials still challenge the scientific field to better understand the mechanisms behind these outstanding beneficial effects. One important point is certainly a modification of CV risk factors by GLP-1 therapy. Blood pressure reduction, weight loss and improved blood lipids are likely to contribute to the observed favourable CV outcomes. However, the fact that GLP-1 receptor agonists have anti-inflammatory and anti-oxidant effects and other direct effects on cells and tissues of the CV system (e.g. epigenetic changes) should not be underestimated.

Cardiovascular outcome trials of selected GLP-1 agonists
Trial ELIXA LEADER SUSTAIN-6 EXSCEL REWIND HARMONY
Lixisenatide Liraglutide Semaglutide Exenatide Dulaglutide Albiglutide
Primary outcome 1.02 (0.89–1.17) P < .001 for noninferiority P = .81 for superiority CV death, MI, stroke 0.87 (0.78–0.97) P < .001 for noninferiority P = .01 for superiority CV death, MI, stroke 0.74 (0.58–0.95) P < .001 for noninferiority P = .02 for superiority CV death, MI, stroke 0.91 (0.83–1.00) P < .001 for noninferiority P = .06 for superiority CV death, MI, stroke 0.88 (0.79–0.99) P = .026 for superiority CV death, MI, stroke, UA 0.78 (0.68–0.90) P < .0001 for noninferiority P = .0006 for superiority CV death, MI, stroke
Secondary outcome 1.0 (0.90–1.11) P = .96 CV death, MI, stroke, UA, HF hosp., revascul. 0.88 (0.81–0.96) P = .005 CV death, MI, stroke, UA or HF hosp., revascul. 0.74 (0.62–0.89) P = .002 CV death, MI, stroke, UA or HF hosp., revascul. n/a n/a 0.78 (0.69–0.90) P = .0005 CV death, MI, stroke, urgent revascul. For UA, individual components of the primary endpoint, CV death/hospital admission because of heart failure
CV death 0.98 (0.78–1.22) P = .85 0.78 (0.66–0.93) P = .007 0.98 (0.65–1.48) P = .92 0.88 (0.76–1.02) 0.91 (0.78–1.06) P = .21 0.93 (0.73–1.19) P = .578
All cause-death 0.94 (0.78–1.13) P = .5 0.85 (0.74–0.97) P = .02 1.05 (0.74–1.50) P = .79 0.86 (0.77–0.97) 0.90 (0.80–1.01) P = .067 0.95 (0.79–1.16) P = .644
HF hospitalisation 0.96 (0.75–1.23) P = .75 0.87 (0.73–1.05) P = .14 1.11 (0.77–1.61) P = .57 0.94 (0.78–1.13) 0.93 (0.77–1.12) a a Hospital admission for heart failure or urgent visit.
P = .46
0.85 (0.70–1.04) b b Composite of death from cardiovascular causes or hospital admission for heart failure, ELIXA trial (Pfeffer et al., 2015 ), LEADER trial (Marso, Daniels, et al., 2016 ), SUSTAIN-6 trial (Marso, Bain, et al., 2016 ), EXSCEL trial (Holman et al., 2017 ), REWIND trial (Gerstein et al., 2019 ), HARMONY trial (Hernandez et al., 2018 ).
P = .113
Reduction of glycated haemoglobin −0.27 pps (−0.31 to −0.22) P < .001 −0.40 pps (−0.45 to −0.34) 0.5 mg semaglutide: −0.7 pps 1 mg semaglutide: −1.0 pps −0.53% (−0.57 to −0.50) P < .001 −0.61% (−0.65 to −0.58) P < .0001 Difference at 8 months: −0.63% (−0.69 to −0.58) difference at 16 months: −0.52% (−0.58 to −0.45)
  • Note: GLP-1 receptor agonists revealing a CV benefit relative to standard care are marked in bold.
  • Abbreviations: CV death, cardiovascular death HF, heart failure hosp., hospitalisation MI, myocardial infarction n/a, not available pps, percentage points revascul., revisualisation UA, unstable angina.
  • a Hospital admission for heart failure or urgent visit.
  • b Composite of death from cardiovascular causes or hospital admission for heart failure, ELIXA trial (Pfeffer et al., 2015 ), LEADER trial (Marso, Daniels, et al., 2016 ), SUSTAIN-6 trial (Marso, Bain, et al., 2016 ), EXSCEL trial (Holman et al., 2017 ), REWIND trial (Gerstein et al., 2019 ), HARMONY trial (Hernandez et al., 2018 ).

GLP-1 binds to the GLP-1 receptor, a GPCR that stimulates the adenylyl cyclase pathway in pancreatic cells, resulting in insulin synthesis and subsequent release to the bloodstream (Drucker et al., 1987 ). Thereby, GLP-1 and GLP-1 receptor agonists primarily exert their effects on blood glucose control of T2DM patients. Today, plenty of studies have shown that GLP-1 acts on several organs and tissues. In some cases, it is not clear whether these effects are directly mediated via the GLP-1 receptor, but the GLP-1 receptor has been shown to be expressed on many cell types, also in the CV system. Figure 2 provides information on the transcripts per million (TPM) for GLP-1 receptor gene (GLP1R) expression in human tissues located in the CV system (blood, heart and vessels). As a reference, the highest expression can be found in the pancreas with 4.7 TPM, whereas 1.7 TPM have been detected in the atrial appendage of the heart and no transcripts in aortic tissue.

Besides the various expression patterns in rodent models and humans, the mechanistic exploration of direct/indirect CV effects of GLP-1 is complicated by the fact that many GLP-1 receptor antibodies and antisera exhibit suboptimal sensitivity and a lack of specificity (Panjwani et al., 2013 ).

In the present review, we will summarise the recent mechanistic evidence for CV protective effects of GLP-1 receptor agonists, beyond glycaemic control. Special focus will be laid on pathways activated by direct GLP-1 receptor interaction and the evidence given for predominant cell types within the CV system susceptible to GLP-1 receptor activation.


Results

GLP-1R expression in human prostate cancer is inversely associated with cancer progression

As we reported previously 10 , GLP-1R is observed in prostate cancer tissue in non-diabetic individuals and colocalizes with P504S, a prostate cancer marker. Interestingly, as shown in Figure 1a, expression levels of GLP-1R were decreased in advanced prostate cancer cases categorized by Gleason score 13 . When the 30 patients were divided into three groups according to Gleason score, expression of GLP-1R in prostate cancer was significantly decreased in advanced prostate cancer patients with high Gleason scores compared with early-stage prostate cancer patients (Figure 1b).

Forced expression of GLP-1R attenuates prostate cancer cell proliferation

To elucidate the effect of GLP-1R expression on prostate cancer, we overexpressed GLP-1R in prostate cancer cells. In our previous report 10 , endogenous GLP-1R expression was negligibly detected in ALVA-41 cells. In the present study, we overexpressed human GLP-1R in ALVA-41 cells using a lentiviral vector. As shown in Figure 1c, GLP1R gene expression was abundantly detected in ALVA-41 cells transfected with the lentiviral vector carrying the human GLP1R gene (ALVA-41-GLP-1R cells) compared with LNCaP cells that express endogenous GLP-1R. However, GLP-1R expression was not detected in ALVA-41 cells transfected with the empty lentiviral vector (ALVA-41-control cells). Furthermore, immunohistochemistry of GLP-1R confirmed significant membranous GLP-1R protein expression in ALVA-41-GLP-1R cells (Figure 1d). The functional effectiveness of overexpressed GLP-1R was demonstrated by intracellular cAMP induction in ALVA-41-GLP-1R cells stimulated with Ex-4 (Figure 1e).

We next examined the anti-proliferative effect of GLP-1R in ALVA-41 cells. As shown in Figure 2a, the number of ALVA-41-GLP-1R cells was slightly, but significantly, reduced compared with ALVA-41-control cells without GLP-1R agonist treatment. In addition, Ex-4 decreased the number of ALVA-41-GLP-1R cells in a dose-dependent manner, as shown by the growth curve in Figure 2b. However, ALVA-41-control cells did not respond to Ex-4 (Figure 2c). Consistent with the growth curve data, bromodeoxyuridine incorporation assays showed that the proliferation of ALVA-41-GLP-1R cells was significantly decreased compared with that of ALVA-41-control cells (Figure 2d). In addition, Ex-4 attenuated ALVA-41-GLP-1R cell proliferation in a dose-dependent manner, but had no impact on ALVA-41-control cell proliferation (Figure 2e). Similar to our previous report using LNCaP cells 10 , GLP-1R activation did not induce apoptosis of ALVA-41-GLP-1R cells (Figure 2f).

