# Neuropeptide 26RFa (QRFP) is a key regulator of glucose homeostasis and its activity is markedly altered in obese/hyperglycemic mice

Gaëtan Prévost, Arnaud Arabo, Marie-Anne Le Solliciec, Justine Bons, Marie Picot, Julie Maucotel, Hind Berrahmoune, Mouna El Mehdi, Saloua Cherifi, Alexandre Benani, et al.

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Gaëtan Prévost, Arnaud Arabo, Marie-Anne Le Solliciec, Justine Bons, Marie Picot, et al. Neuropeptide 26RFa (QRFP) is a key regulator of glucose homeostasis and its activity is markedly altered in obese/hyperglycemic mice. AJP - Endocrinology and Metabolism, 2019, 317 (1), pp.E147-E157. 10.1152/ajpendo.00540.2018. hal-02330443

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The neuropeptide 26RFa (QRFP) is a key regulator of glucose
homeostasis and its activity is markedly altered in
obese/hyperglycemic mice

Gaëtan Prévost¹,², Arnaud Arabo¹, Marie-Anne Le Sollicec¹, Justine Bons¹,², Marie Picot¹, Julie Maucotel¹, Hind Berrahmoune¹,², Mouna El Mehdi¹, Saloua Cherifi¹, Alexandre Benani³, Emmanuelle Nédélec³, Moïse Coëffier⁴,⁵, Jérôme Leprince¹, Anneli Nordqvist⁶, Valéry Brunel⁷, Pierre Déchelotte⁴,⁵, Hervé Lefebvre¹,², Youssef Anouar¹, Nicolas Chartrel¹,*

¹Normandie Univ, UNIROUEN, INSERM U1239, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication (DC2N), 76000 Rouen, France

²Normandie Univ, UNIROUEN, Rouen University Hospital, Department of Endocrinology, Diabetes and Metabolic Diseases, 76000 Rouen, France

³Center for Taste and Feeding Behaviour, CNRS (UMR6265), INRA (UMR1324), Université de Bourgogne-Franche Comté, Dijon, France.

⁴Normandie Univ, UNIROUEN, INSERM U1073 "Nutrition, Inflammation and dysfunction of gut-brain axis", 76000 Rouen, France

⁵Normandie Univ, UNIROUEN, Rouen University Hospital, Department of Nutrition, 76000 Rouen, France

⁶Cardiovascular Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, SE-431 83 Mölndal, Sweden

⁷Normandie Univ, UNIROUEN, Rouen University Hospital, Department of Biochemistry, 76000 Rouen, France

**Running head:** the neuropeptide 26RFa and chronic hyperglycemia

*Corresponding author: Dr Nicolas Chartrel, Normandie Univ, UNIROUEN, INSERM U1239, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication (DC2N), 76000 Rouen, France

Phone: (33)235 14 6686; Fax: (33)23514 6946

e-mail address: nicolas chartrel@univ-rouen.fr

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35 **ABSTRACT**

36 Recent studies have shown that the hypothalamic neuropeptide 26RFa regulates glucose
37 homeostasis by acting as an incretin, and increasing insulin sensitivity. In this study, we
38 further characterized the role of the 26RFa/GPR103 peptidergic system in the global
39 regulation of glucose homeostasis using a 26RFa receptor antagonist, and also assessed
40 whether a dysfunction of the 26RFa/GPR103 system occurs in obese hyperglycemic mice.

41 Firstly, we demonstrate that administration of the GPR103 antagonist reduces the global
42 glucose-induced incretin effect and insulin sensitivity whereas, conversely, administration of
43 exogenous 26RFa attenuates glucose-induced hyperglycemia. Using a mouse model of high-
44 fat diet-induced obesity and hyperglycemia, we found a loss of the anti-hyperglycemic effect
45 and insulinotropic activity of 26RFa, accompanied with a marked reduction of its insulin-
46 sensitive effect. Interestingly, this resistance to 26RFa is associated with a down-regulation of
47 the 26RFa receptor in the pancreatic islets, and insulin target tissues. Finally, we observed that
48 the production and release kinetics of 26RFa after an oral glucose challenge is profoundly
49 altered in the high-fat mice. Altogether, the present findings support the view that 26RFa is a
50 key regulator of glucose homeostasis whose activity is markedly altered under
51 obese/hyperglycemic conditions.

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53 **Key words:** 26RFa; incretin; glucose homeostasis; diabetes; obesity

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The neuropeptide 26RFa, also referred to as QRFP (for pyroglutamilated RFamide peptide), is the latest member of the RFamide peptide family discovered (5,9,16). 26RFa and its N-extended form 43RFa were identified as the cognate ligands of the human orphan G-protein-coupled receptor, GPR103 (6,9,16,30). This peptidergic system is abundantly expressed in the hypothalamus and is involved in the control of feeding behaviour (3,4,5,30). Indeed, 26RFa (or 43RFa) stimulates food intake (5,20,21,30), and the neuropeptide exerts its orexigenic activity via a modulation of the NPY/POMC network in the arcuate nucleus (20). These observations have been recently confirmed by data obtained from 26RFa-deficient mice showing that the knockout (KO) mice are lean and hypophagic, and that, under a high-fat (HF) diet, the 26RFa KO mice show a lower weight gain than the wild-type animals (22). In line with this, a more sustained orexigenic activity of 26RFa/43RFa has been reported in wild type mice submitted to a HF diet (21,27). Moreover, the expression of prepro26RFa mRNA is up-regulated in the hypothalamus of genetically obese *ob/ob* and *db/db* mice, and rodents submitted to a HF diet (27,30). Altogether, these findings support the notion that 26RFa could play a role in the development and maintenance of the obese status (7).

