The adhesion GPCR GPR116/ADGRF5 has a dual function in pancreatic islets regulating somatostatin release and islet development

Röthe, Juliane; Kraft, Robert; Ricken, Albert; Kaczmarek, Isabell; Matz-Soja, Madlen; Winter, Karsten; Dietzsch, André Nguyen; Buchold, Julia; Ludwig, Marie-Gabrielle; Liebscher, Ines; Schöneberg, Torsten; Thor, Doreen

doi:10.1038/s42003-024-05783-9

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Published: 16 January 2024

The adhesion GPCR GPR116/ADGRF5 has a dual function in pancreatic islets regulating somatostatin release and islet development

Communications Biology volume 7, Article number: 104 (2024) Cite this article

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Abstract

Glucose homeostasis is maintained by hormones secreted from different cell types of the pancreatic islets and controlled by manifold input including signals mediated through G protein-coupled receptors (GPCRs). RNA-seq analyses revealed expression of numerous GPCRs in mouse and human pancreatic islets, among them Gpr116/Adgrf5. GPR116 is an adhesion GPCR mainly found in lung and required for surfactant secretion. Here, we demonstrate that GPR116 is involved in the somatostatin release from pancreatic delta cells using a whole-body as well as a cell-specific knock-out mouse model. Interestingly, the whole-body GPR116 deficiency causes further changes such as decreased beta-cell mass, lower number of small islets, and reduced pancreatic insulin content. Glucose homeostasis in global GPR116-deficient mice is maintained by counter-acting mechanisms modulating insulin degradation. Our data highlight an important function of GPR116 in controlling glucose homeostasis.

Introduction

Pancreatic islets are composed of different cell types: endocrine cells, which secrete hormones required for maintaining glucose homeostasis, and non-endocrine cells such as endothelial cells and pericytes. Different endocrine cell types are distinguished within pancreatic islets. The insulin-secreting beta cells and the glucagon-secreting alpha cells represent about 80% and 15% of the islet cell population, respectively, whereas the 5% somatostatin-secreting delta cells constitute the third largest cell population in mouse and human pancreatic islets¹. Because of the low amount of pancreatic islets and the short half-life time, it is assumed that somatostatin secreted from pancreatic delta cells only exhibits paracrine functions on other islet cell types^2,3. Somatostatin receptors are expressed in beta and alpha cells suggesting that somatostatin prevents over-secretion of insulin and glucagon after nutrient stimulation⁴. In mouse islets, delta cells are mainly found in the islet’s periphery. However, delta cells have a neuron-like shape and, therefore, may reach a high number of other islet cell types for paracrine regulation⁵. So far, little is known about the regulation of somatostatin secretion besides nutrient-dependent release. It is well-established that G protein-coupled receptors (GPCRs) fine-tune hormone secretion in pancreatic alpha and beta cells⁶. Similar function has also been described for somatostatin release, e.g., by muscarinic acetylcholine M1 receptor activation⁷. Recently, it was demonstrated that the inhibitory effect of ghrelin on insulin secretion is mediated by somatostatin secreted from pancreatic delta cells^2,3. Furthermore, a negative feedback effect on insulin secretion mediated by urocortin-3 and corticotropin-releasing hormone receptor 2 expressed on delta cells has been shown⁸. These recent results suggest that pancreatic delta cells are important for the paracrine regulation network in pancreatic islets.

Besides the endocrine cells, also endothelial cells have assigned functions in the regulation of islet functionality. Dysfunction of endothelial cells results in reduced insulin secretion and beta cell loss⁹. Several studies focused on the importance of endothelial cells on beta cell development. Thereby, it was shown that endothelial cells secrete growth factors driving beta cell proliferation¹⁰ and endocrine pancreatic differentiation¹¹. Recent work demonstrated that islet GPCRs are also involved in those developmental processes^12,13,14. However, the complete processes of islet development as well as differentiation towards the different endocrine cell types are not well understood.

Several studies have shown that the repertoire of GPCRs expressed in pancreatic islets is much broader than previously assumed^2,3,15,16. Numerous adhesion GPCRs (aGPCRs) including latrophilins (LPHN1/ADGRL1, LPHN2/ADGRL2, and LPHN3/ADGRL3), GPR56/ADGRG1, and GPR116/ADGRF5 are among well-expressed GPCRs. Adhesion GPCRs form a separate class of GPCRs, which is widely uncharacterized regarding ligands and physiological functions mainly because of their extraordinary size¹⁷. A milestone in investigating their physiological function was the discovery that these receptors can be activated by a tethered agonist^18,19.

GPR116 was initially described as an unusual GPCR with a large N terminus composed of immunoglobulin domains²⁰. It was also demonstrated that GPR116 carries an internal agonist and peptides derived from this sequence can be used for receptor activation in vitro and ex vivo^21,22. GPR116 is highly expressed in lung endothelial cells regulating surfactant secretion and its deficiency results in lipid and protein accumulation as well as macrophage activation and inflammation in the alveols^23,24,25,26. Besides lung tissue, GPR116 is abundantly expressed in endothelial cells involved in the vasculature development of the nervous system and in cardiovascular functions^27,28. Moreover, several studies have described involvement of GPR116 in carcinogenesis and cancer metastasis^29,30,31,32. Adipocyte-specific knock-out (ko) of GPR116 suggested a role in metabolic processes^33,34.

In the present study, we show that GPR116 is expressed in pancreatic delta cells modulating somatostatin secretion upon activation. Furthermore, GPR116 deficiency reduces the size and number of beta cells and, subsequently, the insulin content of the endocrine pancreas. Our data demonstrate an important role of GPR116 in glucose homeostasis necessary for assembling fully functional pancreatic islets.

