GLP-1 receptor agonism and GIP receptor antagonism induce substantial alterations in enteroendocrine and islet cell populations in obese high fat fed mice

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Introduction
Dysregulation of the incretin hormone system in obesity and related diabetes has profound effects on metabolic function.Specifically, reduced glucagon-like peptide-1 (GLP-1) secretion and impaired GLP-1 and, especially glucose-dependent insulinotropic peptide (GIP) receptor signalling leads to diminished insulin secretion, impaired glucose uptake and increased hepatic glucose production [1].The blunted GLP-1 response also contributes to decreased satiety and increased appetite, further exacerbating weight gain and obesity [2].It follows that long-acting drugs mimicking the action of GLP-1 are now clinically approved for both obesity and type 2 diabetes [3].
In terms of clinically exploitable effects of GIP in obesity and diabetes, the picture is less clear.Thus, activation and inhibition of GIP receptor (GIPR) signalling impart significant metabolic benefits, both alone [4,5] and in combination with GLP-1 receptor (GLP-1R) activation [6,7].Notably however, there is now a suggestion that chronic GIPR agonism leads to receptor desensitisation, thereby mimicking the observed benefits of GIPR antagonism [8,9].This is in keeping with the early idea that GIP plays a role in obesity [10] and with observations that GIPR knockout (KO) mice fed a high fat diet are resistant to obesity [11].Further to this, there are important species-specific divergences within the GIP system that need to be considered, with the human GIP sequence acting only as a comparatively weak partial agonist in rodent systems [12].Consequently, mouse GIP(3− 30) (mGIP (3− 30)), but not human GIP(3− 30) (hGIP (3− 30)), is demonstrated to be a highly effective molecule to inhibit GIPR activity in mice [13], with hGIP(3− 30) more applicable in the human setting [14].
Therefore, in the current study we have employed sustained injection of either exendin-4 (Ex-4) to activate GLP-1Rs, or mGIP (3− 30) to antagonise GIPRs, in our model system.Although both approaches are known to bring about well-known improvements in metabolism, enteroendocrine cellular adaptations in response to these interventions has not been examined in detail.In this regard, there is clear evidence for both pancreatic islet and enteroendocrine cell (EEC) adaptations in obesity and diabetes, that may be linked to disease onset and progression [15].As such, changes in both islet beta-cell and EEC numbers as well as secretory function have been documented [16].Thus, the primary objectives of the current study were to investigate the impact of (Ex-4) and mGIP (3− 30) therapy on intestinal morphology and hormone content as well as gut cell hormone immunoreactivity profiles in mice fed a high fat diet (HFD).Secondary objectives included validation of benefits of the treatment interventions on metabolic control as well as assessment of pancreatic islet architecture.Together these studies will help uncover the influence of changes in intestinal morphometry in relation to the positive effects of GLP-1R agonism and GIPR antagonism under situations of high fat feeding.

Animals
Female Swiss NIH mice (4-6 weeks old, Envigo, UK) were housed individually in air-conditioned room at 22 ± 2 • C with 12 h light and dark cycle and ad libitum access to standard rodent diet (10% fat, 30% protein and 60% carbohydrate; Trouw Nutrition, Northwich, UK) and drinking water.NIH mice are an outbred strain of mice and therefore more closely resemble the genetic differences present within the human population, with use of female mice confirming lack of sex-specific dimorphic effects of Ex-4 and mGIP (3− 30), since previous studies with these peptides have largely employed male mice [13,17].At 9 weeks of age, mice were fed a HFD (45% fat, 35% carbohydrate and 20% protein; 26.15 kJ/g, Special Diet Services, UK) for 14 weeks, with a separate group maintained in normal diet (ND) throughout and serving as gold standard controls.Following 14 weeks high fat feeding to induce obesity and insulin resistance as confirmed by hyperinsulinaemia in these mice, the three groups of HFD mice (n = 6 per group) were administered either twice daily i.p. injections of saline vehicle (0.9% NaCl), Ex-4 or mGIP(3− 30) (both at 25 nmol/kg body weight) for 21 days, with the final ND group also receiving daily saline vehicle injections.Power calculations were conducted based on changes of ileal hormone content between ND and HFD groups, with n = 6 considered appropriate to achieve significantly meaningful results.All experiments were conducted under the UK Animals (Scientific Procedures) Act 1986 & EU Directive 2010/63EU, approved by the UK Home Office under project licence PPL2902 and University of Ulster Animal Welfare and Ethical Review Body (AWERB).