Accumulating evidence over the last decade supports the role of neuropeptides controlling feeding behavior such as neuropeptide Y (NPY), orexins, ghrelin, corticotropin-releasing factor (CRF) or apelin, in the peripheral regulation of glucose homeostasis (14,15,23,32,33). This has led to the new concept that hypothalamic neuropeptides may serve as a link between energy and glucose homeostasis, and therefore, they represent potential therapeutic targets for the treatment of diabetes and obesity (28,29). In this context, the potential role of 26RFa in the control of glucose homeostasis was investigated. It was found that 26RFa/43RFa and GPR103 are expressed in pancreatic islets, as well as in various insulin-secreting cell lines
(11,26), and that 26RFa and 43RFa prevent cell death and apoptosis of β cell lines and isolated human pancreatic islets (11). It has been shown that 26RFa is abundantly expressed by the gut and that i.p. administration of the neuropeptide attenuates glucose-induced hyperglycemia by increasing plasma insulin via a direct insulinotropic effect on the pancreatic β cells, and also by increasing insulin sensitivity (26). Furthermore, it was reported that an oral glucose challenge induces a massive secretion of 26RFa by the gut into the blood, strongly suggesting that this neuropeptide plays an important role in the regulation of glycemia by acting as an incretin (26).

The incretin effect is known to be markedly reduced in diabetic patients (18), although most studies have reported that levels of GLP-1 and gastric inhibitory polypeptide (GIP) in response to oral glucose were similar in diabetic and non-diabetic subjects (8,13,18). This incretin defect is notably characterized by a decreased, albeit partially preserved, insulinotropic potency of GLP-1 and an almost complete loss of insulin secretion to GIP (18). In this context, the objective of the present study was twofold: (1) to further characterize the role of 26RFa/GPR103 peptidergic system in the global regulation of glucose homeostasis (via an oral glucose tolerance test) using a 26RFa receptor antagonist (namely 25e) (10); and (2) to determine whether a dysfunction of the 26RFa/GPR103 system occurs in obese/hyperglycemic conditions.

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# MATERIAL AND METHODS

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*Animals*. Male C57Bl/6 mice (Janvier laboratory, Le Genest-Saint-Isle, France), weighing 22-25 g, were housed at 5 animals per cage, with free access to standard diet (U.A.R., Villemoisson-sur-Orge, France) and tap water. They were kept in a ventilated room at a temperature of 22±1°C under a 12-h light/12-h dark cycle (light on between 7 h and 19 h). All the experiments were carried out between 09.00 h and 18.00 h in testing rooms adjacent to the animal rooms. Animal manipulations were performed according to the European Community Council Directive of November 24th 1986 (86:609: EEC), and were conducted by authorized investigators.

*High-fat (HF) model*. Mice were submitted to a dietary challenge at two months of age by using a standard chow (U8200G10R; energy density: 2 830 kcal/kg; Scientific Animal Food & Engineering (SAFE), Augy, France) or a HF diet (U8978; energy density: 5 283 kcal/kg; SAFE). Experiments were performed after three months of dietary challenge. Our Institutional Animal Use and Care Committee (CENOMAX, Agreement of the Ministry of Research n°54) approved the experimental procedures (Agreement n°: #11752).

*Peptides and chemicals*. Mouse 26RFa (ASGPLGTLAEELSSYSRRKGGFSFRF-NH₂) was synthezised (0.1 mmol scale) by the solid phase methodology on a Rink amide 4-methylbenzhydrylamine resin (Biochem, Meudon, France) by using a 433A Applied Biosystems peptide synthesizer and the standard fluorenylmethoxycarbonyl (Fmoc) procedure. The synthetic peptide was purified by reversed-phase HPLC on a 2.2 × 25-cm Vydac 218TP1022 C₁₈ column (Alltech, Templemars, France) by using a linear gradient (10-50% over 50 min) of acetonitrile/trifluoroacetic acid (99.9 : 0.1 ; v/v) at a flow rate of 10 mL/min. Analytical HPLC, performed on a Vydac 218TP54 C₁₈ column (0.46 × 25-cm), showed that the purity of the peptides was greater than 99%. The identity of the synthetic
peptide was confirmed by mass spectrometry on a JEOL model AX-500 mass spectrometer.

The GPR103 antagonist 25e was synthesized as previously described (10). Insulin and exenatide were purchased from Eli Lilly (Neuilly-sur-Seine, France).

*Blood glucose and insulin measurements in mice.* For i.p. glucose tolerance test and glucose-stimulated insulin secretion assay, mice were fasted for 16 h with free access to water and then injected i.p. with glucose (1.5 g/kg) and 26RFa (500 µg/kg) or PBS solution. For measurements of basal glycemia, mice were fed ad libitum and injected i.p. with 26RFa (500 µg/kg) or PBS solution. Blood plasma glucose concentrations were measured from tail vein samplings at various times using an AccuChek Performa glucometer (Roche Diagnostic, Saint-Egreve, France). Plasma insulin concentrations were determined using an AlphaLISA insulin detection kit (ref AL204c, Perkin Elmer, Courtableuf, France). For insulin tolerance test, mice were fasted for 6 h and injected i.p. with 0.75 units/kg body weight of human insulin (Eli Lilly, Neuilly-sur-Seine, France) and 26RFa (500 µg/kg) or the GPR103 antagonist 25e (5 mg/kg), or PBS. For oral glucose tolerance test, mice were fasted for 16 h before the test with free access to water and injected i.p. with the GPR103 antagonist 25e (5 mg/kg) or PBS 30 min before a 2 g/kg oral glucose load. For the experiments with 26RFa, the peptide (500 µg/kg) was injected (i.p.) just before the glucose load. Blood plasma glucose and insulin concentrations were measured from tail vein samplings at various times and plasma samples for 26RFa concentration measurements were obtained from decapitation just before or 30 and 120 min after the glucose challenge, and then assayed for 26RFa.