Results

GPR116 is expressed in pancreatic islets

Re-analysis of own RNA-sequencing (RNA-seq) data¹⁶ of mouse pancreatic islets revealed similarly high expression of several aGPCRs as reported for other receptors modulating insulin and glucagon secretion (Fig. 1a). Among these, pancreatic functions of the aGPCRs Gpr56, Lphn1, Bai3, and Lphn3 have already been shown^35,36,37,38. Another aGPCR, Gpr116, has been implicated in metabolic processes^33,34, but not specifically in pancreatic islet function. Quantitative PCR (qPCR) analysis confirmed the abundant expression of Gpr116 in mouse pancreatic islets (Fig. 1b). To test for cell type-specific expression of Gpr116, we performed analysis of different cell lines representing alpha (αTC1-9), beta (MIN6, INS-1), and delta (QGP-1) cells derived from pancreatic islets (Fig. 1b). Interestingly, we found highest expression in the delta cell line QGP-1, whereas expression in αTC1-9, MIN6, and INS-1 cells was significantly lower. This is in line with two published RNA-seq data sets of sorted mouse pancreatic islet cells^2,3, which we re-analyzed to compare the expression of Gpr116 in alpha, beta, and delta cells (Supplementary Experimental Procedures). Data from both sources revealed a significantly enriched expression in delta cells (Supplementary Fig. 1a).

**Fig. 1: Expression of GPR116 and structurally related aGPCRs in pancreatic islet cells.**

To confirm the expression of Gpr116 in delta cells we used in situ hybridization combined with antibody staining in pancreas sections (Fig. 1c–e). Here, Gpr116 expression was clearly visible in somatostatin-expressing cells, but only in a small amount of insulin- and glucagon-positive cells (Fig. 1d, e). Additionally, Gpr116 expression was observed in an additional subset of islet cells co-expressing Cd34 (Fig. 1c) indicating its presence in endothelium cells.

GPR116 signals via Gα_q/11 proteins inducing Ca²⁺ transients in pancreatic islets

GPR116 belongs to the subfamily VI of aGPCRs, which includes the structurally related GPR110, GPR111, and GPR115³⁹. Gpr110, Gpr111, and Gpr115 exhibited only minor expression in pancreatic islets (Supplementary Fig. 1b). Similar to other aGPCRs¹⁸, a peptide (p116) derived from an intramolecular amino acid sequence, the so-called Stachel sequence, has been demonstrated to activate GPR116^21,22. Prior to the activation of endogenously expressed GPR116, the agonistic property of p116 was verified in different control experiments (see Supplementary Results and Supplementary Fig. 1c–g). To control for unspecific peptide effects, we used a scrambled peptide (p116sc) as control in all experiments.

Next, the signaling properties of endogenous GPR116 were investigated in isolated primary islets. Islets were exposed to p116 (specific agonist), p116sc (peptide control), and carbachol (CCh, agonist of muscarinic acetylcholine receptors and established positive control), and Ca²⁺ responses were imaged. As expected, CCh application induced a strong Ca²⁺ elevation in most parts of the islets (Fig. 2a). Ca²⁺ transients were also elicited by p116, but appeared more scattered within the islets (Fig. 2a). The control peptide p116sc did not induce significant Ca²⁺ responses (Fig. 2a, b). Quantification of Ca²⁺ signals induced by p116 or p116sc within responsive regions revealed a specific activation of GPR116 in distinct pancreatic islet cells (Fig. 2c). Stimulation with ghrelin which activates the G_q/11 protein-coupled growth hormone secretagogue receptor/ghrelin (GHS-R) receptor⁴⁰, and subsequent application of p116 elicited Ca²⁺ signals with partially overlapping localization (Fig. 2d). Ghrelin-induced Ca²⁺ signals can be used to identify pancreatic delta cells in primary islets⁴¹ and responses upon both, ghrelin and p116, were absent in insulin-expressing beta cells (Fig. 2e, f). Therefore, the lack of GPR116-mediated Ca²⁺ signals in beta cells is in line with the expression pattern of Gpr116.

**Fig. 2: GPR116 signaling in mouse pancreatic islets.**

Taken together, our results demonstrate that GPR116 is also functionally expressed in a subset of pancreatic islet cells.

GPR116 regulates somatostatin secretion from pancreatic islets

To test functionality of Gpr116 expression in delta cells, we determined somatostatin secretion upon GPR116 activation under high-glucose conditions. As shown in Fig. 2g, secretion of somatostatin is significantly increased (2.7-fold at 2 mM p116 yielding an EC₅₀ of 950 µM ± 124 µM) after incubation of islets with p116, whereas p116sc did not induce secretion. To test whether the p116-induced somatostatin release is mediated via activation of the Gα_q/11 protein/phospholipase C signaling pathway, pancreatic islets were incubated with the G_q inhibitor FR900359⁴² which completely abolished somatostatin secretion upon p116 application (Fig. 2h). Time-dependent measurement of somatostatin release revealed an immediate (within 1 min) and a prolonged increase (within 30 min) of somatostatin secretion after stimulation with p116 under high-glucose conditions (Fig. 2i). Whereas the GPR116-independent release showed a steady increase within 30 min, p116 caused a significant increase already after 5 min of incubation (Fig. 2i). These data demonstrate that activation of GPR116 augments glucose-induced somatostatin secretion over a time period of 30 min.

Taken together, our results identify GPR116 as a novel receptor modulating somatostatin secretion from pancreatic delta cells.