Forced expression of GLP-1R attenuates cell cycle progression through inhibition of SKP2 and upregulation of p27Kip 1

We next examined the mechanism by which overexpressed GLP-1R attenuated ALVA-41 cell proliferation. First, we carried out cell cycle analysis by flow cytometry. As shown in Figure 3a, ALVA-41-GLP-1R cells in G0/G1 phase were increased and those in S phase were decreased compared with ALVA-41-control cells. Furthermore, Ex-4 treatment decreased not only S phase entry, but also G2/M phase transition of ALVA-41-GLP-1R cells (Figure 3b). Consistent with the apoptosis assay (Figure 2f), the sub-G1 fraction was not observed after Ex-4 treatment, further supporting suppression of apoptosis. Notably, significantly increased G0/G1 cells were observed in ALVA-41-GLP-1R cells compared with ALVA-41-control cells in Figure 3a, but not in Figure 3b. This might be an experimental limitation, because the experiment shown in Figure 3b involved a 12-h longer incubation time. We found that exendin (9–39), a GLP-1R antagonist (Figure 3c) and inhibitor of protein kinase A (Figure 3d), significantly counteracted Ex-4-induced attenuation of cell proliferation, indicating that Ex-4 inhibited cell proliferation through the activation of GLP-1R and cAMP-protein kinase A signaling, a canonical pathway of GLP-1R. In our previous report using LNCaP cells, Ex-4 attenuated cell proliferation through inhibition of ERK 10 . However, ERK was not activated in ALVA-41 cells (Figure 3e). To confirm the anti-proliferative effect of intracellular cAMP induced by GLP-1R, we carried out bromodeoxyuridine assay with forskolin, which is a ubiquitous activator of eukaryotic adenylyl cyclase, to increase the cAMP level. As shown in Figure 3f, forskolin significantly decreased cell proliferation in ALVA-41 cells and further reduction of cell proliferation was observed in ALVA-41-GLP-1R cells.

Because G0/1 arrest was induced by GLP-1R activation in ALVA-41 cells, we carried out further experiments focusing on cell cycle regulators. Western blotting showed no significant differences in Rb protein phosphorylation and cyclin D1 expression between ALVA-41-GLP-1R and ALVA-41-control cells without Ex-4 treatment (Figure 4a,b). However, p27Kip1, a negative regulator of the G0/1-to-S phase transition, was significantly increased in ALVA-41-GLP-1R cells compared with ALVA-41-control cells (Figure 4c). Furthermore, Ex-4 treatment significantly decreased Rb phosphorylation (Figure 4d) and cyclin D1 expression (Figure 4e), and significantly increased p27Kip1 expression (Figure 4f) in ALVA-41-GLP-1R cells, but not in ALVA-41-control cells. Because p21Kip1 protein levels are post-translationally regulated by SKP2 ubiquitin ligase, we next examined SKP2 messenger ribonucleic acid expression by quantitative RT–PCR. As shown in Figure 4g, SKP2 gene expression was decreased significantly by Ex-4 in ALVA-41-GLP-1R cells, but not in ALVA-41-control cells.

Forced expression of GLP-1R attenuates prostate cancer growth in vivo independent of glucose metabolism

To determine the anti-prostate cancer effect of overexpressed GLP-1R in vivo, we implanted ALVA-41-GLP-1R or ALVA-41-control cells, which stably express cytomegalovirus-luciferase, as reported previously 16 , into athymic nude mice. Four weeks after subcutaneous implantation of ALVA-41 cells into the flank region of mice, tumor formation was visualized by in vivo imaging of the fluorescence intensity derived from cytomegalovirus-luciferase in ALVA-41 cells just before being euthanized (Figure 5a). The tumor growth measured by the fluorescence intensity of ALVA-41-GLP-1R cells was decreased compared with that of ALVA-41-control cells without Ex-4, but it was not statistically significant. However, Ex-4 treatment significantly reduced the tumor growth of ALVA-41-GLP-1R cells compared with that of ALVA-41-control cells without Ex-4 (Figure 5b). In resected tumors, the calculated tumor growth of ALVA-41-GLP-1R cells was attenuated, but it was not statistically significant (Figure 5c). However, the tumor weight of ALVA-41-GLP-1R cells was significantly decreased compared with that of ALVA-41-control cells without Ex-4, and Ex-4 treatment significantly decreased the tumor weight of ALVA-41-GLP-1R cells compared with that of ALVA-41-control cells treated with Ex-4 (Figure 5d). During the experimental period, serum glucose levels and bodyweights were not significantly different between the four groups (Figure 5e,f).


GLP-1 Safety

The most common side effects or adverse events associated with GLP-1 therapy is nausea, and occasionally vomiting, followed by diarrhea/constipation. For the majority of subjects, the extent of nausea generally diminishes over time, and most subjects do not report ongoing nausea after several months of therapy with a GLP-1 R agonist. Diarrhea has also been reported in subjects taking GLP-1 R agonists. Other reported AEs may include hypoglycemia, particularly if a GLP-1R agonist is used together with an insulin secretagogue, such as a sulfonylurea. Injection site reactions, including itching and skin rashes have also been described. Less commonly, allergic reactions have been reported. An enormous amount of literature, describing both preclinical clinical studies, has addressed the topic of incretin-based safety. Some of the data is summarized below.

The United States Food and Drug Administration announced March 14 2013 it is investigating reports of pancreatitis and unpublished preclinical findings related to potential precancerous findings in studies of the pancreas using incretin-based therapies. The European Medicines Agency also investigated, in the spring and summer of 2013, the link between GLP-1 action and possible development of pancreatitis and cancer. EMEA concluded in July 2013 that " In conclusion, the results of the study by Butler et al are not considered to constitute a new safety signal for the GLP 1 based therapies with respect to pancreatic safety. This is further supported by the review of available preclinical and clinical data". The Assessment report for GLP-1 based therapies is a public document. Moreover, in a joint summary paper published in the NEJM on February 27 2014, authors from bth EMEA and FDA summarized independent reviews of data from academic labs, the FDAs own analyses, and review of all relevant clinical trial data. The authors stated that:

Both agencies agree that assertions concerning a causal association between incretin-based drugs and pancreatitis or pancreatic cancer, as expressed recently in the scientific literature and in the media, are inconsistent with the current data. FDA and EMA have not reached a final conclusion at this time regarding such a causal relationship. Although the totality of the data that have been reviewed provided reassurance, pancreatitis will continue to be considered a risk associated with these drugs until more data are available. FDA and EMA believe that the current knowledge is adequately reflected in the product information or labeling, and further harmonisation among products is planned in Europe

Many of the studies that have informed our understanding of incretin safety, and in some cases, generated considerable debate surrounding scientific veracity, are summarized below.

Pancreatitis and Pancreatic cancer

Clinical perspectives

To review the May 2007 AGA criteria for diagnosis and management of acute pancreatitis, see AGA Institute medical position statement on acute pancreatitis. The diagnosis of acute pancreatitis in subjects with diabetes may be more challenging than in non-diabetic subjects due to a much greater proportion of asymptomatic diabetic subjects that exhibit increased circulating levels of lipase and amylase, without other classic symptoms or signs or imaging abnormalities associated with pancreatitis. Increased variability and abnormalities in pancreatic enzyme concentrations in otherwise asymptomatic subjects with type 2 diabetes Diabetes Metab Syndr Obes. 20125:419-24

The vast majority of epidemiologic studies assessing possible links between GLP-1 R agonists and DPP-4 inhibitors have been retrospective database analyses, which have multiple scientific limitations. Meier & Nauck reported a meta-analyses for the available data describing pancreatitis events in phase 3 trials for DPP-4 inhibitors and GLP-1 R agonists. The odds ratio for acute pancreatitis with a GLP-1 R agonist was 1.39, whereas for exposure to a DPP-4 inhibitor, it was 1.07, however the total number of patients studied and absolute event rates were small (2.1 events of pancreatitis per 1,000 patient years), greater numbers of subjects followed for longer periods of time would be required to increase the statistical power of the conclusions. Risk of pancreatitis in patients treated with incretin-based therapies Diabetologia. 2014 Apr 11.