*Insulin secretion by the EndoC-βH1 cells.* Human β cells (EndoC-βH1), a kind gift from Dr R. Scharfmann (INSERM U1016, Institut Cochin, Paris, France), were grown in DMEM containing 2.8 mM glucose and supplemented with 2% heat-inactivated fetal bovine serum, 2-mercaptoethanol (50 µM), nicotinamide (10 mM), selenite sodium (6.7 ng/ml), transferin (5.5 µg/ml), penicillin (100 mg/l) and streptomycin (100 mg/l), in a humidified atmosphere of 5%
149 CO2, 95% air at 37° C. Concurrently, some wells containing the EndoC-βH1cells were
150 incubated in the same medium containing a solution of palmitate (10 mM, P9767-5G, Sigma
151 Aldrich) for four days. Before the experiments, the culture medium was removed and EndoC-
152 βH1 cells (4.3x10⁵ cells/well) were preincubated in a Krebs-Ringer bicarbonate buffer
153 containing 0.2% BSA and 2.8 mM glucose (low glucose, LG) for 1 h at 37° C (period P1) to
154 evaluate basal insulin secretion. The culture medium was then removed and replaced by the
155 same culture medium (LG) or a culture medium with 16.5 mM of glucose (high glucose, HG)
156 added or not with 26RFa (10⁻⁶ M) and the GLP-1 analogue exenatide (5 10⁻⁷ M) for 1 h
157 (period P2). Insulin secreted in the culture medium was measured using a commercial ELISA
158 kit (Mercodia Insulin ELISA, Uppsala, Sweden) and was expressed as the ratio between
159 secretion during the P2 period/secretion during the P1 period.

160 Immunohistochemical procedure. Deparaffinized sections (15-µm thick) of duodenum and
161 pancreas of control or HF mice were used for immunohistochemistry. Tissue sections were
162 incubated for 1 h at room temperature with rabbit polyclonal antibodies against 26RFa (3)
163 diluted 1:400 or GPR103 (#NLS1922; Novus Biologicals, Littleton, CO) diluted 1:100. The
164 sections were incubated with a streptavidin-biotin-peroxydase complex (Dako Corporation,
165 Carpinteria, CA), and the enzymatic activity was revealed with diaminobenzidine. Some
166 slices were then counterstained with hematoxylin. Observations were made under a Nikon E
167 600 light microscope.

168 Quantitative PCR. Total RNA from the skeletal muscle, adipose tissue, liver and
169 duodenum of HF and control mice, and from EndoC-βH1 cells was isolated as previously
described (25). Relative expression of the 26RFa, GPR103, GLP-1 receptor, GLUT-1 and
170 GLUT-2 genes was quantified by real-time PCR with appropriate primers (Table 1). GAPDH
171 or cyclophilin A were used as internal controls for normalization. PCR was carried out using
172 Gene Expression Master Mix 2X assay (Applied Biosystems, Courtaboeuf, France) in an ABI
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Prism 7900 HT Fast Real-time PCR System (Applied Biosystems). The purity of the PCR products was assessed by dissociation curves. The amount of target cDNA was calculated by the comparative threshold (Ct) method and expressed by means of the 2-ΔΔCt method.

*26RFa radioimmunoassay.* Quantification of 26RFa in plasma and tissue samples was carried out using a specific radioimmunoassay (RIA) set up in the laboratory that has been previously described in detail (3). For the RIA procedure, each plasma sample was diluted (1:1) in a solution of water/TFA (99.9:0.1; v/v). The duodenum fragments were immersed for 15 min in boiling 0.5 M acetic acid, homogenized at 4°C using a glass potter homogenizer and centrifuged (6,000 g, 4°C, 30 min). Diluted plasma and supernatant fluid of tissue extracts were pumped at a flow rate of 1.5 ml/min through one Sep-Pak C₁₈ cartridge. Bound material was eluted with acetonitrile/water/TFA (50:49.9:0.1; v/v/v) and acetonitrile was evaporated under reduced pressure. Finally, the dried extracts were resuspended in PBS 0.1 M and assayed for 26RFa.

*Statistical analysis.* Statistical analysis was performed with Statistica (5th version). A student *t*-test or ANOVA for repeated measures were used for comparisons between two groups. A post-hoc comparison using Tukey HSD was applied according to ANOVA results. Statistical significance was set up at *p* < 0.05.

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193 **RESULTS**

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195 *Consequence of blocking 26RFa action on glucose and insulin tolerance in control mice.*

196 Administration of the 26RFa receptor antagonist, 25e, 30 min before an oral glucose tolerance
test resulted in a significant increase of the glycemia between 30 and 60 min after the oral
197 glucose challenge (Fig. 1A), as assessed by the calculation of the areas under the curve (Fig.
198 1A *insert*). Plasma insulin concentrations tended to be slightly higher in animals that received
199 the GPR103 antagonist, but without reaching the statistical significance (Fig. 1B).
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201 Administration of 25e during an insulin tolerance test significantly reduced insulin
202 sensitivity from 15 to 90 min after the i.p. injection of insulin (p<0.05-p<0.001) (Fig. 1C) as
203 assessed with a statistical difference of the areas under the curve of p=0.07 between 25e-
204 treated mice and animals that did not receive the GPR103 antagonist (Fig. 1C *insert*).

205 *Effect of a 26RFa injection on blood glucose and plasma insulin levels following an oral*
206 glucose challenge.

207 We completed the experiments using the 26RFa receptor antagonist, by performing oral
208 glucose tolerance tests with administration of exogenous 26RFa. In these experiments, the
209 hyperglycemic effect induced by the glucose challenge was significantly reduced by the i.p.
210 injection of 26RFa (Fig. 1D), but without any significant alteration of plasma insulin levels
211 (Fig. 1E).