Islet from GPR116-deficient mice display a lack of p116-induced somatostatin release and a reduced insulin content

To prove that the peptide-induced somatostatin secretion is mediated by GPR116, we used a previously established germline ko mouse strain with a complete deletion of exon 17 (exon annotation based on the mouse GRCm38/mm10 assembly) resulting in a non-functional receptor²³ (Supplementary Results and Supplementary Figs. 2a–d). Thus, we measured GPR116-induced somatostatin secretion from wt and ko pancreatic islets. As expected, somatostatin secretion from wt islets was increased after p116 stimulation in a concentration-dependent manner whereas p116sc did not have a significant effect (Fig. 3a). In GPR116 ko islets, p116 was ineffective to liberate somatostatin even at high peptide concentration (2 mM) indicating that peptide-induced somatostatin secretion depends on the expression of a functional GPR116 (Fig. 3a).

**Fig. 3: Constitutive GPR116 ko mice show reduced insulin secretion and decreased insulin content in pancreatic islets.**

Next, we investigated the amounts of somatostatin and insulin secreted from isolated pancreatic islets of ko and wild-type (wt) mice after incubation with low- and high-glucose solution. We observed no changes in glucose-induced somatostatin secretion between wt and ko pancreatic islets (Fig. 3b). Interestingly, the amount of insulin released from ko pancreatic islets was slightly decreased under low-glucose conditions and significantly reduced under high-glucose conditions compared to wt islets (Fig. 3e). Moreover, while the content of somatostatin was unchanged in ko compared to wt islets (Fig. 3c), the total amount of insulin was significantly reduced in ko pancreatic islets (60% of wt islets) (Fig. 3f). However, relative glucose-induced somatostatin and insulin secretion, normalized to the total amounts of the respective hormones, were not changed in ko pancreatic islets compared to wt islets (Fig. 3d, g).

In general, the ko mice did not display an obvious metabolic phenotype. Neither male nor female mice showed a difference in body weight at an age of 14 to 16 weeks (Supplementary Fig. 2e). To analyze the physiological impact of the reduced insulin secretion onto glucose homeostasis in constitutive GPR116 ko animals, glucose tolerance tests were performed in overnight-fasted wt and ko mice. We found significantly increased blood glucose levels after overnight fasting in GPR116 ko mice compared to wt animals (Fig. 3h). However, no obvious differences between wt and ko mice were detected in glucose tolerance tests (Fig. 3h). Insulin tolerance tests, performed after 6 h fasting, also displayed no significant differences between wt and ko mice despite an initial difference in fasting blood glucose levels (Fig. 3i).

Our data demonstrates that the p116-induced somatostatin secretion is specific for GPR116 and that the receptor is required for adequate insulin production and, as a result, for sufficient glucose-induced insulin secretion. However, the reduced amount of insulin does not result in an impaired glucose tolerance.

Delta cell-specific GPR116 activation mediates somatostatin release

To specify the role of GPR116 in pancreatic delta cells, Gpr116^f/f ²³ mice were crossed to Sst-IRES-Cre transgenic mice (Jackson Laboratories, #018973). Islets isolated from delta cell-specific ko animals show transcripts of Gpr116 with deleted exon 17 and a reduced amount of full-length receptor (Supplementary Fig. 2f, g) indicating the feasibility of this approach. Furthermore, using the specific GPR116 peptide agonist p116, we could trigger somatostatin release from wt pancreatic islets, however, delta cell-specific ko of GPR116 completely abolished this signal (Fig. 4a).

**Fig. 4: Characterization of delta cell-specific *Gpr116* ko mice.**

When analyzing glucose-induced somatostatin and insulin secretion, we found that deletion of functional GPR116 in pancreatic delta cells neither changed the amounts of secreted hormones nor the content (Fig. 4b, c for somatostatin, Fig. 4e, f for insulin). Furthermore, glucose responsiveness evaluated by the normalized values was comparable for wt and ko animals for both hormones (Fig. 4d, g).

This is also reflected in vivo with no major differences when performing glucose and insulin tolerance tests (Fig. 4h, i).

Using a delta cell-specific ko mouse model we demonstrate that activation of GPR116 expressed in pancreatic delta cells increases somatostatin release. However, the observed reduced insulin content in pancreatic islets as well as the increased fasting blood glucose levels of constitutive GPR116-deficient mice is not caused by the absence of GPR116 expressed in delta cells.

GPR116 deficiency reduces insulin secretion in vivo and alters insulin metabolization

Constitutive GPR116 ko leads to increased fasting blood glucose levels already under chow diet (see above). To investigate the mechanisms resulting in this phenotype, we further evaluated metabolic characteristics of these ko animals. To analyze whether the reduced islet insulin secretion is also observed in vivo, we measured plasma insulin content in overnight fasted ko and wt mice at 0, 10, and 20 min after intraperitoneal injection of glucose (Fig. 5a) and no significant differences were observed. However, when analyzing C-peptide plasma concentration, we found significantly reduced amounts after 20 min of glucose administration (Fig. 5b) indicating that the reduced islet insulin secretion also leads to a reduced amount of released insulin in vivo. As this did not translate into different plasma insulin levels, we speculated that insulin metabolization might be affected by GPR116 ko. The insulin-degrading enzyme (IDE) is expressed in hepatocytes and functions as the key enzyme in insulin degradation. Thus, we analyzed Ide expression in liver tissue (Fig. 5c) and isolated hepatocytes (Fig. 5d), however, no differences were observed. Next, we determined activity of IDE in hepatocytes isolated from ko and wt mice and detected a reduced activity in GPR116 ko animals (Fig. 5e). Furthermore, insulin expression was significantly higher in hepatocytes isolated from ko animals compared to wt siblings (Fig. 5f). Together, reduced degradation and increased production of insulin in hepatocytes could be responsible for adjusting plasma insulin levels as a result of dysregulated glucose levels. Indeed, we found an increased insulin expression in isolated wt hepatocytes when stimulated with high glucose (Supplementary Fig. 3a).

