Immunodetection of selected pancreatic hormones under intragastric administration of apelin-13, a novel endogenous ligand for an angiotensin-like orphan G-protein coupled receptor, in unweaned rats

In 1998, Tatemoto et al. (30) utilised bovine stomach tissue samples to discover endogenous ligands for the orphan G-protein-coupled apelin receptor (APJ), leading to the isolation and purification of a novel peptide named apelin. This peptide, a member of the adipokines group, has since been recognised as an essential ligand for APJ. The subsequent exploration of this peptide has revealed that its 77-amino-acid pre-proprotein undergoes intricate processing, generating multiple biologically active apelin isoforms with varying lengths ranging from 12 to 36 amino acid residues, each exerting a spectrum of diverse pharmacological effects (28).

Through a series of experimental studies, it has been unequivocally demonstrated that all apelin isoforms, acting via the APJ receptor, are crucial in the regulation of a spectrum of pivotal physiological processes, those related to maturation and aging being among them (28). Investigations in mice have unveiled an age-dependent decline proportional to maturation (24) in the expression levels of apelin and its receptor in the kidneys, lungs and liver. Intriguingly, mice with knockout of either the apelin gene or the APJ receptor gene manifested accelerated aging symptoms in the aforementioned organs. Significantly, upon restoration of normal apelin levels, a marked improvement in the overall health status of these individuals was observed, suggesting the potential of apelin as an anti-aging factor (24).

Building upon research conducted using animal disease models, it was postulated that apelin isoforms, particularly apelin-13, exert therapeutic effects in various disorders of the nervous system, cardiovascular system, respiratory system, gastrointestinal tract and kidneys, and in metabolic diseases associated with glucose metabolism (3, 28). In the course of most of these systemic diseases, the beneficial effects of apelin were dose-dependent and contingent on the mode of administration. These effects largely centred on the attenuation of caspase-3 levels and pro-inflammatory factors, leading to the mitigation of ongoing inflammation and reduction of apoptosis (3). However, the precise regulatory role of apelin remains enigmatic, a definition of that role not having been aided by the recently uncovered negative effect of this compound: under certain circumstances, apelin could also function as an activator of pro-inflammatory cytokines and promote tumour growth and angiogenesis (34). In sum, these observations suggest the potential dual nature of apelin, whereby its isoforms might exert harmful and detrimental effects under specific conditions.

The objective of this investigation was to elucidate the impact of intragastric administration of apelin-13 on the regulation of critical pancreatic hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, in a cohort of unweaned rat subjects. The utilisation of unweaned rats as the model organism is of particular significance, given that this developmental stage is characterised by increased metabolic plasticity and endocrine adaptability. Such attributes render this age group especially relevant for the study of long-term physiological outcomes. This study aimed to delineate the alterations in secretion profiles of these pivotal pancreatic hormones after apelin-13 administration. By so doing, the research sought to shed light on the prospective modulatory effects that apelin signalling could exert within the complex network of endocrine interactions that dictate metabolic homeostasis during the formative phases of life. The insights derived from this investigation stand to augment our current understanding of apelin’s roles in metabolic and developmental physiology.

The primary objective of our study was to evaluate the effects of intragastric administration of apelin-13 on the expression of key pancreatic hormones in unweaned rats. Our results revealed significant alterations in pancreatic islet architecture, along with modulatory effects on alpha and beta cells. However, it is crucial to contextualise these findings within the broader scientific discourse on apelin’s role in pancreatic physiology.

Extensive research has established that apelin acts as an inhibitor of insulin secretion. This finding is further supported by studies on incubated insulinoma cells (11) and genetically modified mice lacking the apelin gene, in which the subjects showed not only elevated levels of insulin secretion but also symptoms of glucose intolerance (8). Interestingly, work by Kapica et al. (13) identified apelin in pancreatic vesicles but not in islets, setting apelin in contrast to the APJ receptor, which is present in both islets and pancreatic ducts. Complementing these observations, research carried out by Ringström et al. (25) confirmed the presence of apelin in specific pancreatic islet cells, namely the alpha and beta cells of rats. The biological impact of apelin appears to be modulated by both the administration route and the age of the test subjects. To illustrate, a pair of experiments by Antushevich et al. (1) employed intragastric and intraperitoneal administration of apelin, and although their study focused on the exocrine part of the pancreas, their results should be mentioned. These studies revealed age-related differences in physiological responses: young weaned rats displayed an uptick in lipase activity without any concomitant changes in overall protein levels or the activities of trypsin and amylase within the pancreatic homogenate, while adult rats experienced an increase in both total protein content and enzymatic activities, including those of lipase, trypsin and amylase.