Pancreatitis rates have been reported in subjects randomized to either saxagliptin (SAVOR TIMI53) or alogliptin (EXAMINE) in cardiovascular outcome studies. Pancreatitis events were independently adjudicated. The number of events of all pancreatitis (acute and chronic) were similar in saxagliptin vs. placebo-treated subjects (mean follow up period of 2.1 years, 16,884 person-years in the saxagliptin
group and 16,761 person-years in placebo group ). Although slightly more events of acute pancreatitis and fewer cases of chronic pancreatitis were reported in subjects randomized to saxagliptin, the event rates were low, and the small differences did not reach statistical significance. Between 80-90% of patients with pancreatitis had other classical predisposing risk factors and the time to onset of pancreatitis was not different across treatment groups. Fewer events of pancreatic cancer were reported in subjects randomized to saxagliptin. Incidence of Pancreatitis and Pancreatic Cancer in a Randomized Controlled Multicenter Trial (SAVOR-TIMI 53) of the Dipeptidyl Peptidase-4 (DPP-4) Inhibitor Saxagliptin Diabetes Care. 2014 Jun 9. pii: DC_132546

Adverse event reporting has raised an association between use of Exenatide and pancreatititis, an inflammation of the pancreas that can, in rare cases be quite severe, and even fatal. There is limited information about the true incidence of pancreatitis in patients with diabetes but most studies consistently show

2-fold increased rate pf panceatitis in diabetic subjects. A retrospective claims database analysis from Amylin Pharmaceuticals Inc (now BMS) suggested that patients with diabetes have a 2.8-fold greater risk of developing pancreatitis and a 1.9-fold greater risk of developing biliary tract disease the relative risk for pancreatitis was highest in younger patients les than 45 years of age- See Increased Risk of Acute Pancreatitis and Biliary Disease Observed in Patients with Type 2 Diabetes: a Retrospective, Cohort Study Diabetes Care 2009 May32(5):834-8 Analysis of hospitalized patients diagnosed with pancreatitis in a large health insurance data base was carried out using data assessed from June 2005-2008. Patients were analyzed by the type of anti-diabetic therapy they were using, with a specific focus on patients taking exenatide vs sitagliptin. Acute pancreatitis ocurred in 0.13% and 0.12% of patients taking exenatide vs. sitagliptin , respectively, rates comparable to patients on metformin/glyburide as outlined in Use of a claims-based active drug safety surveillance system to assess the risk of acute pancreatitis with exenatide or sitagliptin compared to metformin or glyburide Curr Med Res Opin. 2009 Apr25(4):1019-27.

Adverse events, including pancreatitis and pancreatic cancer, were assessed in a pooled meta-analysis of 25 clinical studies of sitagliptin (7,726 patients on sitagliptin vs. 6,885 non-exposed control subjects) ranging from 12 weeks to 2 years. Mean exposure to drug across the entire pooled analsysi was 284 days 32% of patients were treated with sitagliptin for at least 1 year, and 8% received sitagliptin for 2 years. The incidence of all reported malignancies, including pancreatic cancer was similar across treatment groups, however too few events were reported to calculate 95% confidence intervals. Angioedema was not more common after exposure to sitagliptin , and there was no difference in reported rates of pancreatitis. Rash was more commonly reported in sitagliptin -treated subjects Safety and Tolerability of Sitagliptin in Type 2 Diabetes: Pooled Analysis of 25 Clinical Studies Diabetes Ther. 2013 May 23

Wenten and colleagues assessed the relative risk of pancreatitis using a health care claims database in 482,034 eligible patients who had started a new medication for the treatment of type 2 diabetes 24,237 initiated exenatide twice daily and 457,797 initiated another anti-diabetic medication. Patients starting exenatide were felt to have more advanced forms of diabetes. No significant association was found between exenatide use and the development of diabetes. Relative risk of acute pancreatitis in initiators of exenatide twice daily compared with other anti-diabetic medication: a follow-up study. Diabet Med. 2012 Nov29(11):1412-8

Singh et al analyzed pooled health care claims administrative database data for cases of acute pancreatitis for diabetic subjects with administrative codes suggestive of inpatient pancreatitis, and compared rates of multiple variables, including exenatide or sitagliptin use, to rates reported for randomly selected controls. Pancreatitis reporting rates were

2.2-fold higher among recent users (prescriptions written, use not verified) of exenatide or sitagliptin . The duration of diabetes for cases vs. control was not reported. The authors chose not to report relative pancreatitis rates for patients treated with any other diabetes therapy (insulin, metformin, SUs, TZDs etc). Glucagonlike Peptide 1&ndashBased Therapies and Risk of Hospitalization for Acute Pancreatitis in Type 2 Diabetes Mellitus JAMA Intern Med. 2013():1-6. doi:10.1001/jamainternmed.2013.2720

Case reports of pancreatitis have also been described, and the FDA has provided updates through correspondence in the literature and periodic safety alerts, as outlined in FDA NEJM Pancreatitis and Information for healthcare professionals about Exenatide and pancreatitis .

Elashoff and colleagues reviewed case reports of adverse events submitted to the Adverse Event Reporting System Database from Q1 2004 through Q2 2009 with the aim of detecting cases of a) pancreatitis b) thyroid cancer and pancreatic cancer and c) all cancers linked to associated use of sitagliptin (DPP-4 inhibitor) or exenatide ( GLP-1 R agonist) for the treatment of type 2 diabetes. A brief FDA AERS overview provides independent information about the utility of the AERS database. Control rates were ascertained by Elashoff by querying rates for cancer, pancreatitis, and other pre-defined events that may or may not be associated with use of other anti-diabetic drugs rosiglitazone (Avandia), nateglinide (Starlix), repaglinide (Prandin), and glipizide. Control events that were also queried for use in this analysis were back pain, urinary tract infection, chest pain, cough, and syncope. The authors discarded the use of pioglitazone from their AERS analysis, since it was noted to be associated with an elevated reporting rates for control events not known to be associated with pioglitazone in other database analyses. Reported rates of pancreatitis were six-fold higher for exenatide and sitagliptin for pancreatitis and reported event rates in this analysis for pancreatic cancer was noted to be 2.9-fold greater with exenatide and 2.4-fold greater with sitagliptin. More thyroid cancer was reported in patients using exenatide and case report rates for "all other cancers" were higher with sitagliptin. See Increased Incidence of Pancreatitis and Cancer Among Patients Given Glucagon Like Peptide-1 Based Therapy Gastroenterology. 2011 Jul141(1):150-6. A number of epidemiological surveys and data base analyses reveal an increased incidence of multiple neoplasms, including pancreatic cancer in subjects with T2DM Association between diagnosed diabetes and self-reported cancer among U.S. adults: findings from the 2009 Behavioral Risk Factor Surveillance System Diabetes Care. 2011 Jun34(6):1365-8

A retrospective case control study of 268,561 subjects with diabetes revealed that over a several year period (2007-2009),

2.6% of subjects reported use of exenatide . Overall, hospitalization rates for acute pancreatitis were higher in subjects who did not use exenatide . A similar analysis of claims for pancreatic cancer revealed no significant association between exenatide use and pancreatic cancer diagnosis Exenatide therapy and the risk of pancreatitis and pancreatic cancer in a privately insured population Diabetes Technol Ther. 2012 Oct14(10):904-11

An overview of the safety issues and current understanding of the risk:benefit ratio for the use of GLP-1 R agonists and DPP-4 inhibitors was published in the July 2011 issue of JCEM The safety of incretin-based therapies review of the scientific evidence J Clin Endocrinol Metab 2011 96(7)2027-2031

A meta-analysis of serious adverse events reported during evaluation of clinical trials of at least 24 weeks duration for DPP-4 inhibitors (sitagliptin, saxagliptin, linagliptin, vildaglitpin, dutogliptin) focused on reporting of cancers, pancreatitis, and cardiovascular (MACE) events. No signal was reported for cancer or pancreatitis, whereas MACE events were relatively reduced in subjects exposed to DPP-4 inhibitors, however the short term nature of these studies precludes definitive conclusions. Safety of dipeptidyl peptidase-4 inhibitors: a meta-analysis of randomized clinical trials Curr Med Res Opin. 2011 Nov27 Suppl 3:57-64

Similarly, an independent meta-anlysis of 25 clinical trials using the GLP-1 R agonists exenatide or liraglutide found no conclusive evidence that the use of these agents was associated with a significantly increased risk of acute pancreatitis or cancer, compared to active comparators in the same trials A meta-analysis of serious adverse events reported with exenatide and liraglutide: acute pancreatitis and cancer Diabetes Res Clin Pract. 2012 Nov98(2):271-84

Butler and colleagues reported histopathological findings in pancreata obtained from organ donors with diabetes treated with (8) or without incretin-based therapy (12) vs. findings in non-diabetic controls (12). All subjects had been treated with incretin-based therapy ( sitagliptin (7) or exenatide (1) for at least a year, however the duration of exposure was not provided, and the interval between comencement of life support, death, procurement of the pancreas, and last exposure to an incretin agent was not described. There were substantial differences in regard to age (58), sex (6 M, 2F) and duration of diabetes (12) for pancreata from subjects exposed to incretins, vs control DM groups (4M, 8F), age (35) and 8 years of previous diabetes. All of the incretin-treated subjects but one were on 2 or more anti-diabetic medications whereas 5 DM controls were taking no anti-diabetic medications and 6 were treated with only 1 agent. Three of the control subjects (2 described in the paper, one in the database) yet none of the incretin-treated subjects had a history of diabetic ketoacidosis. The beta cell mass and pancreatic weight and age for the control diabetic subjects was generally much lower than for the diabetic pancreases from subjects treated with incretin therapy. Pancreatic intraepithelial neoplasia (PanIN) was scored histologically by a pathologist blinded to the clinical data. Donor subjects treated with incretin therapy exhibited an increase in pancreatic mass of 40% Beta cell mass was decreased by 55% in diabetic control subjects, whereas incretin-treated subjects exhibited a 6-fold increase in beta cell mass relative to diabetic controls and a 3-fold increase in beta cell mass vs. non-diabetic controls. This increase was largely secondary to an increase in beta cell number rather than size. The pancreatic fractional area immunopositive for glucagon was increased