**Fig. 5: Analysis of insulin metabolism in constitutive GPR116 ko mice.**

Since differences in fasting blood glucose levels hint towards changes in liver gluconeogenesis, we performed pyruvate tolerance tests in overnight-fasted animals. Here, ko mice showed a significant increase in blood glucose levels 20 and 30 mins after pyruvate injection indicating a higher gluconeogenesis rate compared to wt animals (Fig. 5g). Therefore, we analyzed expression of key enzymes of glycolysis and gluconeogenesis in liver samples of wt and ko mice but found no differences (Fig. 5h).

GPR116 ko alters pancreatic islets

The previous results show that deletion of GPR116 specifically in pancreatic delta cells is not the cause for the observed change in insulin content of constitutive GPR116 ko animals. To find the cause of this phenotype, we quantified the amounts of insulin-positive beta cells and somatostatin-positive delta cells. Thereto, we performed immunofluorescence staining of sections of mouse pancreas with specific antibodies against insulin and somatostatin (Fig. 6a, b). The analysis revealed a decreased number of insulin-positive beta cells in GPR116 ko mice while the amount of somatostatin-positive delta and glucagon-positive alpha cells did not significantly differ between wt and ko mice (Fig. 6c and Supplementary Fig. 3b, c).

**Fig. 6: GPR116 deficiency affects islet number, insulin-positive area, and insulin expression.**

Furthermore, trends towards smaller total islet area in pancreatic sections and to a decreased total number of pancreatic islets in ko mice were observed (Figs. 6d and 6e). In-depth analysis showed a significantly reduced number in small islets with a diameter of < 100 µm (Fig. 6f and Supplementary Fig. 3d).

Quantitative PCR analysis of adult mice revealed that expression of the insulin genes Ins1 and Ins2 was significantly lower in ko islets compared to wt islets (Fig. 6g, h). Also, somatostatin expression is significantly reduced in ko islets compared to wt islets, whereas glucagon expression remained unchanged (Supplementary Fig. 3e, f). Interestingly, we observed the opposing expression pattern in pancreatic tissue from neonatal P1 mice. Here, ko animals showed significantly higher insulin gene expression (Fig. 6i, j). This data suggests that GPR116 is already involved in the development of pancreatic islets leading to a reduced amount of beta cells as well as pancreatic islets.

Discussion

Mammalian pancreatic islets consist of five different endocrine cell types - alpha, beta, delta, epsilon, and PP cells⁴³ - as well as endothelial cells⁴⁴. While the most abundant insulin- and glucagon-secreting beta and alpha cells have been intensively studied, less attention has been paid to the other cell types. However, paracrine crosstalk between pancreatic cells may influence islet cell composition and hormone secretion. For example, it is well established that somatostatin released by pancreatic delta cells negatively regulates both, insulin and glucagon secretion^45,46. Currently, the importance of somatostatin secreted by delta cells is discussed with respect to insulin-induced hypoglycemia in diabetic models⁴⁷. Furthermore, interactions of pancreatic beta cells and endothelial cells have been proposed⁴⁸.

Here, we describe expression of GPR116, a member of the adhesion GPCR class, in pancreatic islets. Besides its well-studied function in lung surfactant regulation, GPR116 is also involved in endothelial function and metabolic processes^{23,24,25,27,33,34}. However, due to its high expression in pancreatic islets^2,3,15,16, we assumed a role of GPR116 in metabolic processes beyond modulating insulin sensitivity in adipocytes^33,34. In contrast to rhodopsin-like GPCRs the role of aGPCRs in pancreatic islets is not well-established, however, some aGPCRs have been shown to regulate islet function and/or hormone secretion. GPR56 has been implicated in beta cell viability and insulin secretion in pancreatic islets via collagen III interaction^35,36. LPHN3 and BAI3 activation have been linked to reduction of insulin secretion^37,38, whereas the activation of LPHN1 increases the release of insulin³⁷. Because most aGPCRs are still orphan receptors, elucidating their physiological roles is severely limited. Previous work on GPR116 identifying synthetic peptides derived from the Stachel sequence of the receptor suitable for activation^21,22 and a knock-out mouse line enabled us to study the function of GPR116 in pancreatic islets.

The here presented data clearly demonstrate that abundant Gpr116 expression is found in somatostatin-positive islet cells, while there is only marginal overlap with insulin or glucagon (Fig. 1) suggesting a role in pancreatic delta cells. This is supported by RNA-seq data of sorted pancreatic cells^2,3 indicating a high expression of GPR116 in pancreatic delta cells but not alpha or beta cells. According to a delta cell-dependent function we found that activation of GPR116 using a Stachel-derived peptide results in a robust somatostatin release which could be blocked using the Gα_q/11 inhibitor FR900359⁴² (Fig. 2g, h) indicating that the effect of somatostatin release is G_q/11-protein dependent. These data confirm the reported GPR116-mediated G-protein signaling in vitro and ex vivo^21,22,32. Because of the observation that GPR116 is expressed by another islet cell type, we used a constitutive and delta cell-specific Gpr116 ko mouse model²³ to demonstrate that the modulation of somatostatin secretion is mediated by delta-cell GPR116 (Figs. 3a and 4a). Therefore, we conclude that activation of GPR116 induces somatostatin release via a G_q/11 protein/Ca²⁺-mediated mechanism. Thus, GPR116-dependent regulation of somatostatin secretion shows similarity to the signaling of the ghrelin receptor expressed in delta cells^2,3. Unfortunately, the endogenous signal for triggering somatostatin release via GPR116 still remains unknown. Recently, the hepatokine FNDC4 has been identified to activate GPR116 leading to an increase in intracellular cAMP³⁴. However, the increase of somatostatin secretion is a result of the G_q protein pathway which is not activated via the FNDC4 / GPR116 axis. It is tempting to speculate that the immunoglobulin-like domains of the large N terminus of GPR116 mediates GPR116 activation by cell-cell contacts. Also, aGPCRs have been shown to be activated by cell-matrix molecules like collagen and laminin^49,50,51 or by mechanical forces^51,52,53. Even though there has no mechano-activation of GPCRs been shown on pancreatic islets, it has been reported that glucose stimulation might cause an increase in volume and, therefore, membrane tension in pancreatic cells^54,55.