Tissue processing
At the end of the treatment period, pancreatic and intestinal tissues were extracted from mice and fixed for 48 h in paraformaldehyde (4% w/v in phosphate buffered saline (PBS)) to preserve cellular architecture by cross-linking proteins.Tissues were then processed in an automated tissue processor which involved dehydrating in 70-100% ethanol followed by xylene immersion to remove wax before paraffin embedding.Embedded tissues were then cut at 5 µm sections on a microtome (Shandon Finesse 325, Thermo Scientific) and placed on poly-L-lysine coated slides [18].

Immunohistochemistry
To assess immunoreactive staining for insulin, glucagon, PYY, somatostatin, GLP-1, Ki-67 and TUNEL (Table 1), as appropriate, ileum and pancreatic sections were dewaxed in histoclear for 30 mins, before being rehydrated with decreasing concentrations of ethanol.Sections were blocked with 2.5% bovine serum albumin (BSA) and then incubated with designated primary antibody (Table 1) overnight.Importantly, the specificity of all primary antibodies for use in immunohistochemistry has been previously validated, and subsequently successfully employed in our laboratory [19,20].On day 2, sections were then rinsed in PBS and incubated with suitable secondary antibody (Alexa Fluor® 594 for red and Alexa Fluor® 488 for green; Table 1) for 1 h at 37 • C.After a PBS wash, slides were then incubated with DAPI for 15 mins at 37 • C [18].Finally, sections were mounted on coverslips using antifade mounting media before being viewed at 40x magnification using an Olympus IX51 inverted microscope and photographed using a DP70 digital camera system.

Image analysis
Image J software was used to assess total ileal crypt depth and villi length using the straight-line function.Total number of cells positive for GIP, GLP-1 and PYY, along with their counts in respective villi and crypt areas, were counted using the multi-point and polygon function.Cell F software was used to analyse images for assessment of pancreatic islet, beta-and alpha-cell areas.For islet cell co-localisation studies, PPY and GLP-1 detection in alpha or somatostatin cells was determined by counting cells with positive PPY or GLP-1 and glucagon/somatostatin immunoreactivity and expressed as % of total alpha or somatostatin cells, as appropriate.For beta-cell proliferation, insulin and Ki67 positive cells were counted whereas for apoptosis, insulin and TUNEL positive cells were counted, as described previously [21].

Biochemical analysis
At the end of the study, non-fasting plasma glucose was directly measured from the cut tip on the tail vein of conscious mice using a hand-held Ascencia Contour blood glucose meter (Bayer Healthcare, Newbury, Berkshire, UK) at 10:00 h.For plasma insulin analysis, blood samples were collected into chilled fluoride/heparin glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany) and immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) for 1 min at 13,000 g and stored at − 20 • C, prior to determination of insulin concentrations by a modified insulin radioimmunoassay [22].Intestinal tissues were then excised and immediately snap frozen in liquid nitrogen and stored at − 80 • C. Subsequently, for hormone content analysis tissues were homogenised using RIPA lysis buffer and 0.1% bovine lung protease inhibitor cocktail (Sigma-Aldrich, Dorset, UK).Homogenised tissues were centrifuged at 664 g for 20 mins at 4 • C, prior to analysis of total intestinal PYY (rat PYY ELISA, ORB441862-BOR, Stratech Scientific), GLP-1 (GLP-1 total ELISA, EZGLP-1 T-36 K, Millipore) and GIP (rat/mouse GIP ELISA, EZRM-GIP-55 K, Millipore) according to individual manufacturer's instructions.Total intestinal protein content of the samples was assessed using the Bradford protein assay.