2.8 fold in subjects treated with incretins, largely due to increased numbers of islet alpha cells. Enlarged islets andincreased numbers of insulin-positive and glucagon -positive cells adjacent to duct structures were more commonly observed in pancreatic from donor subjects treated with incretins. The single pancreas from an exenatide -treated individual exhibited marked alpha cell hyperplasia. A 1.5 cm glucagon -producing alpha cell tumor was detected in the pancreas from a subject treated with sitagliptin glucagon -producing microadenomas were also obsrved in 2 additional subjects and hyperplastic glucagon -immunopositive islets were detected in 7/8 pancreases from subjects that received incretin therapy. More islet cells exhibiting dual immunopositivity for both insulin and glucagon were detected in pancreata from incretin-treated subjects. Pancreatic intraepithelial neoplasia lesions (low grades 1&2, not broken out separately) were more common in pancreata exposed to incretins. Although the authors repeatedly speculate that reduced glucagon secretion arising secondary to incretin therapy likely accounts for the alpha cell hyperplasia they observed, they did not provide a reference linking partial reduction of glucagon secretion to the development of alpha cell hyperplasia. Marked Expansion of Exocrine and Endocrine Pancreas with Incretin Therapy in Humans with increased Exocrine Pancreas Dysplasia and the potential for Glucagon-producing Neuroendocrine Tumors Diabetes published ahead of print March 22, 2013, doi:10.2337/db12-1686. A commentary on this paper appeared online April 17 2013, raising many similar issues in regard to challenges in the interpretation of the data reported Incretin Therapy and Islet Pathology &ndash A Time for Caution Diabetes published ahead of print April 17, 2013, doi:10.2337/db13-0520

Extensive critical appraisal of the Butler pathology paper has been published by independent research groups. Harja and colleagues noted extensive differences between cases and controls, including differences in age of onset of diabetes, age of subjects analyzed, sex of subjects, inclusion of subjects with T1D (ketoacidsos, autoantibodies), missing data for pancreatic weight, and failure to note abnormalities of glucagon staining in control pancreata An analysis of characteristics of subjects examined for incretin effects on pancreatic pathology. Diabetes Technolo Therap 2013 Aug15(8):609-18 Bonner-Weir and colleagues re-examined the same pathological slides, where available, and raise multiple concerns about the interpretation of the data. In addition to the multiple significant differences between "controls" vs. cases noted above, they also highlight major methodological issues that challenge the validitiy of the data, including large variability in the intensity of chromogen Fast Red staining and problems with the methodology used to calculate endocrine cell area via hematoxylin staining. They note that the difference in pancreatic weight amongst groups is driven by one extreme outlier with a huge pancreas of 204 g, a measurement more than 2 standard deviations from the mean and a finding approximated by only one of 1238 pancreases donated for islet isolation in the Brussels database. As noted by Harja, the authors question the findings of alpha cell hyperplasia and they note that alpha cell hyperplasia and glucagon + microaadenomas may be common finding in control pancreata, increasingly observed in older pancreas specimens. Similarly, they note concerns with failure to correct for age in the analysis of PAnIn lesions. They conclude that most of the major conclusions made are not supported by the data and no meaningful conclusions can be drawn from the existing slides. Re-analysis of study of pancreatic effects of incretin therapy: Methodological deficiencies Diabetes Obes Metab. 2014 Jan 8 .

Potentially confounding interpretation of many of the findings reported in this paper, PanIN lesions are known to occur with increasing frequencey in older subjects The prevalence of pancreatic intraepithelial neoplasia in pancreata with uncommon types of primary neoplasms Am J Surg Pathol. 2006 Jan30(1):36-41 and Pancreatic intraepithelial neoplasms in the normal appearing pancreas: on their precise relationship with age Hepatogastroenterology. 2008 May-Jun55(84):1103-6. Furthermore, it has been elegantly described previously that a large number of histological changes develop in the endocrine pancreas, inluded large increases in beta cell replication, and increased replication of glucagon-, somatostatin-, and CA19.9-positive cells, as described in pancreata procured from young organ donors subjected to prolonged (more than 3 days) life support ventilation prior to death Beta-cell replication is increased in donor organs from young patients after prolonged life support Diabetes. 2010 Jul59(7):1702-8. doi: 10.2337/db09-1698. Moreover, the presence of positive anti-islet antibodies in several control diabetic subjects, as well as the likelihood of T1DM in at least 3 of the control diabetic subjects, together with data from the same authors illustrating that pancreatic mass is reduced in subjects with antibodies and further reduced in T1DM subjects, greatly complicates interpretation of the data. Pancreas organ weight in individuals with disease-associated autoantibodies at risk for type 1 diabetes JAMA. 2012 Dec 12308(22):2337-9.

Does acute or sustained GLP-1 receptor activation modify gallbladder function in diabetic subjects? Keller et al examined gallbladder emptying in normal fasting healthy subjects without a previous history of gallbaldder dysfunction in a randomized double blind crossover study, with administration of exenatide 10ug, or placebo, with and without CCK infusion to stimulate gallbladder emptying. Exenatide significantly reduced GB emptying, with no change in the diameter of the pancreatic or bile ducts. Effect of exenatide on cholecystokinin-induced gallbladder emptying in fasting healthy subjects Regul Pept. 2012 Sep 6.

Waser and colleagues used a combination of in situ ligand autoradiography with 125I- GLP-1 (7&ndash36)amide, and immunocytochemistry using a GLP-1 R-selective monoclonal antibody, to study GLP-1 R expression in normal and neoplastic human pancreas and thyroid. GLP-1 R was predominantly localized to islet beta cells and within ductal neuroendocrine cells in the pancreas. GLP-1 R immunopositivity was not detected in ductal cells or in 88 pancreatic adenocarcinomas however PanIN 1/2 lesions did exhibit GLP R+ cells (6/20 cases). Low level staining was observed in human acinar cells. GLP R expression was not detected in normal human thyroid, in papillary thyroid cancer, or in 10 cases from MTC related to MEN2. However 2/10 sporadic medullary thyroid cancers did exhibit GLP GLP-1R-immuopositive cells. See Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas Mod Pathol. 2014 Sep 12. doi: 10.1038/modpathol.2014.113

Smits et al examined the profiles of plasma amylase and lipase after acute infusion of exenatide (50 ng/min in 30 min, followed by continuous infusion of 25 ng/min) in human subjects with type 2 diabetes, studied both in the fasting state and following consumption of a meal. Plasma levels of amylase were significantly higher after exenatide , whereas plasma levels of lipase trended higher but were not significantly different within the 280 minute study period. Acute plasma amylase increase after glucagon-like peptide -1 receptor agonist exenatide administration in Type 2 diabetes Diabet Med. 2017 Apr34(4):591-592

Smits et al reported a number of exocrine pancreas-related outcomes in a small number of patients (n=19)with type 2 diabetes treated with liraglutide or sitagliptin for 12 weeks. Small and transient increases in pancreatic enzymes were deected with both drugs that were not sustained after 12 weeks. Pancreatic intraduodenal fluid secretion was slightly increased with sitagliptin but not liraglutide , and pancreas volume assessed by imaging was not changed. Pancreatic Effects of Liraglutide or Sitagliptin in Overweight Patients With Type 2 Diabetes: A 12-Week Randomized, Placebo-Controlled Trial Diabetes Care. 2016 Dec 20. pii: dc160836. doi: 10.2337/dc16-0836

Preclinical Data

How useful are rodents for studies of pancreatic pathology? Chadwick and colleagues report an extensive analysis of pancreatic lesions detected in three different strains of commonly studied rats, Sprague Dawley (SD), Zucker Diabetic fatty rats (ZDF) and rat insulin promoter-human islet amyloid polypeptide transgenic rats HIP). Rats (not treated with any drugs), n=36 per group at the start of the study, were analyzed after 4 months on a regular chow or high fat diet. SD rats gained more weight on the HFD, whereas HIP rats were slightly heavier on regular chow. 50% of the ZDF rats became unwell over time at 17-23 weeks of age. Plasma levels of amylase and lipase were higher at baseline in ZDF rats, and remained higher, relative to levels in the other rat strains. Plasma levels of MCP-1 and IL-6 varied substantially over time in all groups) and no correlation was noted between amylase, lipase, MCP-1 or IL-6 and histological analysis of findings in the exocrine pancreas (histology was reviewed by 3 separate pathologists). End of study pancreatic weights (normalized to body weight) were decreased in ZDF, SD-HFD and HIP rats, relative to other groups. Pancreatic exocrine inflammation (reduced size and number of acinar cells, necrosis, fibrosis, inflammatory cell infiltrate, more prominent ductal epithelial cells) was a common finding (up to 70% of all SD rats, 39% in HIP rats and 6% in ZDF rats) in all groups. Some SD rats also exhibited peri-islet inflammation. Abnormal ductal histology, including pancreatic duct glands (PDGs) was seen in 7/37 SD and ZDF rats, common enough to be considered a "normal histological feature" in these animals. No difference in histology was noted in when comparing head, body or tail of pancreas.