Interestingly, using the constitutive knock-out of GPR116 we observed several changes regarding the pancreatic islets which are not connected to the receptor’s function in delta cells. Such, insulin content which was significantly reduced in GPR116 ko islets leading to a reduced glucose-stimulated insulin secretion. However, relative insulin secretion (in relation to insulin content) was not different in wt and ko mice (Fig. 3), indicating no alteration of the secretion process itself. Furthermore, ko animals display increased fasting blood glucose already under chow diet. This phenotype was also not observed using the delta-cell specific ko mouse model. Despite the reduced amount of secreted insulin, we did not observe altered glucose or insulin tolerances when comparing wt and ko mice. This seems to be contrary to previous work, where an adipocyte-specific GPR116 ko resulted in an impaired glucose tolerance^33,34. However, a germline GPR116 ko may mask this adipocyte-specific phenotype because of summative results in the regulation of the glucose homeostasis. Furthermore, GPR116 ko mouse lines are differently generated. In the mouse line used in our study only the G protein-mediated signaling is fully abolished (Supplementary Fig. 2). Because we still detect significant cell surface expression of the receptor’s N terminus, we cannot exclude trans-signaling (N terminus as ligand or interaction partner for other proteins). This is a specific feature of aGPCR which can display cis- and trans-signaling abilities with different physiological functions^56,57.

As the reduced amount of insulin content as well as the reduced glucose-induced insulin secretion is contradictory to the results of the glucose tolerance test, we also analyzed plasma insulin and C-peptide content after glucose administration (Fig. 5a, b). Interestingly, we found comparable insulin amounts but significantly reduced C-peptide in GPR116 ko mice. Our further work, therefore, concentrated on the compensating actions regulating plasma insulin. Here, we find reduced IDE activity connected to increased expression of proinsulin in hepatocytes (Fig. 5). Since proinsulin is a known inhibitor of IDE⁵⁸, we conclude that the increased expression of proinsulin in hepatocytes regulates the degradation of insulin resulting in wt-like glucose tolerance.

Furthermore, our immunofluorescence analysis revealed a reduced number of islets and beta cells in GPR116 ko mice which likely accounts for the reduced insulin content and, subsequently, insulin secretion. This phenotype is clearly independent of GPR116 function in delta cells. The reduction in insulin might be the cause for the observed increased fasting blood glucose levels in whole-body ko mice, however, with Gpr116 expressed in several tissues including liver, we cannot exclude a non-islet cause for the increased fasting blood glucose levels. However, in situ hybridization revealed Gpr116 mRNA expressed in Cd34 positive cells. High expression of Gpr116 has been shown for endothelial cells in general²⁷ but also for islet endothelial cells⁵⁹. Furthermore, Gpr116 is amongst the 10 highest expressed GPCRs during islet development (E18)¹⁴ indicating that it is involved in developmental processes. Interestingly, when analyzing insulin expression in pancreas of newborns at postnatal day 1, we observed increased insulin expression in ko animals (Fig. 6e, j). Such high insulin levels during islet development have been shown to induce apoptosis in beta cells⁶⁰, which may explain the reduced number of insulin-positive cells in GPR116-deficient animals. Therefore, one can assume that GPR116 is important during islet development mediating an anti-apoptotic effect on beta cells. Taking all data together, we clearly demonstrate that delta-cell expressed GPR116 activation induces secretion of somatostatin. Furthermore, we show that whole-body lack of GPR116 leads to a mild phenotype of increased blood glucose levels caused by increased gluconeogenesis. However, with expression of GPR116 in several tissues, we currently cannot clarify which tissue-specific function of GPR116 leads to this phenotype. Similarly, we find an effect of GPR116 onto islet development. However, at the current stage we cannot distinguish how GPR116 is involved in this process. We could speculate that loss of the receptor in an endocrine precursor cell or the lack in endothelial cells is responsible for the observed phenotype, however, it does not necessarily need to be located in one of these cell types.

In summary, our data implies that GPR116 has different functions in pancreatic islets: in adult pancreatic islets, delta cell-expressed GPR116 can augment somatostatin secretion after receptor activation. In addition, GPR116 is required for the development of fully functional islets by influencing islet number, amount of beta cells as well as insulin expression, and subsequently, insulin content. This strengthens the idea that GPR116 has a dual function in fine-tuning pancreatic islet development and endocrine functionality.

Methods

Material

All standard chemicals were purchased from Sigma-Aldrich Chemie GmbH or C. Roth GmbH + Co. KG. If not mentioned otherwise, reagents, cell culture materials, and kits were obtained from Thermo Fisher Scientific. Primers were synthesized from Seqlab or Thermo Fisher Scientific. Peptide synthesis was carried out by the Core Unit Peptide Technology²².

Ca²⁺ imaging

Fura-2-based Ca²⁺ imaging was performed in transfected HEK293T cells (Supplementary Experimental Procedures) and intact pancreatic islets using a monochromator-based imaging system, and the imaging software TILLvisION 4.0 (T.I.L.L. Photonics). Emitted fluorescence (excited at 340 and 380 nm) was acquired at intervals of 2 seconds and corrected for background fluorescence. HEK293T cells and islets were loaded with 5 µM fura-2 AM in standard bath solution containing 140 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 3 mM glucose for 30 or 60 min. HEK293T cells (25,000 cells/well) were seeded into 24-well plates on poly-L-lysine coated glass cover slips and were transiently transfected with plasmids encoding for GPR116 and GFP the next day. Ca²⁺ imaging was carried out 72 h post transfection. HEK293T cells were exposed to p116sc, p116 (1 mM each), and ATP (100 µM) in standard bath solution. Pancreatic mouse islets (3 - 4 islets per coverslip) were transferred one day after preparation into 12-well plates on poly-L-lysine coated glass cover slips. 48 h after seeding islets were exposed to 1 mM p116sc, 1 mM p116, and 100 µM carbachol (CCh) in standard bath solution.