Statistical analysis
GraphPad PRISM (version 5.0) software was used to perform statistical analysis.Values are expressed as mean ± S.E.M. Comparative analyses between groups were carried out using a one-way ANOVA with Bonferroni's post hoc test.There was no inclusion and exclusion criteria applied.Groups of data were considered to be significant if p < 0.05.

Effects of sub-chronic Ex-4 and mGIP(3− 30) treatment on metabolic parameters in HFD mice
As expected, the saline treated HFD group of mice exhibited a significant (p < 0.01) increase in body weight when compared to ND controls (Table 2).Body weight was decreased (p < 0.05) by both Ex-4 and mGIP(3− 30) administration on day 21, that was related to reduced (p < 0.05) energy intake when compared to HFD control mice (Table 2).Blood glucose levels were decreased (p < 0.05) by Ex-4, but not mGIP (3− 30), when compared to HFD control mice (Table 2).Terminal plasma insulin concentrations were increased (p < 0.05) in HFD mice, with Ex-4 significantly decreasing (p < 0.05) circulating insulin when compared to HFD controls (Table 2).

Effects of sub-chronic Ex-4 and mGIP(3− 30) treatment on ileal GIP, GLP-1 and PYY cells in HFD mice
Fig. 2A, 3A & 4A depict representative images of ileal tissue from each treatment group stained for GIP, GLP-1 and PYY, respectively.HFD increased (p < 0.05) the number of GIP positive ileal cells as a whole, and specifically within crypts (Fig. 2B,C), but interestingly not in villi (Fig. 2D).Whilst mGIP (3− 30) treatment had no obvious effect on these parameters, HFD mice treated with Ex-4 had similar levels of GIP positive ileal cells as ND controls when assessed per mm 2 of ileum (Fig. 2B), crypt (Fig. 2C) or villi (Fig. 2D).Numbers of GLP-1 positive ileal cells were not different between all groups of mice in the overall ileum and related crypts (Fig. 3B,C), however treatment with Ex-4 or mGIP(3− 30) decreased (p < 0.05) numbers of GLP-1 positive villi cells whereas high fat feeding alone had no significant effect on this parameter (Fig. 3D).Total (Fig. 4B) and villi (Fig. 4D) numbers of PYY immunoreactive ileal cells were increased (p < 0.01) in HFD mice but returned to normal levels by both treatments, with no difference in overall and villi numbers of PPY positive cells in HFD mice treated with Ex-4 or mGIP (3− 30) when comparted to ND controls (Fig. 4B,D).In addition, Ex-4 increased (p < 0.05) numbers of positively stained PYY cells in crypts when compared to ND mice (Fig. 4C), whereas all other groups of mice had similar levels of PYY immunoreactivity in ileal crypts (Fig. 4C).

Effects of sub-chronic Ex-4 and mGIP(3− 30) treatment on pancreatic islet morphology in HFD mice
Saline and mGIP(3− 30) treated HFD mice had increased (p < 0.05p < 0.01, respectively) islet and beta-cell areas when compared to ND mice, whereas these factors were similar to ND controls in Ex-4 treated HFD mice (Fig. 5A,B).All groups of HFD mice presented with significantly (p < 0.05-0.01)elevated alpha-cell area (Fig. 5C).Islet PYY cell area remained unchanged across all groups of mice (Fig. 5D), whilst Ex-4 treatment increased (p < 0.05) delta-cell area when compared to ND controls (Fig. 5E).When viewed as a whole, the percentage of beta-cells was increased (p < 0.01) by mGIP(3− 30) treatment (Fig. 5F), with a corresponding decrease (p < 0.05) in the percentage of alpha-cells (Fig. 5G).As expected, HFD mice had higher levels (p < 0.01) of glucagon positively stained cells within the centre of islets, which was unaffected by mGIP(3− 30) but returned to ND levels by Ex-4 (Fig. 5H).In terms of beta-cell turnover, neither high fat feeding nor the treatment interventions had any significant impact on beta cell proliferation or apoptosis rates (Fig. 5I,J).Representative images of islets stained for insulin and glucagon from all groups of mice are shown within the supplementary material.