Roy and colleagues reported summary data for more than 70 preclinical toxicology studies examining the effects of saxagliptin and exenatide in rodents (up to 2 years) rabbits, dogs, and non-human primates (up to 12 months) at very high levels of clinical exposure. No histological evidence for drug-related pancreatitis or pancreatic neoplasms was noted in these studies The Glucagon-like Peptide-1-Based Therapeutics Exenatide and Saxagliptin Did Not Cause Detrimental Effects on the Pancreas in Mice, Rats, Dogs, and Monkeys Diabetes Obes Metab. 2014 Mar 26. doi: 10.1111/dom.12294

Koehler and colleagues have directly examined whether GLP-1 receptor activation modifies the proliferation or survival of human pancreatic cancer cells that express the endogenous GLP-1 receptor. The effect of Exenatide on human pancreatic cancer cell growth and survival was examined in studies using three different human pancreatic cancer cell lines that express the endogenous GLP-1 receptor. Although Exenatide was able to activate several different signal transduction pathways in these cells, no significant effects of Exenatide were observed on stimulation of cell growth or cell survival in vitro. Furthermore, Exenatide had no effect on cell killing induced by multiple different cytotoxic agents in vitro. Tumor growth was also studied in vivo in mice treated with daily injections of Exenatide following implantation of human pancreatic tumors into nude mice. Exenatide did not stimulate cell proliferation or expansion of tumor mass in vivo. See Activation of Glucagon-Like Peptide-1 Receptor Signaling Does Not Modify the Growth or Apoptosis of Human Pancreatic Cancer Cells Diabetes 2006 55: 1369-1379.

Pancreatic Protein Synthesis In subsequent studies, Koehler explored the link between GLP-1 R signaling and increased pancreatic mass in mice. The data show that while the mass of the pancreas increases by

10% in mice treated with exenatide or liraglutide , DNA content or cell proliferation or edema do not change-rather, activation of GLP-1 R signaling leads to increased protein synthesis in the pancreas. Furthermore, exendin-4 rapidly increased phosphorylation of proteins such as EIF2a, S6, and 4EBP1, all involved in control of protein synthesis. These effects of GLP-1 R agonists required a functional GLP-1 receptor, were absent in Glp1r -/- mice and were eliminated by co-treatment of mice with rapamycin, an inhibitor of protein synthesis. Furthermore, although the relative abundance of the majority of proteins, including amylase and lipase, was not different after treatment with GLP-1 R agonists, a number of proteins important for translation/protein syntesis were selectively increased after exendin-4 administration. Se e Glucagon-like peptide-1 receptor agonists increase pancreatic mass by induction of protein synthesis Diabetes published ahead of print October 2, 2014,doi:10.2337/db14-0883

Consistent with localization of Glp1r expression to the mouse and human exocrine pancreas, Hou et al demonstrated that Glp1r expression was identified in isolated pancreatic acini, and GLP-1 directly stimulated amylase release and cyclic AMP production, findings abolished in experiments using pancreatic tissue from Glp1r -/- mice. Loss of the Glp1r in Glp1r -/- mice did not perturb pancreatic morphology, or the basal expression/localization of amylase or phalloidin proteins in acinar cells similarly basal mRNA transcripts levels for amylase, lipase, elastase, chymotrypsin, and ribonuclease was comparable in pancreatic lysates from Glp1r -/- vs. Glp1r +/+ mice. Native GLP-1 (30 pM-3 nM) stimulated amylase release from isolated acini from Glp1r -/- but not from Glp1r +/+ mice wheres VIP, A23187, CCK and cyclic AMP agonists (but not carbachol) produced comparable amylase secretion from Glp1r -/- and. Glp1r +/+ mice. No GLP-1 -dependent potentiation of CCK-mediated amylase stiumulation was detected The Glucagon-Like Peptide-1 Receptor is Present in Pancreatic Acinar Cells and Regulates Amylase Secretion through cyclic AMP Am J Physiol Gastrointest Liver Physiol. 2015 Nov 5:ajpgi.00293.2015. doi: 10.1152/ajpgi.00293.2015

The putative relationship between GLP-1 receptor activation, exenatide , sitagliptin and the expression of genes and proteins that modules the risk of pancreatitis was examined in WT and Glp1r -/- mice. WT mice on a regular diet, a high fat diet, or following induction of diabetes with STZ, were treated, for both acute periods, and for several months with different GLP-1 R agonists, metformin, or sitagliptin. Although GLP-1 receptor activation with exendin-4 ( exenatide ) or liraglutide regulates gene and protein expression in the exocrine pancreas, GLP-1 receptor activation prior to or following the induction of experimental pancreatitis with the CCK analogue careulein had no effect on the development of experimental pancreatitis. Therapy with GLP-1 R agonists actually induced a gene/protein expression program that would be predicted to be protective agaonst the development of pancreatitis. Moreover the molecular, biochemical and histological course and severity of pancreatitis was comparable in Glp1r -/- vs. Glp1r +/+ mice. See GLP-1 receptor activation modulates pancreatitis-associated gene expression but does not modify the susceptibility to experimental pancreatitis in mice Diabetes published ahead of print June 9, 2009, doi:10.2337/db09-0626

Forest and colleagues examined 300 ZDF diabetic rats treated with placebo, metformin, or 3 doses of sitagliptin for up to 14 weeks. No histological evidence for pancreatic inflammation, proliferation in the ductual inflammation, or neoplasia, was noted, following analysis of more than 5,000 sections of the pancreas Characterization of the Exocrine Pancreas in the Male Zucker Diabetic Fatty Rat Model of Type 2 Diabetes Mellitus Following 3 Months of Treatment with Sitagliptin Endocrinology en.2013-1781

Nyborg and colleagues examined the chronic effects of liraglutide in 3 species, rats, mice and monkeys for up to 2 years, exposed to a range of liraglutide doses. Histological abnormalities were quantified in liraglutide - vs. placebo treated groups encompassing hundreds of animals. Monkeys were doses for up to 87 weeks with no evidence for preneoplastic lesions, pancreatitis, or pancreatic cancer despite exposure to plasma liraglutide concentrations up to 60 fold higher than those obtained in clinical studies in humans. Rats were treated for up to 2 years. No significant inflammatory or preneoplastic lesions were detected in the pancreata of mice, rats, and monkeys. The Human GLP-1 Analog Liraglutide and the Pancreas: Evidence for the Absence of Structural Pancreatic Changes in Three Species Diabetes. 2012 May61(5):1243-9.

Ellenbroek treated normal diet- or high fat fed mice with liraglutide for 1 or 6 weeks. Increased acinar cell proliferation and size was detected in mice on a normal chow diet, but not in mice on a high fat diet after 6 weeks of twice daily liraglutide . Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice Diabetologia. 2013 Jun 16

Tatarkiewicz and colleagues examined the effects of exenatide in normal and diabetic mice treated with continuous exenatide infusions for 4 weeks and in mice and rats treated with acute exenatide . Experimental pancreatitis was induced using caerulein or sodium taurocholate and plasma markers of inflammation and pancreatic histology was examined. Acute or chronic exenatide administration had no effect on plasma amylase or lipase in the absence of inflammation, and in some experiments, plasma amylase and lipase actually decreased after exenatide treatment, in the presence or absence of pancreatitis. Exenatide had no consistent effect on inflammatory mediators, and at the highest doses employed, actually decreased histological markers of inflammation in both experimental models. The rates of ductal proliferation were also examined and were unchanged after exenatide treatment in ob/ob mice however exenatide increased the expression of the protective gene, PAP. See Exenatide does not evoke pancreatitis and attenuates chemically-induced pancreatitis in normal and diabetic rodents Am J Physiol Endocrinol Metab. 2010 Oct 5