212 *Effect of 26RFa on basal glycemia, glucose-induced hyperglycemia and insulin tolerance.*

213 The high-fat (HF) diet challenge induced a significant (p<0.001) weight gain right from the
214 first month of treatment (Fig. 2A). After three months, the HF mice exhibited a weight of
215 43.00 ± 0.65 g, whereas mice fed a standard chow weighted 27.00 ± 0.85 g (Fig. 2A).
216 Similarly, plasma glucose was significantly increased (p<0.01) after one month of HF diet
217 (Fig. 2B). After 3 months, the HF mice exhibited a fasting glycemia of 2.11 ± 0.06 g/l,
whereas that of the mice fed a standard chow at the same time was 1.16 ± 0.05 g/l (p<0.001; Fig. 2B).

The impact of 26RFa was investigated on basal glycemia in mice fed a standard chow and in HF mice. As illustrated in figure 2C, D, i.p. administration of 26RFa did not affect significantly basal plasma glucose levels during the 90-min period of the test in the two diet conditions. By contrast, an IPGTT performed in the two groups of mice revealed that 26RFa significantly attenuated (p<0.05) the hyperglycemia induced by an i.p. glucose challenge in mice fed a standard chow during the first 30 min of test, whereas this anti-hyperglycemic effect of the neuropeptide was totally abolished in HF mice (Fig. 2E, F). I.p. injection of 26RFa also significantly potentiated (p<0.05) insulin-induced hypoglycemia during the 90 min following the insulin challenge in mice fed a standard chow as illustrated by the quantification of the areas under the curves (Fig. 3A, C). By contrast, the beneficial effect of 26RFa on insulin sensitivity was strongly attenuated in HF mice (Fig. 3B, C). Quantification of GPR103 mRNA in insulin target tissues revealed a significant decrease (p<0.05) in the expression of the 26RFa receptor in the muscle and the adipose tissue of HF mice (Fig. 3D). No expression of GPR103 was detected in the liver of the two groups of animals (Fig. 3D).

*Consequence of blocking 26RFa action on glucose and insulin tolerance in HF mice.*

Administration of the GPR103 antagonist 25e during an oral glucose tolerance test in HF mice had no significant effect on plasma glucose and insulin levels (Fig. 4A, B) in contrast to what was observed in mice fed a standard chow (Fig. 1A, B). i.p. injection of 25e had no significant impact on insulin sensitivity during an insulin tolerance test in HF mice (Fig. 4C) in contrast to the insulin-resistant effect of the GPR103 antagonist observed in mice fed a standard chow (Fig. 1C). In addition, administration of 26RFa had no effect on the hyperglycemia induced by the oral glucose load (Fig. 4D) nor on plasma insulin levels (Fig. 4E).
Effect of 26RFa on insulin production. The effect of 26RFa on insulin production was investigated *in vivo* and *in vitro*. An acute glucose-stimulated insulin secretion test showed that injection of 26RFa significantly stimulated glucose-induced insulin production in mice fed a standard chow (p<0.05; Fig. 5A), whereas this insulinotropic effect of the neuropeptide was totally abolished in HF mice (Fig. 5B). Concurrently, the impact of 26RFa on insulin secretion by pancreatic β cells was examined using the human insulin-secreting cell line, EndoC-βH1, that were treated with palmitate to mimic conditions of lipotoxicity. As illustrated in figure 5C, addition of 26RFa in the culture medium significantly stimulated insulin secretion by untreated EndoC-βH1 cells incubated in LG (p<0.05) or HG (p<0.01) conditions. The GLP-1 analogue, exenatide, used as a positive control, also significantly stimulated insulin secretion (p<0.01) by the untreated EndoC-βH1 cells (Fig. 5C). Conversely, when the cells were treated for 96 h with palmitate, the insulinotropic effect of 26RFa was totally abolished whatever the glucose concentration in the culture medium (Fig. 5D). By contrast, exenatide still significantly stimulated insulin secretion by palmitate-treated cells (p<0.01, Fig. 5D). Comparison of insulin secretion between native and palmitate-treated EndoC-βH1 cells revealed that, in non-stimulated conditions, basal insulin release was not affected in palmitate-treated cells (Fig. 5E). By contrast, under stimulatory conditions (26RFa or exenatide), insulin secretion was dramatically altered in palmitate-treated cells as compared to native cells (Fig. 5E). Finally, expression of GLUT-1, GLUT-2, the GLP-1 receptor and GPR103 was evaluated in native and palmitate-treated EndoC-βH1 cells (Fig. 5F). GLUT-1 and the GLP-1 receptor mRNA levels were not altered by the lipotoxic treatment of the cells (Fig. 5F). By contrast, expression of GLUT-2 was significantly decreased (p<0.05) in palmitate-treated cells and that of GPR103 was also lowered (p=0.06; Fig. 5F). Consistent with this latter observation, immunohistochemical observation of pancreas of mice fed a
267 standard chow and HF mice revealed a decrease in the intensity of the GPR103 labeling in the
268 pancreatic islets of HF mice (Fig. 5G, H).

269 *Effect of the high fat diet on gut 26RFa production.* We first investigated the expression
270 and distribution of 26RFa in the duodenum of HF mice and mice fed a standard chow using a
271 Q-PCR approach combined with immunohistochemistry (Fig. 6A, B). The expression of
272 26RFa was significantly lowered (p<0.01) in the duodenum of HF mice as compared to mice
273 fed a standard chow (Fig. 6A). In agreement with this finding, the immunohistochemistry
274 revealed an important decrease in the number of enterocytes immunostained with the 26RFa
275 antibody in the duodenum of HF mice (Fig. 6B).