The conducted study revealed a significant reduction in the pancreatic islet area in rats treated with apelin-13, this decreasing by 47.52% compared to the control group. Additionally, the average islet diameter decreased by 32.43%. Importantly, islet density remained unchanged, suggesting that the reduction in islet size was likely due to a decrease in cell numbers rather than cell size. Further analyses of cells secreting the key pancreatic hormones insulin and glucagon revealed an increase in the proportion of glucagon-secreting cells (alpha cells) in medium-sized islets and insulin-secreting cells (beta cells) in large-sized islets compared to the control group. This was accompanied by an increase in the density of alpha cells in these islet size subgroups and a decrease of beta cell density in small and medium-sized islets. This increase contributed to the absence of a significant reduction in overall islet density, despite a notable decline in the density of the cells which typically dominate the islets, i.e. beta cells. Nevertheless, it is noteworthy that the overall islet density displayed indications of a discernible trend, which means that its values pointed to differences which were very close to statistically significant between the control group and the experimental group (P-value = 0.056–0.097). While other variables did not demonstrate significant changes (except for small decreases in the density of delta cells), the morphometric measurements implied that the constancy of the proportion of cells secreting somatostatin and pancreatic polypeptide in the medium and large islets may be misleading. This can be attributed to the reduced islet area, leading to the plausible assumption that despite stable proportional cell numbers, there may have been a concurrent reduction in the absolute number of cells secreting these hormones.

Similar findings were reported in neonate rats with streptozotocin-induced diabetes. Gallego et al. (9) documented a reduced islet area and an increased proportion of alpha cells. However, the percentage of beta cells differed from ours, as a decrease was observed in their study. These outcomes are likely attributable to beta cell necrosis induced by streptozotocin, an antibiotic with a selective affinity for beta cells commonly used for diabetes induction (7). Interestingly, in our study, no discernible changes were found in F cells and barely any in delta cells upon apelin-13 treatment across all types of pancreatic islets. This observation contrasts with the suggestion of a recent study that apelins, including apelin-13, may exert some modulatory influence on the secretion of somatostatin and pancreatic polypeptide (25); however, because in our case only a decrease in delta cell density in small islets was observed, our results are inconclusive. Several factors could contribute to this divergence in results. One particularly noteworthy consideration is the age of the rats used in our investigation. It is important to recognise that the metabolic system of the adopted unweaned rat model is still in a formative stage of development. This nascent state could render these systems less responsive or sensitive to changes induced by apelin-13. Furthermore, the specific developmental timing of the rats, which were not fully mature, may have acted as a significant limiting factor on the observed physiological changes. It is plausible that the delta and F cells in these juvenile rats had not yet reached a developmental stage where they could interact effectively with apelin-13. It could also be hypothesised that there may have been differences in receptor expression or availability at this developmental stage, leading to reduced or altered functionality of apelin-13 with respect to these cell types. However, we opted for unweaned rats as our experimental subjects for several compelling reasons. Firstly, this developmental stage allowed the stabilisation of apelin levels, as it is a period where the concentration of apelin acquired from maternal milk begins to diminish. Maintaining a consistent level of apelin was crucial for the integrity of our study design. Secondly, the existing literature suggests that the weaning stage serves as a pivotal moment for the maturation of pancreatic cells (29). We sought to examine whether supplying exogenous apelin in the dosage applied during the period leading up to weaning would expedite the maturation and stabilisation of pancreatic cells, potentially influencing insulin levels in the following stages of development. As the study was conducted on young, unweaned rats suckling maternal milk, the intragastric administration of apelin in this study was the most physiological method of delivering the hormone during this period of life.

To gain the advantage of a model lending itself to stabilisation of apelin levels, it was necessary to accept a limitation this imposed on the study. The use of very young and small animals limited the volume of blood obtained during sample collection and made it impossible to measure hormone or glucose levels in the blood. The insufficient quantity of blood precluded accurate measurements of hormonal and glucose concentrations, impeding a comprehensive analysis of physiological responses.

Our study proved a remarkable increase in mitotic activity within small and medium-sized islets – specifically, a 102.78% increase in small islets and a 125.50% increase in medium-sized islets – after apelin administration. Furthermore, our findings demonstrated a 32.47% decrease in apoptosis in small islets after apelin administration. This substantial augmentation in mitotic rates suggests that apelin may serve as a regulatory molecule, bolstering mitotic activity while inhibiting apoptotic pathways in the pancreatic islet cells. In vitro studies revealed that apelin could have divergent effects on cell proliferation, by either promoting it (14) or inhibiting it (2), with the outcome depending on the specific cell line. Apelin also had a significant impact on cell proliferation and death processes in other tissues, as it inhibited apoptosis and stimulated proliferation (2). The reduction in apoptosis aligns with the research by Liu et al. (15), who highlighted the anti-apoptotic role of the apelin/APJ system. This could potentially confer a protective role on apelin, particularly in the context of metabolic diseases where islet cell dysfunction is a hallmark, such as type 2 diabetes. The study by Chen et al. (6) suggested that long-term treatment with apelin had a protective effect on pancreatic islet beta cells in rats with type 1 and 2 diabetes.