RNA isolation, reverse transcriptase PCR, and quantitative real-time PCR

RNA was isolated from pelleted cells or 80–180 pancreatic islets with ReliaPrep^TM RNA Cell Miniprep System (Promega) or PicoPure^TM RNA Isolation Kit, respectively. Tissue RNA from pancreas and liver was isolated using SV Total RNA Isolation System (Promega) according to the manufacture’s protocol. RNA concentration and quality were determined using the NanoDrop technology (Peqlab). Reverse transcription was carried out with Superscript II Reverse Transcriptase using oligo (dT) and random hexamer primers following the manufacturer’s instructions.

Quantitative real-time PCR (qPCR) was performed by Platinum SYBR Green qPCR SuperMix-UDG with the MxPro3000 instrument (Agilent). The qPCR setup was as follows: each reaction (20 µl) contained of 10–30 ng cDNA, 10 µM primers, 6.25 µl SYBR Green and 0.5 µl of reference dye ROX (1:10 diluted in water). All samples were run in triplicates using the following protocol: 2 min at 50 °C, 2 min at 95 °C, 45 cycles of 95 °C for 15 s and 60 °C for 30 s. Subsequently, melt curves were recorded (50–95 °C, 0.5 °C increment, 10 s/step). Quantitative PCR primer pairs are summarized in Supplementary Table 1. Measured Ct values were normalized to the reference gene (β-actin, Actb) to calculate ΔCt values as relative expression for the genes of interest.

Animals

Mice were bred under specific pathogen-free conditions on 12 h light/12 h dark cycle, 21 °C, and 55% humidity with ad libitum access to food and water. All experiments were conducted in accordance with European Directive 2010/63/EU on the protection of animals used for scientific purposes and were performed with permission from the Animal Care and Use Committee (ACUC# T24/16; T19/18; TVV02/15; TVV43/18) and the Government of the State of Saxony, Germany. We have complied with all relevant ethical regulations for animal use. All mice were between 10 and 16 weeks and were matched to age and sex.

Mice strains and genotyping

MIP-GFP (B6.Cg-Tg(Ins1-EGFP)1Hara/J) express GFP under the control of mouse insulin 1 promoter leading to GFP expression in pancreatic beta cells⁶¹. Sst-Cre (B6N.Cg-Ssttm2.1(cre)Zjh/J) are knock-in animals with Cre recombinase expression in somatostatin-positive cells. Both mice strains were obtained from Jackson laboratories (#006864 and #018973).

The generation of GPR116 ko and GPR116-floxed mice has been described previously²³. Delta cell-specific GPR116 deletion was obtained by crossing GPR116-floxed and Sst-Cre mice. Thereby, mice heterozygous for Cre recombinase and homozygous for GPR116-floxed represent ko animals. The corresponding wt mice also contain heterozygous Cre recombinase but the wt background at the GPR116 locus.

Tail biopsies were digested overnight with proteinase k (500 µg/ml) in lysis buffer (1 M Tris-HCl, 10% SDS, 0.5 mM EDTA, 5 M NaCl, pH 8). An ethanol precipitation protocol was used for extraction of genomic DNA followed by genotyping PCR.

MIP-GFP mice were genotyped using 5’-AAGTTCATCTGCACCACCG and 5’-TCCTTGAAGAAGATGGTGCG by applying the following protocol: denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 60 s and a final amplification step of 72 °C for 10 min. Genotyping of GPR116 mice was performed by PCR with following primers: 5’-GTGACAAACCCTGTAGACA, 5’-AAACCCAAACAAGAGGAGGA, and 5’-TACTTGTGTGATGGGGACAT. PCR protocol was used for genotyping as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 62 °C for 45 s, and 72 °C for 30 s and a final amplification step of 72 °C for 10 min. For genotyping of GPR116-floxed mice genomic DNA was amplified using 5’-GTGACAAACCCTGTAGACA and 5’-AGAAAATCTCTCCCCTGC for 35 cycles (95 °C for 4 min initial denaturation; 95 °C for 30 s, 52 °C for 45 s, and 72 °C for 90 s; 72 °C for 10 min for final amplification). Determination of Sst-Cre was performed with the following primers: 5’-GGGCCAGGAGTTAAGGAAGA, 5’-TCTGAAAGACTTGCGTTTGG, and 5’-TGGTTTGTCCAAACTCATCAA using a touch-down protocol (95 °C for 5 min initial denaturation; 94 °C for 45 s, 10 cycles 65 °C to 60 °C for 45 s followed by 28 cycles of 60 °C, and 72 °C for 90 s; 72 °C for 10 min).

Islet isolation

Pancreatic islets were isolated from mice sacrificed by cervical dislocation. Collagenase p solution (0.5 mg/ml) was dissolved in ice-cold Krebs-Ringer buffer (KRB: 115 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.56 mM CaCl₂, 20 mM NaHCO₃, 10 mM Hepes, 0.1% BSA, 5 mM glucose, pH 7.3) and was injected via the common bile duct into pancreas. Afterwards, distended pancreas was digested in shaking water bath at 37 °C for 12–15 min and isolated islets were cultured in RPMI + 10% FBS and 100 units/ml penicillin, and 100 μg/ml streptomycin overnight at 37 °C and 5% CO₂ in a humidified atmosphere. For Ca²⁺ imaging, islets were cultured up to 3 days.