276 The impact of a 2 g/kg oral glucose load on plasma glucose, insulin and 26RFa, and on
277 26RFa content in the duodenum was investigated in HF mice and mice fed a standard chow
278 (Fig. 6C-G). In mice fed a standard chow, the oral glucose challenge induced an important
279 increase of the glycemia at 30 min, followed by a decrease at 120 min (Fig. 6C). The HF mice
280 exhibited a similar glycemic profile although plasma glucose levels were significantly higher
281 all along the test (p<0.05-0.01; Fig. 6C). Measurement of plasma insulin during the OGTT
282 indicated that insulinemia was always higher in HF mice as compared to mice fed a standard
283 chow, as assessed by the point by point statistical analysis (p<0.05-0.001) and the
284 measurement of the area under the curve (p<0.01) (Fig. 6D). Plasma 26RFa profile in mice
285 fed a standard chow was very similar to that observed for glycemia and insulinemia, with an
286 increase of plasma 26RFa levels observed at 30 min followed by a decrease at 120 min (Fig.
287 6E). In HF mice, plasma 26RFa concentration was similar to that of mice fed a standard chow
288 during the first 30 min of the test, but the obese/hyperglycemic mice exhibited a dramatic rise
289 in plasma 26RFa levels at 120 min (p<0.05) that was not observed in mice fed a standard
290 chow (Fig. 6E), as underlined by the significant difference of the areas under the curves (Fig.
291 6F). Quantification of 26RFa content in the duodenum showed a significant decrease (p<0.01)
292 of the neuropeptide production in HF mice under basal condition (at T0) (Fig. 6G). During the
293 oral glucose tolerance test, 26RFa content in the duodenum exhibited a profile closely related
294 to that of plasma 26RFa in the two groups of mice with, in particular, a highly significant rise
295 of duodenal 26RFa production (p<0.001) 120 min after the oral glucose challenge in HF mice
296 (Fig. 6G).

297
In the first part of the present study, we took advantage of the first disclosed GPR103 antagonist (10) that can be used as an *in vivo* tool to decipher the role of 26RFa in glucose homeostasis. We have previously demonstrated that 26RFa could be considered as a novel incretin (26). This conclusion was based on experiments carried out by our team showing that 26RFa is secreted by the gut in response to an oral glucose load, and that the neuropeptide stimulates insulin secretion from pancreatic β cells, leading to an anti-hyperglycemic effect (26). However, without a specific antagonist molecule, it is difficult to isolate the proper role of 26RFa on the glycemic status from the well-known effects of the other incretins such as GLP-1 and GIP during an oral glucose challenge, which is recognized as the gold standard test to assess the effects of incretin hormones. Here, we further characterized the role of 26RFa in the global regulation of glucose homeostasis using a GPR103 antagonist with DMPK properties suitable for *in vivo* experiments, namely 25e (10). In our set of experiments, administration of 25e induced a robust hyperglycemic effect during an oral glucose load in mice fed a standard chow. We also found that administration of the 26RFa receptor antagonist significantly reduces insulin sensitivity, confirming that one mechanism of action of the 26RFa/GPR103 system to control glucose homeostasis is to increase insulin sensitivity in addition to its incretin activity (26). There was a tendency for 25e to increase plasma insulin levels during a glucose challenge. We hypothesize that this may reflect the occurrence of a compensatory mechanism to counteract the insulin-resistant effect of the GPR103 antagonist. However, this increase of insulin secretion is not sufficient to maintain normoglycemia, suggesting that blockade of 26RFa action also alter the insulin secreting capacity of the pancreatic islets. Conversely, we show that exogenous administration of 26RFa during an oral glucose load significantly reduces the hyperglycemia without any significant alteration of the
323 plasma insulin levels. In this experiment, it is important to note that, in fact, we have analyzed
324 the effects of both exogenous and endogenous 26RFa. Thus, it seems that exogenous
325 administration of 26RFa could potentiate the antihyperglycemic effect already induced by
326 endogenous 26RFa production. We may speculate that the decrease of the glycemia induced
327 by exogenous 26RFa is rather linked to an alteration of the insulin sensitivity than insulin
328 secretion since no modification of plasma insulin levels was observed. Together, these
329 findings confirm our previous data (26) and support the view that the 26RFa/GPR103 system
330 plays a crucial role to sustain normoglycemia in stimulatory conditions.

331 The second objective of the present study was to investigate whether an obese/chronic
332 hyperglycemic status is associated with a dysfunction of the 26RFa/GPR103 system. For this
333 purpose, we submitted mice to a HF diet for a period of three months. Measurement of weight
334 and glycemia revealed that the HF mice were actually obese and intolerant to glucose. 26RFa
335 did not affect basal plasma glucose levels neither in the HF mice nor in control animals. This
336 observation is consistent with the incretin status of the neuropeptide previously reported (26).
337 By contrast, we found that the anti-hyperglycemic effect of 26RFa observed during a glucose
338 tolerance test in control mice was lost in HF mice. We previously showed that the anti-
339 hyperglycemic effect of 26RFa was the result of both increased insulin sensitivity in target
340 tissues and a direct stimulation of insulin production by the pancreatic β cells (26). Here, we
341 show that during an insulin tolerance test, the beneficial effect of 26RFa on insulin sensitivity
342 is significantly attenuated. This was probably due to the fact that the expression of GPR103
343 mRNA was down-regulated in muscle and adipose tissues of HF mice compared to controls.