A subsequent study examined the effects of treating Zucker diabetic fatty rats with a range ( 6, 40 and 250 µg/kg/day ) of exendin-4 doses, twice daily for 13 weeks. No significant effects of exendin-4 treatment were noted in assessments of lipase concentrations, pancreatic weight, pancreatic histology, ductal cell proliferation or apoptosis. No evidence of drug-induced pancreatitis in rats treated with exenatide for 13 weeks Diabetes Obes Metab. 2012 Nov 19. doi: 10.1111/dom.12040

Analysis of the biological activity of exendin-4 in guinea pig pancreatic exocrine slices revealed that exendin-4 stimulates cyclic AMP release through incompletely characterized mechanisms as outlined in Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J Biol Chem. 1992 Apr 15267(11):7402-5. Subsequent experiments revealed that although exendin-4 alone did not stimulate amylase release, exendin-4 potentiated amylase release induced by CCK, carbamylcholine, bombesin or a calcium ionophore, A23187 Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini. Regul Pept. 1992 Sep 2241(2):149-56. These interactions of exendin-4 with pancreatic acinar cells are also seen with native GLP-1 , which increased cyclic AMP release in comparable studies, actions which were blocked by the GLP-1 R antagonist exendin(9-39) similarly, binding sites for both exendin-4 and GLP-1 were detected in experiments using radiolabelled peptides and guinea pig pancreatic acinar cells Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4. J Biol Chem. 1992 Oct 25267(30):21432-7. and Use of 125I-[Y39]exendin-4 to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig. Regul Pept. 1994 Aug 3153(1):47-59

There is very little information available about the possible effects of GLP-1 /exendin-4 on the gall bladder or biliary tract. Preclinical studies in rats demonstrate that GLP-1 /exendin-4 stimulates cholangiocyte growth, and that proliferating cholangiocytes may be capable of actually synthesizing GLP-1 Glucagon-like peptide-1 and its receptor agonist exendin-4 modulate cholangiocyte adaptive response to cholestasis. Gastroenterology. 2007 Jul133(1):244-55. Subsequent studies demonstrated that exendin-4 is also capable of exerting anti-apoptotic effects on rat cholangiocytes cultured in vitro, and in a bile duct ligation/toxin (Ccl4)-induced model of cholangiocyte apoptosis in vivo Exendin-4, a Glucagon-Like Peptide 1 receptor agonist, protects cholangiocytes from apoptosis. Gut. 2008 Oct 1. [Epub ahead of print]. Cholangiocytes from rats with bile duct ligation express Pdx-1 and GLP-1 R activation enhances nuclear Pdx1 protein expression in rat cholangiocytes. IGF-1 expression was also induced following exendin-4 treatment of rat cholangiocytes and the ability of exendin-4 to induce IGF-1 and VEGF expression and cholangiocyte proliferation was impaired following Pdx1 knockdown Pancreatic Duodenal Homeobox-1 de novo expression drives cholangiocyte neuroendocrine-like transdifferentiation J Hepatol. 2010 Oct53(4):663-70. As noted below in the section on cancer, GLP-1 R activation reduced the growth and enhanced the apoptosis of human cholangiocarcinoma cell lines both in vitro and in mice in vivo Effect of the GLP-1 Analog Exendin-4 and Oxaliplatin on Intrahepatic Cholangiocarcinoma Cell Line and Mouse Model. Int J Mol Sci. 2013 Dec 1314(12):24293-304.

The putative mechanism through which GLP-1 R agonists may act on the exocrine pancreas remains uncertain, and pancreatitis has not been commonly described in hundreds of preclinical studies of GLP-1 R agonists or DPP-4 inhibitors. Infusion of GLP-1 inhibits pancreatic exocrine secretions in short term studies of normal human subjects Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci. 1993 Apr38(4):665-73. These effects are likely indirect, due to the effects of GLP-1 on reduction of gastric emptying, and hence reduced transit of food into the small bowel.

Treatment of Sprague Dawley rats with exendin-4 once daily for 75 days produced no changes in amylase, but a 2-fold increase in serum lipase was noted, in associated with histological changes consistent with mild pancreatitis in some acini. Remarkably, the exendin-4-treated rats exhibit a profound reduction in body weight gain, with the control rats starting at

250 grams and ending up at 600 grams after 9 weeks, whereas rats treated with exendin-4 started at

240 grams and ended up at

390 grams. No other anti-diabetic agents were included as controls, and no pair-fed controls were studied to assess the effects of marked weight loss alone, a know risk factor for pancreatitis, in the study. The authors stated that "the histological differences observed between controls and exendin-4-treated animals were not very striking". Some exendin-4-treated rats exhibited morphological changes consistent with acinar fibrosis, and focal acinar degeneration and inflammation, as outlined in Biochemical and histological effects of exendin-4 (exenatide) on the rat pancreas Diabetologia. 2009 Sep 13. [Epub ahead of print]. In an accompanying Editorial, Butler and colleagues summarized the available evidence, and based on their interpretation of the available animal data, recommended that therapy with GLP-1 mimetics only be used in conjunction with metformin Glucagon-like peptide-1 therapy and the exocrine pancreas: innocent bystander or friendly fire? Diabetologia. 2009 Nov 6. [Epub ahead of print]. Transgenic over-expression of exendin-4 has also been associated with the development of an immune response against the preproexendin-4 transgene but not exendin-4 alone in mice, in association with tissue infiltrates involving multiple organs, including the, heart, liver, kidney and pancreas Lymphocytic infiltration and immune activation in metallothionein promoter-exendin-4 (MT-Exendin) transgenic mice Diabetes. 2006 Jun 55 (6): 1562-70

Matveyenko and colleagues demonstrated adverse effects of sitagliptin on pancreatic histology in HIP-transgenic rats with overexpression of human amylin in the beta cells of transgenic animals. Groups of rats were treated with either sitagliptin alone, metformin, or both drugs together. Significantly increased ductal turnover was observed in non-treated transgenic rats, and sitagliptin increased whereas metformin decreased ductal turnover. Sitagliptin was also associated with increased ductal metaplasia, and one sitagliptin-treated rat developed focal pancreatitis. See Beneficial endocrine but adverse exocrine effects of sitagliptin in the human islet amyloid polypeptide transgenic rat model of type 2 diabetes: interactions with metformin Diabetes. 2009 Jul58(7):1604-15.

In a subsequent study Pancreatic duct replication is increased with obesity and type 2 diabetes in humans Diabetologia. 2009 Oct 21. [Epub ahead of print], the same authors studied rates of ductal replication in pancreata from autopsy studies of 45 humans subdivided into 4 groups lean, obese, non-diabetic, and subjects with type 2 diabetes. Obesity (BMI greater than 27) was associated with a 10-fold increase in the rate of ductal replication as assessed by staining with Ki67. Cytokeratin was used as a marker for ductal cells. Lean subjects with type 2 diabetes exhibited a 4-fold increase in ductal cell replication compared to lean non-diabetic controls. Obese individuals with diabetes did not exhibit a further increase in ductal cell replication. The autopsy series represents a subgroup selected from a previously published analysis of beta cell mass and replication Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes Diabetes. 2003 Jan52(1):102-10. The authors suggest a note of caution in regard to therapeutic strategies based on incretin action that are used to treat human diabetics as these agents ( GLP-1 R agonists and DPP-4 inhibitors) have been shown to increase ductal replication in rodents. Intriguingly, a similar analysis of pancreatic ductal turnover in human subjects subjected to one or 2 pancreatic surgeries, who exhibited progressive increases in blood glucose to the frankly diabetic range, did not reveal any increases in pancreatic ductal replication Partial pancreatectomy in adult humans does not provoke beta-cell regeneration Diabetes. 2008 Jan57(1):142-9. Although the Butler paper Pancreatic duct replication is increased with obesity and type 2 diabetes in humans Diabetologia. 2009 Oct 21. [Epub ahead of print] and Editorial Glucagon-like peptide-1 therapy and the exocrine pancreas: innocent bystander or friendly fire? Diabetologia. 2009 Nov 6. [Epub ahead of print] speculates that GLP-1 may induce chronic pancreatitis leading to enhanced ductal cell replication and an increased risk of pancreatitis, careful quantitative analysis of pancreatic tissue from 43 human subjects with chronic pancreatitis did not reveal any differences in ductal replication between subjects with pancreatitis vs. control subjects without pancreatitis. These authors speculate that the high rates of ductal turnover seen in many rodent studies may not be representative of ductal replication rates in humans. See Figure 6 in Reduced pancreatic volume and beta-cell area in patients with chronic pancreatitis Gastroenterology. 2009 Feb136(2):513-22.