Discussion
Obesity and related diabetes detrimentally alter the incretin hormone system, with compelling data to demonstrate reduced secretion of GLP-1 alongside excessive circulating GIP levels that subsequently leads to desensitisation of action [1].These observations corroborate the benefits of sustained GLP-1R activation [23] or inhibition of GIPR signalling in obesity-diabetes in terms of body weight loss [24].
Importantly, whilst the anti-obesity effects of GLP-1R agonism or GIPR antagonism have been largely established in male mice [25,26], the current work confirms lack of sex-based dimorphic impact with either intervention.Effects on body weight appeared to be at least partly related to inhibition of appetite in both treatment groups, that is certainly characteristic of GLP-1R agonism [27,28].Whilst there are recent reports of appetite suppressive effects of GIPR signalling upregulation [29,30] a GIPR antibody was demonstrated to inhibit food consumption in obese mice [31], whereas others report no effect on feeding following GIPR blockade in rodents [32].In addition, GIPR KO  A. Sridhar et al. mice have been shown to present with both reduced [33] and unaffected [34,35] energy intake, further complicating matters.Given that the impact of mGIP(3− 30) on decreasing energy intake was more prominent than Ex-4 in the current study, it is clear that further investigation into the effects of GIP on energy homeostasis is required.Indeed, multiple regulatory pathways and heterogeneity of central GIPR signaling has recently been noted in relation to GIP effects on appetite control [36].Ex-4 marginally decreased circulating blood glucose, but mGIP(3− 30) was without effect, which does correspond with changes in circulating insulin concentrations on day 21.This might imply improved insulin action in Ex-4, but not mGIP(3− 30), treated HFD mice, but further assessment of insulin-induced cellular glucose uptake would be required to confirm this.Moreover, although non-fasting glucose values can be used as part of the criteria for diagnosing diabetes [37], it is not employed in isolation and also prone to meal induced fluctuations, which should also be considered when interpreting these data.
Obesity is known to have deleterious effects on the integrity and morphometry of intestinal EECs [38].However, there is surprisingly limited knowledge on the influence of sustained GLP-1R agonism or GIPR antagonism on such factors.In the ileum of HFD mice, both Ex-4 and mGIP(3− 30) treatment enlarged villi length indicating expansion of the absorptive surface, highlighting positive effects of both treatments on digestion, as has been previously observed for PYY (3− 36) [39].In good agreement with others, HFD prominently reduced crypt depth [40], an effect that was fully reversed by Ex-4, but not mGIP (3− 30), therapy.This may suggest differential effects of the treatments on EEC secretions, or on production of new epithelial cells that will ultimately line crypts and villi [41].However, such differences may also simply  reflect the very rapid turnover rate of EECs [42].
More in-depth investigation of ileal cell immunoreactivity revealed elevations of GIP and PYY cell populations in HFD mice, with no discernible impact on GLP-1 positive cell numbers.This is likely a reflection of the importance of PYY [43], and especially GIP [35,44,45], signalling on lipid metabolism, which would typically be more pronounced in HFD mice.Thus, GIP and PYY possess established biological actions to improve glucose homeostasis and satiety, respectively [46,47], and their elevated ileum immunoreactivity in HFD mice may represent an adaptive response to promote improved metabolic state in the face of sustained excess fat intake.Moreover, high fat feeding can lead to altered differentiation of EEC stem cells towards expression of more GIP positive cells [48].mGIP(3− 30) further enhanced GIP cell immunoreactivity within the ileum compared with ND mice, possibly as an adaptive response in the face of prolonged GIPR inhibition.Differences in the immunodetection of ileal GLP-1 and PYY in HFD mice is intriguing, given these hormones are thought to be co-localised and secreted from the same vesicles [49].Thus, although the magnitude of alterations were relatively small, HFD mice had clear elevations in PYY, but not GLP-1, immunoreactive ileal cells.That said, both treatment options did concomitantly reduce GLP-1 and PYY cell populations within ileal villi.This is also notable considering that EEC L-cell density is thought to be greater in intestinal crypts than villi [50].There was a small, but significant, increase in numbers of PYY positive cells in the crypts of Ex-4 treated HFD mice, which may merit further investigation.In that respect, EECs can modify hormone expression along the crypt-to-villus length [51], with upregulation of GLP-1R signalling potentially impacting this phenomenon.