344 In the *in vivo* experiments, we found that the stimulatory effect of 26RFa on insulin
345 production was totally lost in the HF mice. We have previously shown that 26RFa exerts a
346 direct insulinotropic effect on pancreatic β cells via activation of GPR103 (26). In this study,
347 we used a human β pancreatic cell line that expresses GPR103 (i.e., EndoC-βH1cells) to
further explore the direct effects of 26RFa on insulin secretion. We show that 26RFa robustly stimulates insulin release by these cells. Treatment of the EndoC-βH1cells with palmitate, that mimics conditions of lipotoxicity observed in diabetes, totally abolished the insulinotropic effect of 26RFa. By contrast, the GLP-1 analogue, exenatide, was still able to stimulate insulin release (albeit with a reduced potency) under the same lipotoxic conditions. This latter observation is in agreement with previous studies showing a reduction of GLP-1-induced insulin secretion under conditions of lipo and glucotoxicity induced by a HF diet (12). Finally, we show that the expression of the 26RFa receptor is lowered in the EndoC-βH1cells cultured in lipotoxic conditions as well as in the pancreatic islets of HF mice, suggesting that this decreased expression of GPR103 in β-cells is responsible, at least in part, for the loss of the insulinotropic effect of 26RFa in obese/hyperglycemic conditions. In accordance with these findings, a loss of GIP insulinotropic effect has also been reported in type 2 diabetes (13,19) and down-regulation of the GIP receptor has been proposed as one of the mechanisms underlying the lack of GIP activity in type 2 diabetic patients (1, 17, 24). Similarly, a down-regulation of the GLP-1 receptor has been described in pancreatic β cells under diabetic conditions that may underlie the reduced incretin effect of GLP-1 in diabetes (17). However, some studies suggest that the loss or altered incretin effect in type 2 diabetes is a consequence of the diabetic state and not a primary event and that the incretin defect is mainly a consequence of general β cell dysfunction (8, 13, 17, 18). Together, these observations support the idea that the altered insulinotropic activity of the incretins in diabetes is probably the result of a down-regulation of their receptor expression in β-cells associated with a dysfunction of insulin synthesis and production.

We have previously found that the enterocytes of the duodenum are the main source of peripheral 26RFa and that its release to the general circulation is increased after an oral glucose challenge (26). Here, we show that, after an overnight fasting, the expression and
373 production of 26RFa in the duodenum is significantly decreased in HF mice. This finding,
374 together with the low expression of the 26RFa receptor in insulin target tissues in HF mice,
375 suggests that the 26RFa/GPR103 peptidergic system is markedly altered under obese/chronic
376 hyperglycemic condition. However, we concurrently show that, under stimulatory conditions
377 (an oral glucose load), the duodenum of HF mice keeps its ability to produce and secrete large
378 amounts of 26RFa in the general circulation and is even over-stimulated as compared to
379 control mice. Indeed, in HF mice, plasma 26RFa levels still increase 2 h after the oral glucose
380 load whereas, in mice fed a standard chow, they return to basal values as previously reported
381 (26). Interestingly, this plasma 26RFa profile during the OGTT in HF animals follows that of
382 duodenal 26RFa production suggesting that the duodenum is the main source of circulating
383 26RFa. In addition, these high plasma 26RFa levels 2 h after glucose ingestion might reflect
384 the occurrence of a mechanism of action involving 26RFa production by the gut, aiming at
385 decreasing glycemia. In this context, it would be interesting to investigate whether insulin,
386 whose plasma levels also remain high 2 h after the oral glucose challenge in HF mice, could
387 regulate 26RFa production by the gut as the hormone has already been reported to be able to
388 regulate glucose intestinal absorption, in addition to its peripheral effects (31). It is
389 noteworthy that this discrepancy in the plasma 26RFa profile during an oral glucose tolerance
390 test between normal and chronic hyperglycemic conditions is not found for GLP-1 and GIP
391 (2) and thus seems to be a specificity of 26RFa that deserves further investigation.

392 Finally, we show that administration of the 26RFa receptor antagonist 25e or 26RFa in
393 obese/hyperglycemic mice has no effect on the glycemic response during an oral glucose
394 tolerance test or an insulin tolerance test, nor an insulin secretion during an acute glucose-
395 stimulation insulin secretion test. This observation is in agreement with the blunted 26RFa
396 response we have observed in HF mice and can be linked to the decreased expression of
397 GPR103 in insulin target tissues and in β pancreatic cells of the HF mice.
398 In conclusion, we have confirmed, using a 26RFa receptor antagonist, that the
399 neuropeptide 26RFa is a key regulator of glucose homeostasis by acting as an incretin and
400 improving insulin tolerance. Our data also reveal a loss of the incretin and insulin-sensitive
401 effect of 26RFa in obese/chronic hyperglycemic conditions that is associated with a down-
402 regulation of its receptor. Together, these observations strongly suggest that dysfunction of
403 the 26RFa/GPR103 system participate to the general reduced incretin effect and insulin
404 intolerance observed in diabetes.
405

406 ACKNOWLEDGMENTS

407
408 We are indebted to Dr R. Scharfmann (INSERM U1016, Cochin Institute, Paris, France)
409 for his kind gift of the human pancreatic β cell line, EndoC-βH1.
410

411 GRANTS

412 This work was funded by INSERM (U1239), the University of Rouen, the Institute for
413 Research and Innovation in Biomedecine (IRIB), the "Fondation pour la Recherche
414 Médicale" (DEA 20140629966), the "Société Francophone du Diabète" (R16038EE) and the
415 "Plateforme Régionale de Recherche en Imagerie Cellulaire de Normandie (PRIMACEN)".

416 The present study was also co-funded by European Union and Normandie Regional
417 Council. Europe gets involved in Normandie with European Regional Development Fund
418 (ERDF).

419
420 DISCLOSURES

421
422 The authors declare that there is no conflict of interest that could be perceived as prejudicing

423 the impartiality of the research reported.

424

425 AUTHOR CONTRIBUTIONS

426

427 G.P. and N.C. contributed to the study design and interpretation and wrote the manuscript.

428 MA. LS., J.B., A.A., M.P., J.M. and M.M. contributed to the in vivo experiments on mice.

429 J.B., MP. H.B., M.C. and P.D. contributed to the in vitro experiments on cell lines. S.C. and

430 M.P. contributed to the immunohistochemical experiments. J.B., M.P., V.B. and H.B.

431 contributed to the PCR experiments. A. B. and E. N. performed the insulin assays. J. L. and

432 A. N. produced 26RFa and 25e. H.L. and Y. A. revised and approved the final version of the

433 manuscript. N.C. is the guarantor of this work and, as such, had full access to all the data in

434 the study and takes responsibility for the integrity of the data and the accuracy of the data

435 analysis.