Gier and colleagues examined the effects of exendin-4 on human pancreatic ductal cell replication and on formation and number of dysplastic foci in mice with Kras-induced pancreatic dysplasia. WT rats treated with once daily injections of exendin-4 exhibited a 'relative' increase in pancreatic weight, no pancreatitis, but an expansion of the PDG (pancreatic duct gland compartment), with a 4-fold increase in ductal cell replication. Treatment of the Pdx-1Kras for 12 weeks with exendin-4 significantly increased pancreatic weight, increased the histological evidence for pancreatitis, increased ductal cell proliferation, increased plasma lipase activity, and increased inflammation, fibrosis, and metaplastic changes. Immunohistochemistry demonstrated GLP-1 R-positive ductal cells but no GLP-1 R expression was detected in pancreatic acinar cells. GLP-1 R expression was also detected in PanIN lesions (pancreatic intraepithelial lesions) and exendin-4 increased cyclinD1, ERK1/2 and proliferation of human ductal cells. Concomitant treatment with metformin reduced the effects of exendin-4 on ductal proliferation. Chronic GLP-1 Receptor Activation by Exendin-4 Induces Expansion of Pancreatic Duct Glands in Rats and Accelerates Formation of Dysplastic Lesions and Chronic Pancreatitis in the KrasG12D Mouse Model Diabetes published ahead of print January 20, 2012, doi:10.2337/db11-1109

The incidence of pancreatic cancer is known to be increased in diabetic subjects, and in some studies, the more advanced duration of diabetes (requiring insulin therapy) together with gallstones/gall bladder disease further increased the relative risk in retrospective association studies Gallstones, a cholecystectomy, chronic pancreatitis, and the risk of subsequent pancreatic cancer in diabetic patients: a population-based cohort study J Gastroenterol. 2012 Oct 3. [Epub ahead of print]

On Sept 25 2009, the FDA posted the following notice about case reports of pancreatitis in patients treated with sitagliptin.

Sitagliptin (marketed as Januvia and Janumet) - acute pancreatitis

FDA notified healthcare professionals and patients of revisions to the prescribing information for Januvia (sitagliptin) and Janumet (sitagliptin/metformin) to include information on reported cases of acute pancreatitis in patients using these products. Eighty-eight post-marketing cases of acute pancreatitis, including two cases of hemorrhagic or necrotizing pancreatitis in patients using sitagliptin, were reported to the Agency between October 2006 and February 2009. It is recommended that healthcare professionals monitor patients carefully for the development of pancreatitis after initiation or dose increases of sitagliptin or sitagliptin/metformin. Sitagliptin has not been studied in patients with a history of pancreatitis. Therefore, it is not known whether these patients are at an increased risk for developing pancreatitis and the medication should be used with caution and with appropriate monitoring in patients with a history of pancreatitis. Considerations for healthcare professionals, information for patients, and a Data Summary are provided.

GLP-1 and cancer

Ligumsky and colleagues examined the effects of GLP-1 and exendin-4 on breast cancer growth and survival. Although the classical GLP-1 receptor was not detected in breast cancer cell lines, the GLP-1 R agonists reduced breast cancer cell (MCF-7, MDA-MB231 and MDA-MB-468) growth and increased apoptosis, both in cells cultured in vitro, and in breast cancer tumors propagated in athymic nude mice treated with daily exendin-4 (5 days a week for 6 weeks) or with continuous infusion pumps for 4 weeks in vivo. GLP-1 R agonists increased accumulation of cAMP, and cAMP inhibition reversed the effects of GLP-1 /exendin-4 on breast cancer cell growth. See The peptide-hormone glucagon-like peptide-1 activates cAMP and inhibits growth of breast cancer cells. Breast Cancer Res Treat. 2011 Jun 3.

Koehler and Kain examined the effects of exendin-4 on colon cancer cell growth in cells propagated in vitro, and in syngeneic tumors implanted in mice in vivo. Mouse CT26 colon cancer cells expressed a full length functional GLP-1 R coupled to cAMP activation. Both forskolin and exendin-4 reduced cell growth and increased apoptosis in CT26 cells. Exendin-4 reduced colony formation in soft agar and enhanced apoptosis in tumor cells in mice. The ability of exendin-4 to enhance colon cancer cell death was enhanced in the presence of cytotoxic agents. GLP-1 R activation inhibited Erk1/2 activation and inactivated GSK3, athough different mechanisms. See Glucagon-Like Peptide-1 Receptor Activation Inhibits Growth and Augments Apoptosis in Murine CT26 Colon Cancer Cells Endocrinology. 2011 Jul 19.

Chen and colleagues studied the effects of exendin-4, with and without oxaliplatin, on the growth and apoptosis of human cholangiocarcinoma cells (HuCCT1) in vitro and in mice in vivo. GLP-1 /exendin-4 reduced colony formation and cell proliferation in vitro and exendin-4 treatment once daily for 1 week followed, in some instances, by weekly oxaliplatin administration) of Balb/C mice with subcutaneous HuCCT1 tumors GLP-1 R activation (exendin-4 alone) reduced the growth rate of tumors in vivo, and further reduced tumor growth in mice receiving chemotherapy. These findings were associated with enhanced apoptosis and reduced cell migration of human cholangiocarcinoma cell lines both in vitro and in mice in vivo Effect of the GLP-1 Analog Exendin-4 and Oxaliplatin on Intrahepatic Cholangiocarcinoma Cell Line and Mouse Model. Int J Mol Sci. 2013 Dec 1314(12):24293-304.

Thyroid C cells and calcitonin

Preclinical studies demonstrate that rodent, specifically mouse and rat thyroid calcitonin-producing C cells express a functional GLP-1 receptor. Indeed several studies employing rodent C cell lines demonstrate that GLP-1 R activation in vitro leads to rapid stimulation of calcitonin secretion, actions that are attenuated by blocking the GLP-1 receptor with the antagonist exendin(9-39) Effects of glucagon and glucagon-like peptide-1-(7-36) amide on C cells from rat thyroid and medullary thyroid carcinoma CA-77 cell line Endocrinology. 1996 Sep137(9):3674-80 Effects of glucagon and glucagon-like peptide-1-(7-36) amide on C cells from rat thyroid and medullary thyroid carcinoma CA-77 cell line and Expression of glucagon-like peptide 1 receptor in a murine C cell line: regulation of calcitonin gene by glucagon-like peptide 1 FEBS Lett. 1996 Sep 16393(2-3):248-52. In contrast, functional GLP-1 receptors coupled to calcitonin secretion have not been detected in human C cell lines.

The role and importance of GLP-1 receptor activity in rodent C cells is likely related to the gut-bone axis and control of bone resorption. In rodents, and to some extent in humans many gastrointestinal peptides inhibit bone resorption in the postprandial state Potential role of pancreatic and enteric hormones in regulating bone turnover. J Bone Miner Res. 2005 Sep20(9):1497-506. Indeed administration of the GLP-1 R agonist exendin-4 increases calcitonin gene expression in WT mice, whereas Glp1r -/- mice exhibit increased bone resorption and reduced calcitonin expression in the thyroid The murine glucagon-like peptide-1 receptor is essential for control of bone resorption Endocrinology. 2008 Feb149(2):574-9. Nevertheless, in contrast to the biology in rodents, GIP or GLP-1 do not inhibit bone resorption in human subjects whereas the related peptide GLP-2 is a potent inhibitor of bone resorption Role of gastrointestinal hormones in postprandial reduction of bone resorption J Bone Miner Res. 2003 Dec18(12):2180-9.

Continuous activation of the GLP-1 receptor in rats and to a lesser extent in mice produces C-cell hyperplasia, periodically associated, after sustained administration, with the subsequent development of medullary thyroid cancer. Moreover, acute administration of any GLP-1 R agonist produces a rapid increase in plasma calcitonin levels in rodents. In contrast, studies with monkeys demonstrate a) no increase in plasma calcitonin levels and b) no C-cell hyperplasia and c) no MTC despite continuous prolonged GLP-1 R agonist administration. Similarly, administration of liraglutide once daily to thousands of human subjects with diabetes or obesity did not result in significant increases in plasma calcitonin over time. Taken together, the available data demonstrate a major species-specific difference in the biology of GLP-1 receptor activity in the rodent vs primate thyroid gland. See Glucagon-like peptide-1 receptor agonists activate rodent thyroid C-cells causing calcitonin release and C-cell proliferation Endocrinology 2010 Apr151(4):1473-86. C-cell hyperplasia was not observed following continuous exenatide or intermittent liraglutide dosing for 13 weeks in Glp1r -/- mice, whereas control mice exhibited calcitonin increases and C-cell hyperplasia with exenatide and liraglutide . Evaluation of RET activation using a phospho-RET antibody and immunohistochemistry revealed no evidence for RET activation in the thyroid glands of WT mice treated with liraglutide , however increased levels of the mTOR-dependent S6 protein were detected in the thyroid glands from the same experiments. GLP-1 Receptor Agonists and the Thyroid: C-Cell Effects in Mice Are Mediated via the GLP-1 Receptor and not Associated with RET Activation Endocrinology January 10, 2012 en.2011-1864.