Intriguingly, to the most part, alterations in EEC immunoreactivity were not matched by reciprocal changes in ileal hormone content, the difference presumably reflecting the balance between hormone synthesis and secretion.Thus, HFD mice presented with substantially reduced GIP, GLP-1 and PYY ileal content, that was not associated with reductions in ileal cell immunoreactivity of these hormones.Indeed, the number of GIP and PYY positive cells were increased in HFD mice, with no obvious change in GLP-1 cell immunoreactivity levels.Assessment of hormone plasma levels or intestinal expression may have helped with interpretation, although continual variations of hormone secretion with sustained excess dietary fat could be one likely explanation.As such, this would agree with the notion that prolonged high fat feeding alters the expression and secretory function of EECs [52], together with the inherent rapid turnover of these endocrine cells [42].The only prominent effect of the treatment regimens in this regard was a noteworthy restoration of intestinal GLP-1 content.It follows that a significant part of the metabolic benefits of sustained Ex-4 or mGIP (3− 30) administration in HFD mice is linked to enhanced GLP-1R mediated effects.Unfortunately, we were unable to measure circulating levels of GLP-1, that may have provided more information in this regard.However, in pancreatic islets we did detect increased co-localisation of GLP-1 within alpha-cells following treatment with either Ex-4 or mGIP (3− 30), that would further support this notion.Although the current study focuses predominantly on GLP-1R activation and GIPR inhibition, it would be interesting to assess the impact of combined GLP-1R and GIPR activation on alterations of EEC populations in obese mice, especially given the A. Sridhar et al. relatively recent clinical approval of tirzepatide for obesity [53].Furthermore, a dual acting GLP-1 agonist and GIP antagonist molecule has also been developed with body weight reducing effects in rodents and primates, and is now progressing towards phase II clinical trials [54].
As expected, HFD induced the classic enlargement of islet and betacell areas in mice [55].Somewhat surprisingly, mGIP(3− 30) treatment tended to enhance these islet adaptive responses as a result of increased numbers of beta-cells with a relative decrease in alpha-cells.This being despite the well characterised benefits of GIPR signalling activation to enhance beta-cell proliferation and protect against apoptosis [56].Moreover, prolonged GIPR antagonism is believed to induce beta-cell rest and improve insulin action [57], that would be expected to reduce insulin demand.Yet, we did not detect any obvious effect of mGIP(3− 30) on beta-cell turnover, perhaps indicating a potential effect on islet cell transdifferentiation and alteration of lineage of alpha-and beta-cells, that has been observed previously following modulation of GIPR signalling [58].PYY is evidenced within islets [59], being linked to amelioration of beta-cell function and survival [60].In that respect, it is interesting to note the pronounced elevations of detectable islet PYY in mGIP(3− 30) treated HFD mice, in keeping with their augmented beta-cell area.Ex-4 treatment has a relatively modest impact on basic pancreatic islet histology in HFD mice.However, these mice did present with increased somatostatin staining in islets, implying that upregulation of GLP-1R signalling may possess more pronounced effects on intra-islet signalling pathways [61].In good agreement, Ex-4 treated mice also had significantly reduced numbers of centrally located islet alpha-cells, with the characteristic alpha-cell halo in murine islets known to be particularly important for overall islet signalling and functionality [62].In addition, effects may have been more pronounced with use of an extended treatment schedule, or increasing the doses of Ex-4 and mGIP (3− 30).Finally, there is also a possibility that part of the observed treatment effects could be related, either directly or indirectly, to body weight loss.In this respect, pair-fed studies to mimic Ex-4 and mGIP(3− 30) induced reductions of body weight would be required to help establish if adaptations of ileal and pancreatic islet morphology occur independently of body weight.
From a therapeutic viewpoint, our studies provide evidence that the established metabolic benefits of sustained GLP-1R activation or GIPR inhibition, through twice daily Ex-4 or mGIP(3− 30) administration respectively, are linked to positive modulation of intestinal morphometry and hormone content.Thus, given that certain GLP-1R mimetics are already approved for obesity [63], an aspect of their clinical benefit is likely related to reversal of the detrimental effects of sustained excess calorie intake on EEC populations.Specifically, small alterations in EEC hormone detection following both treatment regimens results in a significant and consistent increase in ileal GLP-1 content.Our studies also demonstrate that mGIP(3− 30) enhanced islet PYY levels, that may support the idea of elevated PYY signalling within pancreatic islets leading to an enhancement of beta-cell mass [60], but this still needs to be corroborated.However, the relative contribution of beneficial effects on ileal and pancreatic islet morphology towards the prominent metabolic advantages of approved GLP-1R mimetics, and GIPR antagonists in preclinical investigation for obesity, still needs to be fully determined.Moreover, aspects of these findings could also be relevant in terms of the exciting evolution of dual-acting drugs that concomitantly modulate GLP-1R and GIPR's [53,54].
These studies were supported by Diabetes UK RD Lawrence Fellowship grant to RCM and Ulster University strategic funding.A. Sridhar et al.