436

437

438
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552

553
554 LEGENDS TO FIGURES

555

556 **Figure 1: Consequence of blocking 26RFa action on glucose and insulin tolerance in**
557 **control mice. (A, B) i.p. injection of the GPR103 antagonist 25e at a dose of 5 mg/kg**
558 significantly increases the blood glucose level during an oral glucose tolerance test (A, insert).
559 During the same test, plasma insulin concentrations tend to be higher all along the test in
560 animals that received the GPR103 antagonist (B), although statistical analysis does not reveal
561 any significant difference between treated and non-treated animals (B insert). (C)
562 Administration of 25e during an insulin tolerance test significantly reduces insulin sensitivity
563 from 15 to 90 min after the i.p. injection of insulin. (D, E) i.p. administration of 26RFa (500
564 µg/kg) reduces significantly the hyperglycemia induced by a 2-g/kg oral glucose challenge
565 (D), but without affecting plasma insulin levels (E). (Inserts) Calculation of the areas under
566 the curves. Data represent means ± SEM of 3 independent experiments (n=10-15/group). *,
567 p<0.05; **, p<0.01; *** p<0.001 vs non-treated animals.

568

569 **Figure 2: Effect of 26RFa on basal glycemia and glucose-induced hyperglycemia. (A, B)**
570 A 3-month high fat diet induces a highly significant weight gain (A) and increase of glycemia
571 (B) right from the first month of treatment as compared to mice fed a standard chow. (C, D)
572 Effect of i.p. administration of 26RFa (500 µg/kg) on basal plasma glucose levels. 26RFa
573 does not alter basal plasma glucose during the 90 min following its injection neither in mice
574 fed a standard chow (C) nor in HF mice (D). (E, F) Effect of i.p. administration of 26RFa
575 (500 µg/kg) during a glucose tolerance test. 26RFa significantly attenuates the hyperglycemia
576 induced by the i.p. injection of glucose (1.5 g/kg) throughout the duration of the test in mice
577 fed a standard chow (E). In HF mice, the anti-hyperglycemic effect of 26RFa during the
578 glucose tolerance test is totally abolished (F). Calculation of the areas under the curves reveal
579 no significant difference between the PBS and the 26RFa groups whatever the type of diet
580 given (inserts). Data represent means ± SEM of 5 independent experiments (n=20/group). ns,
581 non significant; *, p<0.05; **, p<0.01; ***, p<0.001 vs mice fed a standard chow.

582

583 **Figure 3: Effect of 26RFa on insulin tolerance. (A-C)** i.p. administration of 26RFa (500 µg/kg) during an insulin tolerance test significantly potentiates insulin-induced hypoglycemia during the 90 min following the insulin load (0.75 Units/kg) in mice fed a standard chow (A) as illustrated by the quantification of the areas under the curves (C) whereas, in HF mice, the beneficial effect of 26RFa on insulin sensitivity is strongly attenuated (B, C). Data represent means ± SEM of 5 independent experiments (n=20/group). (D) Expression of GPR103 mRNA was determined by quantitative polymerase chain reaction and adjusted to the signal intensity of GAPDH in the muscle, the adipose tissue and the liver. GPR103 mRNA levels are significantly lowered in the muscle and adipose tissue of HF mice as compared to those measured in mice fed a standard chow. No expression of GPR103 is detected in the liver of control or HF mice. Data represent means ± SEM of 3 independent experiments (n=6-9/group). *, p<0.05 vs mice fed a standard chow.

595

596 **Figure 4: Consequence of blocking 26RFa action on glucose and insulin tolerance in HF mice. (A, B)** Administration of the GPR103 antagonist 25e (5 mg/kg) during an oral glucose tolerance test in HF mice has no significant effect on plasma glucose (A) and insulin levels (C), as assessed by the calculation of the areas under the curves (inserts). (C) Similarly, i.p. injection of 25e does not alter insulin sensitivity during an insulin tolerance test in HF mice. (D, E) i.p. administration of 26RFa (500 µg/kg) has no significant effect on the hyperglycemia induced by an oral glucose challenge in HF mice (2 g/kg) (D), nor on plasma

597

598

599

600

601

602
603 insulin levels (E). (Inserts) Calculation of the areas under the curves. Data represent means ± SEM of 3 independent experiments (n=10-12/group).

605

606 **Figure 5: Effect of 26RFa on insulin production. (A, B)** 26RFa (500 µg/kg) stimulates significantly glucose-induced insulin production during an acute glucose (1.5 g/kg)-stimulated insulin secretion test in mice fed a standard chow (A), whereas injection of the neuropeptide has no effect on plasma insulin in HF mice (B). Data represent means ± SEM of 5 independent experiments (n=20/group). (C, D) Effect of 26RFa (10⁻⁶ M) on insulin secretion by the human pancreatic β cell line EndoC-βH1non treated or treated by palmitate for 96 h. 610 611 26RFa significantly stimulates insulin secretion in untreated EndoC-βH1 cells incubated in a 612 low glucose (LG) or a high glucose (HG) medium (C). Conversely, when the cells are treated 613 with palmitate, the insulinotropic effect of 26RFa is abolished whatever the glucose 614 concentration in the culture medium (D). Data represent means ± SEM of 4 independent 615 experiments (n=10/condition). (E) Comparison of insulin secretion between native and 616 palmitate-treated EndoC-βH1 cells. In non-stimulatory conditions, insulin release is not 617 affected in palmitate-treated cells as compared to native cells. By contrast, under stimulatory 618 conditions (26RFa or exenatide), the secreting response of the cells is significantly altered by 619 the palmitate treatment. Data represent means ± SEM of 4 independent experiments 620 (n=10/condition). (F) Expression of the glucose transporters GLUT-1 and GLUT-2, the GLP-621 622 1 receptor (GLP1R) and GPR103 mRNA was determined in native EndoC-βH1 cells and in 623 palmitate-treated EndoC-βH1 cells by quantitative polymerase chain reaction and adjusted to 624 the signal intensity of cyclophilin A. GLUT-1 and the GLP1R mRNA levels are not altered by 625 the lipotoxic treatment of the cells whereas the expression of GLUT-2 and GPR103 is 626 decreased in palmitate-treated cells. Data represent means ± SEM of 3 independent 627 experiments (n=6-9/group). *, p<0.05; **, p<0.01. (G, H) Immunohistochemical labeling of
628 pancreatic islets of control and HF mice with GPR103 antibodies. Representative
629 photomicrographs of pancreatic islets showing a robust decrease in the intensity of the
630 GPR103 labeling in the pancreatic islets of HF mice (H) as compared to the pancreatic islets
631 of mice fed a standard chow (G). Scale bars: 50 µm.