Qualitatively similar results have been detected in preclinical studies with the long acting GLP-1 R agonist dulaglutide, which had no effect on C-cell proliferation, C-cell volume, or serum levels of calcitonin when dosed at relatively high levels ( 8.15 mg/kg twice weekly in male monkeys for 52 weeks which achieved plasma drug levels

500-fold greater than maximal human exposure). Dulaglutide was almost certainly active as the monkeys exhibited a sustained reduction in body weight Effects of Dulaglutide on Thyroid C-Cells and Serum Calcitonin in Male Monkeys Endocrinology. 2015 Apr 10:en20141717 . In contrast, rats were much more susceptible to C-cell proliferation, exhibiting hyperplasia and medullary thyroid carcinoma at lower multiples of drug exposure, whereas WT and RASH2 transgenic mice exhibited minimal C-cell changes after 26 weeks of dulaglutide. Chronic Toxicity and Carcinogenicity Studies of the Long-Acting GLP-1 Receptor Agonist Dulaglutide in Rodents Endocrinology. 2015 Apr 10:en20141722

Nevertheless, the human GLP-1 receptor has been shown, using immunocytochemical techniques to be expressed in subsets of thyroid tumor cells from patients with C cell hyperplasia, medullary thyroid cancer, and a smaller proportion of papillary thyroid cancers. The functional significance of these findings, if any, has not yet been ascertained, and the sensitivity and specificity of the antisera used to detect the GLP-1R in these studies remains uncertain. Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland. J Clin Endocrinol Metab. 2012 Jan97(1):121-31

Boess and colleagues examined GLP-1 R expression and action in rodent and human thyroid glands and in primary thyroid cell cultures. Rat cultures were responsive to TSH, and calcium promoted calcitonin secretion human thyroid cultures were much less responsive and more variable. Glp1r mRNA transcripts were not consistently detected in RNA from rat or human thyroids or from primary thyroid cultures. In situ hybridization co-localized Glp1r and calcitonin RNA in rat thyroid C cells. Exendin-Cy5 detected GLP-1 R binding on the cell surface of transfected cells, but no positive thyroid cells were visualized in binding studies using this probe. Liraglutide and taspoglutide increased calcitonin release in rat thyroid cultures but not in studies with human thyroid cultures. Effect of GLP-1R agonists taspoglutide and liraglutide on primary thyroid C-cells. J Mol Endocrinol. 2013 Mar 5

Pach and colleagues reported the use of an exendin-4-based imaging ligand (Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-Exendin-4) for visualization of recurrent or disseminated medullary thyroid cancer in 4 human subjects with elevated calcitonin levels (two with MEN, two with sporadic MTC). The extent of GLP-1 receptor expresion (mRNA or protein) in human tumor tissue excised from these patients was not evaluated. No control patients without MTC were studied. Glucagon-Like Peptide-1 Receptor Imaging with [Lys (40) (Ahx-HYNIC- (99 m) Tc/EDDA)NH 2 ]-Exendin-4 for the Diagnosis of Recurrence or Dissemination of Medullary Thyroid Cancer: A Preliminary Report Int J Endocrinol. 20132013:384508

Waser and colleagues used in situ radiography and iodinated peptides to study GLP-1 and GIP receptor binding capacity in rodent and human thyroid glands. GLP-1 but not GIP receptor binding activity was easily detected in normal rodent thyroid glands, and in rat medullary thyroid cancer cells GIP receptors were also detected in rat medullary thyroid cancer but not in specimens of C cell hyperplasia. GIP and GLP-1 receptor binding was not detected in normal human thyroid glands. 27% of human medullary thyroid cancers contained GLP-1 receptor binding sites, whereas GIP receptor binding sites were more numerous and higher density, with 89% of medullary thyroid cancers exhibiting GIP receptor positivity. The authors also detected GIP receptors in human TT thyroid cells, that do not express the GLP-1 R. Incretin Receptors in Non-Neoplastic and Neoplastic Thyroid C Cells in Rodents and Humans: Relevance for Incretin-Based Diabetes Therapy Neuroendocrinology. 2011 Sep 2.

Additional longitudinal data from serial measurements of calcitonon levels in over 5,000 individuals with either diabetes or obesity treated with liraglutide was presented by Hgedus et al. Non-stimulated calcitonin measurements were measured every 3 months, in liraglutide vs. control subjects for up to 2 years in 8 phase 3 clinical trials for diabetes, and 1 phase 2 trial for obesity. No clinically significant shift in mean calcitonin levels was observed across the entire study population and no differences were seen in calcitonin levels in subjects treated with exenatide vs. liraglutide . Several cases (6) of C-cell hyperplasia were identified, most with elevated baseline calcitonin levels, and one case of MTC was described in a non- liraglutide -treated patient. Liraglutide did not produce further increases in calcitonin levels even in subjects with baseline CT elevations. See GLP-1 and Calcitonin Concentration in Humans: Lack of Evidence of Calcitonin Release from Sequential Screening in over 5000 Subjects with Type 2 Diabetes or Nondiabetic Obese Subjects Treated with the Human GLP-1 Analog, Liraglutide. J Clin Endocrinol Metab. 2011 Jan 5. [Epub ahead of print]


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Which human cell lines do not express the GLP-1 receptor? - Biology

The cell line was developed from the human Raji cell line. Raji cells constitutively express different antigens on their cell surface. These antigens can be targeted by specific antibodies, especially inducing antibody-dependent cellular cytotoxicity (ADCC).

-Human CD20 is highly expressed in Raji cells. It has no known natural ligands and is considered to be a calcium channel to achieve the best B cell response. In non-Hodgkin B-cell lymphoma or chronic lymphocytic leukemia with monoclonal antibody-mediated therapy (rituximab, ofatumumab, obinutuzumab), human CD20 is a clinically relevant target.

-Human CD19 is highly expressed by Raji cells. As a co-receptor, it lowers the threshold of antigen B cell receptor (BCR) dependent stimulation. Human CD19 is the clinical target of anti-hCD19-CD3 bispecific antibody (blinatumomab) in non-Hodgkin's B-cell lymphoma.

-Human PD-L1 (programmed cell death ligand 1 also known as CD274 or B7-H1) is expressed at low levels by Raji cells. This transmembrane protein is expressed in hematopoietic and non-hematopoietic cells and is induced by proinflammatory cytokines in the tumor microenvironment. Human PD-L1 is a clinically relevant target related to various cancers in mAb-mediated treatments (atezolizumab, durvalumab and avelumab).


Recombinant Human Glucagon-like peptide 1 receptor(GLP1R)

Human Glucagon-like peptide 1 receptor (GLP1R) amino acids 24-463 with a 10xHis-tag at the N-terminus were expressed in vitro E.coli expression system. The resulting protein is the recombinant full-length of mature human GLP1R protein. The purity of this protein is greater than 85% measured by SDS-PAGE analysis. Its calculated molecular weight is 53.6 kDa. This recombinant protein may be used to produce anti-GLP1R antibodies or in the studies of neuroscience.

GLP1R is a G protein-coupled receptor for GLP1 with wide expression in the CNS (central nervous system) and peripheral tissues such as heart, kidney, and adipose tissue (AT). GLP1 binding to GLP1R exerts its biological roles such as promoting glucose-dependent insulin secretion and inhibiting gastric emptying and food intake. A recent study has shown the implication of GLP1R in energy metabolism by directly stimulating mitochondrial bioenergetics and brown AT remodeling.


<p>This section provides information on the quaternary structure of a protein and on interaction(s) with other proteins or protein complexes.<p><a href='/help/interaction_section' target='_top'>More. </a></p> Interaction i

<p>This subsection of the <a href="http://www.uniprot.org/help/interaction%5Fsection">'Interaction'</a> section provides information about the protein quaternary structure and interaction(s) with other proteins or protein complexes (with the exception of physiological receptor-ligand interactions which are annotated in the <a href="http://www.uniprot.org/help/function%5Fsection">'Function'</a> section).<p><a href='/help/subunit_structure' target='_top'>More. </a></p> Subunit structure i

May form homodimers and heterodimers with GIPR.

Manual assertion inferred from sequence similarity to i

Sites

Feature keyPosition(s)Description Actions Graphical viewLength
<p>This subsection describes interesting single amino acid sites on the sequence that are not defined in any other subsection. This subsection can be displayed in different sections ('Function', 'PTM / Processing', 'Pathology and Biotech') according to its content.<p><a href='/help/site' target='_top'>More. </a></p> Site i 121 Interaction with the endogenous ligand GLP-1 By similarity

Manual assertion inferred from sequence similarity to i

Manual assertion inferred from sequence similarity to i

Protein-protein interaction databases

The Biological General Repository for Interaction Datasets (BioGRID)

STRING: functional protein association networks

Chemistry databases

BindingDB database of measured binding affinities


Watch the video: GLP1 (August 2022).