The reduction in apoptosis and an increase in mitosis particularly in small islets, suggested that apelin played a pivotal role in the genesis of new islet cells, which could indicate a specific mechanism by which the apelin/APJ system contributed to cellular survival, thereby maintaining the integrity of pancreatic islets. The smaller the size of the islet, and ipso facto the newer it is, the higher the likelihood that it contains beta cells: the smallest islets are often made up almost entirely of these cells. A more potent protective effect on new islet cells may be imputed to apelin on the basis of its protection of beta cells as Chen et al. (6) suggested. Given the consistent distribution of both small and large pancreatic islets, it seems likely that the smaller islets serve as a resource for regenerating the larger ones, especially when significant damage occurs from inflammation or other harmful events. Lower beta cell viability in small islets corroborates the hypothesis advanced by Tomita (31), who indicated that apoptosis in pancreatic beta cells plays a critical role in the pathogenesis of type 2 diabetes. This notion was also confirmed by our findings demonstrating a significant increase in the percentage of alpha and beta cells following apelin treatment. This correlation underlined apelin’s crucial role in cell proliferation in the pancreatic islets, thereby expanding what is understood of its multifaceted influence on pancreatic physiology. Interestingly, the observed increase in the number of small clusters and single cells expressing insulin and glucagon located outside the pancreatic islets in rats treated with apelin suggested a neogenetic basis. A study by Mezza et al. (18) reported increased expression of single cells and small clusters expressing insulin in pancreatic tissue in individuals with impaired glucose tolerance. These results are consistent with the hypothesis that apelin serves as a key modulatory factor in the generation of new cells in pancreatic islets.

The regeneration of pancreatic islet cells occurs via the self-renewal of existing beta cells and through intra-islet progenitors. Additionally, centroacinar/terminal ductal cells expressing stem cell markers may also contribute to islet regeneration. Separate studies suggested neogenesis via the transdifferentiation of acinar cells post treatment with activin or glucagon-like peptide (21), giving rise to cells that produce insulin and glucagon. The co-expression of pivotal factors – specifically Pdx1, Ngn3 and MafA – within the acinar portion initiated the transformation of acinar cells into fully functional beta cells in mice, indicating the potential for generating islet cells (20). Notably, pancreatic duct epithelial cells express the pro-endocrine marker Ngn3. The robust regenerative capacity of alpha cells is particularly noteworthy, especially concerning changes observed in the glucagon signalling pathway (26).

Numerous studies have explored pregnancy’s impact on regulating beta cell mass in rodents and humans. During rodent pregnancy, primary compensation mechanisms seemed to involve existing beta cell proliferation and enhancements in their functionality, likely including beta cell neogenesis (23). Maternal apelinaemia is more pronounced during pregnancy, with the placenta releasing significant apelin amounts around the 17^th gestational day in rats – this period marks the highest apelin release in both the mother and the foetus, the latter exhibiting double the apelin levels of the mother. By delivery day (the 21^st day), apelin levels have halved in both the foetus and the mother, suggesting a placental source of prenatal apelin aimed at augmenting nutrient transfer, especially glucose, from mother to foetus. In newborns, tissue distribution of apelin and APJ mirrored that in adults (22). Apelin is also richly secreted in colostrum, albeit less in milk, impacting food intake and energy balance and stimulating gastrointestinal tract development.

In rats, apelin and APJ expression levels in the stomach, duodenum and colon peak at birth but significantly decline postnatally. The elevated apelin mRNA levels during birth hint at apelin’s role in postnatal gastrointestinal (GI) tract development. Notably, apelin-containing cells in the rat stomach only emerge around the weaning period and are absent in neonates. It was theorised that the GI tract’s intrinsic apelin production might be dispensable in the early postnatal phase, given the significant apelin levels detected in breast milk (32). The rise in gastric apelin-containing cells post weaning suggested that weaning prompted the synthesis of apelin, stimulated by the shift from breast milk to solid food (33). In our study, administering apelin aimed to restore its GI tract levels to match the natural peak concentration in maternal milk, thus extending its duration of action in the GI tract.

The observed increase in mitosis and decrease in apoptosis following apelin administration have intriguing implications, particularly in the context of tumour growth (5) It is well-established that uncontrolled cell division, represented by increased mitosis and reduced apoptosis, is a hallmark of tumour proliferation. The substantial augmentation in mitotic activity, notably in small and medium-sized islets, underscores apelin’s potential role as a regulatory molecule bolstering cell proliferation (10). This heightened mitotic activity could constitute a crucial mechanism through which apelin influences cell survival, thus maintaining the integrity of pancreatic islets, but also causing uncontrolled cell growth leading to the formation of neoplastic growth. While our study focused on the pancreas, it is conceivable that apelin may exert similar effects in other tissues (16). Consequently, the knowledge gleaned from apelin research could contribute to the development of targeted apelin-blocking therapies aimed at inhibiting tumour growth (12). Understanding the delicate balance of cell division and apoptosis under apelin’s influence is paramount, as this information may be leveraged to develop innovative approaches in cancer research.