632

633 **Figure 6: Effect of the high fat diet on gut 26RFa production. (A, B)** Expression and distribution of 26RFa in the duodenum of control and HF mice. Expression of 26RFa mRNA in the duodenum was determined by quantitative polymerase chain reaction and adjusted to the signal intensity of GAPDH, and its distribution was determined by immunohistochemistry. The expression of 26RFa is significantly lower in the duodenum of HF mice as compared to mice fed a standard chow (A). In agreement with this finding, the immunohistochemistry reveals an important decrease in the number of enterocytes immunostained with the 26RFa antibody in the duodenum of HF mice in comparison to duodenal slices of mice fed a standard chow (B). Scale bars: 50 µm. (C-G) Impact of a 2-g oral glucose load on plasma glucose, insulin and 26RFa, and on 26RFa content in the duodenum of HF mice and mice fed a standard chow. During an oral glucose challenge, the HF mice exhibit a similar glycemic profile to that of mice fed a standard chow, although their plasma glucose levels are significantly higher all along the test (C). Measurement of plasma insulin during the same test reveals that insulinemia is always higher in HF mice as compared to mice fed a standard chow, as assessed by the point by point statistical analysis and the calculation of the areas under the curves (D). In HF mice, plasma 26RFa concentration is similar to that of mice fed a standard chow during the first 30 min of the test, but the obese/diabetic mice exhibit a dramatic rise in plasma 26RFa levels at 120 min that is not observed in mice fed a standard chow (E), as underlined by the significant difference of the areas under the curves (F). Quantification of 26RFa concentration in the duodenum reveals
lower duodenal 26RFa content in HF mice under basal condition (G). During the oral glucose
tolerance test, 26RFa content in the duodenum exhibits a profile closely related to that of
plasma 26RFa in mice fed a standard chow as well as in HF mice with, in particular, a
significant rise of duodenal 26RFa production 30 and 120 min after the oral glucose challenge
in obese/diabetic animals (G). Data represent means ± SEM of 5 independent experiments
(n=18-20/group). *, p<0.05; **, p<0.01; ***, p<0.001 vs mice fed a standard chow. §,
p<0.05; §§, p<0.01 vs T0 min.
A

<figure><img src="image_2.png" /></figure>

B

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C

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D

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E

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A

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B

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C

Control mice

<figure><img src="image_9.png" /></figure>

D

PBS
26RFa (500 µg/kg)

<figure><img src="image_10.png" /></figure>

E

<figure><img src="image_11.png" /></figure>

F

<figure><img src="image_12.png" /><figcaption>Figure 2</figcaption></figure>
A

Control mice

<figure><img src="image_13.png" /></figure>

B

High fat mice

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /></figure>

D

<figure><img src="image_16.png" /></figure>
<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_19.png" /></figure>

- Control
-- GPR103 antagonist

<figure><img src="image_20.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_21.png" /></figure>

Figure downloaded from www.physiology.org/journal/ajpendo at INRA Institut National de la Recherche Agronomique (138.102.192.062) on May 20, 2019.
A

Control mice

B

High fat mice

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

C

Native endoC-βH1 cells

<figure><img src="image_24.png" /></figure>

D

Palmitate-treated endoC-βH1 cells

<figure><img src="image_25.png" /></figure>

E

Native cells
Palmitate-treated cells

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F

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High fat mice

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<figure><img src="image_30.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>High fat mice</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>E</figcaption></figure>

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<figure><img src="image_37.png" /><figcaption>Figure 6</figcaption></figure>

Downloaded from www.physiology.org/journal/ajpendo at INRA Institut National de la Recherche Agronomique (138.102.192.062) on May 20, 2019.
<table><caption>Table 1 - Sequence of the primers used for the Q-PCR experiments</caption><thead><tr><th></th><th>Forward primer</th><th>Reverse primer</th></tr></thead><tbody><tr><td>Human GPR103</td><td>GAAAGGCACCAGGGACTTGT</td><td>CCAGACCACACCTAGCATTGT</td></tr><tr><td>Human GLP-1 rec</td><td>TGAGCATAGGCTGGGGTGTT</td><td>CAGCCGGATAATGAGCCAGT</td></tr><tr><td>Human Glut-1</td><td>TGGCATCAACGCTGTCTTCT</td><td>AGCCAATGGTGGCATACACA</td></tr><tr><td>Human Glut-2</td><td>CTAGTCAGGTGCATGTGCCA</td><td>AGGGTCCCAGTGACCTTATCT</td></tr><tr><td>Mouse GPR103</td><td>GGACGAAGGGCACCATCTT</td><td>CGTCACGATGCTCCAGAACA</td></tr><tr><td>Mouse 26RFa</td><td>TCTGCCGTCCTTACCATCTCA</td><td>TCTCAGGACTGTCCCAAAGGAG</td></tr><tr><td>Mouse GAPDH</td><td>TCCGGACGCACCCTCAT</td><td>CGGTTGACCTCCAGGAAATC</td></tr><tr><td>Human cyclophilin A</td><td>ATGGCACTGGTGGCAAGTCC</td><td>TTGCCATTCCTGGACCCAAA</td></tr></tbody></table>