Islet stimulation and hormone determination

Equilibration of five islets was performed in KRB containing 2.8 mM glucose for 30 min at 37 °C in 96-well plates. If not mentioned otherwise, islets were stimulated with peptide solution or respective controls in KRB for 30 min at 37 °C. Islets were lysed with acid ethanol and supernatant as well as lysates kept frozen at −20 °C until measurement. To measure insulin the AlphaLISA technology (Perkin Elmer) was used according to the manufacturer’s protocol in 384-well OptiPlate microplates (Perkin Elmer) with the EnVision Multilabel Reader (Perkin Elmer). Somatostatin amounts were determined using either AlphaLISA (Perkin Elmer) or ELISA (Phoenix Pharmaceuticals). For reliable measurement protocol adaptions were necessary. For AlphaLISA these were as follows: increasing sample volumes to 35 µl/well, reduced bead volume of 5 µl/well, and an extended incubation period with donor beads of 90 min. For the ELISA measurement the sample incubation time was extended to over-night and the biotinylated peptide was diluted 1:5 to achieve reliable results.

Combined fluorescence in situ hybridization and immunolabeling

The RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, ACD) was used to colocalize multiple RNAs and antigens on the same section. Pancreas samples were immediately fixed in 4% formalin for 24 hours and embedded in paraffin. 10-µm-thick sections were prepared from the paraffin blocks and mounted on Superfrost® slides. For dual labeling of specific Gpr116 and somatostatin RNA, a mixture of 1:50 diluted commercially purchased probes for Gpr116 (318021-C2, ACD) and somatostatin (404631-C3, ACD) was prepared. The freshly diluted probes were then incubated for 2 hours at 40 °C in a manual HybEZ™ II assay hybridization system (ACD) on the organ sections. This was followed by two consecutive amplification steps and labeling of the probes with single Opal fluorophores at a dilution of 1:750 (Akoya Biosciences). Gpr116 was labeled with Opal 570 (OP-001003) and somatostatin was labeled with Opal 690 (OP-001006). Between each RNA labeling step, the sections were thoroughly washed with wash buffer. After completion of RNA detection, either insulin or glucagon was co-detected by immunolabeling. Prior to antigen labeling, sections were treated with blocking buffer (0.3% Tris / 0.3% Triton X-100 / 5% NGS in PBS). Immunostaining with diluted primary rabbit polyclonal antibodies against insulin (3014 S, Cell Signaling Technologies, 1:1000) and glucagon (2760 S, Cell Signaling Technologies, 1:400) was performed overnight at 4 °C. Binding of the primary antibodies was labeled after washing in PBS with a 1:250 diluted secondary Alexa-488-conjugated goat anti-rabbit antibody (A11034, Invitrogen) at room temperature for 1 hour. After another wash, DAPI nuclear counterstaining (ACD) was performed prior to embedding the sections in Dako fluorescent mounting medium (Aligent). LSM 700 confocal laser scanning microscopy was used to evaluate and document the results of fluorescent in situ hybridization and immunolabeling.

Immunofluorescence assessment of islet tissue

Semiautomated quantification of pancreatic endocrine cell masses was essentially performed as described by Golson and coworker⁶² and involved standardized systematic tissue preparation, high-resolution slide scanning and computerized image analysis. In brief, pancreata were removed in total from the retroperitoneal space from lateral to medial under binocular microscopic view. The dissected pancreata were weighted, spread out longitudinally in cassettes, fixed in 4% formaldehyde overnight, dehydrated, and embedded in paraffin. The paraffin blocks were cut 5 µm thick in 10 series each containing sections a 250 µm distance to each other. By that series with 10 to 12 sections per pancreas were yielded. The sections in a series were dewaxed and either HE-stained or indirect immunofluorescence double-labeled for the detection of somatostatin in combination with insulin. Prior to antibody incubation sections were microwaved for antigen retrieval in 10 mM citrate buffer (pH 6.0) for 5 min, and exposed to 3% normal goat serum (NGS) containing 0.3% Triton X-100 for 1 hour for reduction of background staining. Primary antibody incubation was performed with a cocktail including 1:400 diluted rat anti-somatostatin (Abcam) and 1:2,000 diluted rabbit anti insulin (Cell Signaling Technologies) or 1:400 diluted rabbit anti glucagon (Cell Signaling Technologies) antibodies. The primary antibody cocktail was applied in PBS supplemented with 0.5% NGS and 0.03% Triton-X 100 in a moist chamber at 4 °C overnight. Primary antibody binding was probed with a secondary antibody cocktail containing Alexa 568 goat anti rat and Alexa 488 goat anti rabbit antibodies both at a 1:250 dilution in a moist atmosphere at 25 °C for 30 min. After immunofluorescence labeling, sections were thoroughly washed, nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI), coverslipped in Roti®-Mount FluorCare mounting medium and hardened overnight.

Double immunofluorescence-stained sections were fully digitized at 20x magnification using a digital slide scanner (Pannoramic Scan II, 3D HISTECH Ltd.) equipped with a quad band (DAPI/FITC/TRITC/Cy5) filter set. Images of the sections were manually exported from slide scanner data sets (Pannoramic Viewer, Version 1.15.4, 3D HISTECH Ldt.) as TIFF images with pixel dimensions of 0.65 µm. Perimeters of pancreatic islets were manually drawn onto the exported images (GIMP 2.10.2) and exported as a separate image containing all islet masks of the respective fluorescence image. Image analysis was performed with Mathematica (Version 11.1, Wolfram Research, Inc.). Fluorescence images were imported and split into separate color channels. Color correction of red and green image channels was performed by subtracting one from the other. This procedure resulted in red and green images containing only relevant signals without significant background fluorescence. Matching islet mask images were imported and all present islet regions were identified and counted. Image sections of each islet region were extracted from the color corrected images and separately submitted to image segmentation. Green channels (insulin) were segmented using Otsu’s (cluster variance maximization) thresholding method⁶³, while red channels (somatostatin) were segmented using Kapur’s (histogram entropy minimization) thresholding method⁶⁴. Finally, the area of each islet region and its segmented red and green cell content was computed.