Fig. 2 .
Fig. 2. Effect of Ex-4 and mGIP(3− 30) on ileum GIP cell distribution.Parameters were measured after 21 days twice daily treatment with Ex-4 or mGIP(3− 30) (each at 25 nmol/kg bw) in HFD female mice.(A) Representative images of ileum stained for GIP (green) and DAPI (blue).Related quantification of (B) number of GIP positive cells per mm 2 of ileum, (C) number of GIP positive cells per mm 2 of crypt and (D) number of GIP positive cells per mm 2 of villi.White arrows indicate positively stained cells.Values are mean ± SEM (n = 6).*p < 0.05 compared to ND control mice.

Fig. 4 .
Fig. 4. Effect of Ex-4 and mGIP(3− 30) on ileum PYY cell distribution.Parameters were measured after 21 days twice daily treatment with Ex-4 or mGIP(3− 30) (each at 25 nmol/kg bw) in HFD female mice.(A) Representative images of ileum stained for PYY (green) and DAPI (blue).Related quantification of (B) number of PYY positive cells per mm 2 of ileum, (C) number of PYY positive cells per mm 2 of crypt and (D) number of PYY positive cells per mm 2 of villi.White arrows indicate positively stained cells.Values are mean ± SEM (n = 6).*p < 0.05 and **p < 0.01 compared to ND control mice.Δ p < 0.05 compared to saline treated HFD control mice.

Table 1
Target, host and source of primary and secondary antibodies employed for immunofluorescent imaging experiments.
A.Sridhar et al.

Table 2
Effects of Ex-4 and mGIP(3-30) on metabolic parameters after 21 days twice daily treatment in HFD female mice.