Functional in vivo studies

Glucose and pyruvate tolerance tests were carried out with mice fasted overnight (15 - 18 h). Blood glucose levels were measured before intraperitoneal injection of glucose (10 µl/g body weight of 20% glucose solution, Glucosteril, Fresenius Kabi GmbH) or pyruvate (10 µl/g body weight of 10% pyruvate solution). Blood glucose levels were determined at the indicated time points after injection. For insulin tolerance tests mice were fasted for 6 h and blood glucose levels determined. Insulin (1 mU/g body weight human Insuman Rapid, Sanofi) was injected intraperitoneally and blood glucose levels determined at the indicated time points.

Hepatocyte isolation and determination of IDE activity

Primary hepatocytes were isolated from male and female GPR116 ko mice and wt siblings. Isolation of liver cells is based on an in vivo perfusion method described by Seglen in the 1970s⁶⁵ and used today with modifications^66,67,68. Both the portal vein and vena cava are cannulated to perfuse the liver with buffer for calcium depletion and blood washing, followed by perfusion with collagenase. This two-step procedure allows the matrix of the liver parenchyma to be dissolved. The closed liver capsule allows the entire liver to be removed from the animal after perfusion and transferred to buffer. After rupture of the capsule, the liver cells, both hepatocytes and NPCs, are in suspension. To ensure the highest possible purity of the hepatocytes, the NPC fraction should be separated as completely as possible by differential centrifugation steps. IDE activity was determined using SensoLyte® 520 IDE Activity Assay (AnaSpec Inc.) according to the manufactures’ protocol. In brief, 1 × 10⁶ hepatocytes were washed with phosphate-buffered saline and lysed in 1 ml assay buffer. After 15 min incubation on ice, cells were centrifuged and the supernatant stored at -70 °C until use. Then, the supernatant was mixed with IDE substrate solution, incubated for 30 min at 37 °C, and fluorescence intensity was measured at the EnVision Multilabel Reader (Ex/Em = 490 nm/520 nm).

Statistics and reproducibility

For statistical and graphical analyses Prism version 9 (GraphPad Software Inc., San Diego, CA) was used. Comparing two groups, statistical significance was analyzed using the two-tailed t test, comparing two groups at multiple time points an unpaired multiple t test corrected for multiple testing using the Holm-Šídák method was applied. When comparing several datapoints to one reference one-way ANOVA with Dunnet’s multiple comparison test was performed. The applied tests are given in the figure legends. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data supporting the findings of this study have been deposited at Zenodo (https://doi.org/10.5281/zenodo.10459692)⁶⁹ and are publicly available as of the date of publication. All other information is available from the corresponding author on reasonable request.

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Acknowledgements

We thank Petra Krumbholz, Jana Brendler, Lea Fitze, and Natalie Marquar for excellent experimental assistance, Jessica Michelle Jannicke, Irina Seiler, and Tina Steinhardt for outstanding animal care, and Sven Rothemund for peptide synthesis. This research was supported by grants from the European Social Fonds, the German Diabetes Association (DDG) and the German research foundation (CRC1052 project number 209933838, CRC1423 project number 421152132). The publication is funded by the Open Access Publishing Fund of Leipzig University, which is supported by the German Research Foundation within the program Open Access Publication Funding.

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Authors and Affiliations

Rudolf Schönheimer Institute of Biochemistry, Medical Faculty, Leipzig University, Leipzig, Germany
Juliane Röthe, Isabell Kaczmarek, André Nguyen Dietzsch, Julia Buchold, Ines Liebscher, Torsten Schöneberg & Doreen Thor
Carl-Ludwig-Institute for Physiology, Medical Faculty, Leipzig University, Leipzig, Germany
Robert Kraft
Institute of Anatomy, Medical Faculty, Leipzig University, Leipzig, Germany
Albert Ricken & Karsten Winter
Medical Department II – Gastroenterology, Hepatology, Infectious Diseases, Pneumology, University Medical Center, Leipzig, Germany
Madlen Matz-Soja
Division of Hepatology, Clinic and Polyclinic for Oncology, Gastroenterology, Hepatology, Infectious Diseases, and Pneumology, University Hospital, Leipzig, Germany
Madlen Matz-Soja
Novartis Institutes for Biomedical Research, Basel, Switzerland
Marie-Gabrielle Ludwig

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Juliane Röthe
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Contributions

J.R. and D.T. conceived the project, designed the experiments, wrote the manuscript, and prepared the figures. J.R., D.T., R.K., A.R., M.M.-S., I.K., and A.N.D. performed experimental work. K.W. performed the analysis of immunofluorescence images. I.L. provided the GPR116 peptide and established the in vitro functional assays. J.B. generated the GPR116 receptor construct and M.G.L. provided the GPR116 ko mouse model. T.S. discussed the data and contributed to the manuscript. All authors reviewed the manuscript.

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Correspondence to Doreen Thor.

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Röthe, J., Kraft, R., Ricken, A. et al. The adhesion GPCR GPR116/ADGRF5 has a dual function in pancreatic islets regulating somatostatin release and islet development. Commun Biol 7, 104 (2024). https://doi.org/10.1038/s42003-024-05783-9

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Received: 03 November 2023
Accepted: 05 January 2024
Published: 16 January 2024
DOI: https://doi.org/10.1038/s42003-024-05783-9

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