Insulin signaling and pharmacology in humans and in corals

Once thought to be a unique capability of the Langerhans islets in the pancreas of mammals, insulin (INS) signaling is now recognized as an evolutionarily ancient function going back to prokaryotes. INS is ubiquitously present not only in humans but also in unicellular eukaryotes, fungi, worms, and Drosophila. Remote homologue identification also supports the presence of INS and INS receptor in corals where the availability of glucose is largely dependent on the photosynthetic activity of the symbiotic algae. The cnidarian animal host of corals operates together with a 20,000-sized microbiome, in direct analogy to the human gut microbiome. In humans, aberrant INS signaling is the hallmark of metabolic disease, and is thought to play a major role in aging, and age-related diseases, such as Alzheimer’s disease. We here would like to argue that a broader view of INS beyond its human homeostasis function may help us understand other organisms, and in turn, studying those non-model organisms may enable a novel view of the human INS signaling system. To this end, we here review INS signaling from a new angle, by drawing analogies between humans and corals at the molecular level.


Introduction to glucose homeostasis and insulin function in humans
The primary source of energy for most cells in the body is glucose and it is also a substrate for many biochemical reactions (Nakrani, Wineland & Anjum, 2023).Blood glucose levels in the body are maintained and balanced by glucose homeostasis (Da Silva Xavier, 2018), as outlined in Fig. 1.Glucose, as a highly polar molecule, cannot diffuse into the lipid membranes of cells and its transport is therefore facilitated by glucose transporters (GLUTs), a family of 12 members (Navale & Paranjape, 2016).Glucose is phosphorylated upon entering the cell and is broken down through glycolysis, followed by oxidative phosphorylation of pyruvate in the TCA cycle, generating ATP (Fukunaga & Hunter, 1997;Watowich et al., 1999).Alternatively, it is polymerized to glycogen for storage of excess glucose (Nakrani, Wineland & Anjum, 2023).To maintain the balance between these opposing processes resulting in regulated blood glucose levels hormones are produced from a group of multicellular endocrine cells called Islets of Langerhans (Da Silva Xavier, 2018).On average, the human pancreas contains 3.2 million islets.Islets consist of four major types of cells: a-cells, β-cells, δ-cells, and pancreatic polypeptide (PP)-cells (Erlandsen et al., 1976;Da Silva Xavier, 2018).β-cells produce and store insulin (INS) which lowers blood glucose levels, while a-cells produce glucagon (GCG) raising blood glucose concentrations, and δ-cells produce somatostatin (SST) which inhibits the secretion of growth hormones and GCG, while PP cells secrete gastrointestinal and intestinal enzymes (Erlandsen et al., 1976), shown in Fig. 1.

SURVEY/SEARCH METHODOLOGY
Our comprehensive literature search was conducted using an array of scientific databases, including but not limited to PubMed, Google Scholar, and Web of Science.This multi-platform approach was implemented to ensure the broadest possible coverage of available literature.
Our search strategy involved the use of key terms and phrases, carefully chosen for their relevance to the subject matter.These included "insulin signaling," "insulin receptor," "corals," "glucose homeostasis in corals," "coral bleaching," "coral pharmacology," and "coral metabolism."Additionally, we used Boolean operators to refine our searches, combining terms such as "insulin AND corals," "insulin signaling AND corals," "insulin receptor in corals," and "insulin signaling in corals AND diabetes in humans".
To ensure an unbiased and objective review, we utilized a set of pre-determined inclusion and exclusion criteria.Inclusion criteria encompassed peer-reviewed research articles, reviews, reports, and meta-analyses published in English within the past two decades.A particular emphasis was placed on studies published within the past 5 years to maintain a focus on current and emergent findings.We sought to include a comprehensive range of literature, focusing not only on recent studies but also incorporating seminal works that have significantly contributed to the field, regardless of their publication date.Inclusion criteria encompassed peer-reviewed research articles, reviews, reports, and meta-analyses published in English, with a strong emphasis placed on studies that have had a substantial impact on the field.While a special emphasis was placed on literature published within the past 5 years to highlight the most current and emergent findings, we also included older literature, particularly those fundamental to our understanding of insulin signaling in humans and corals.
Exclusion criteria involved literature that did not directly pertain to insulin signaling in either humans or corals, studies not subjected to peer-review, and non-English publications.Furthermore, we considered the citation count of each study, using it as a metric of its impact within the scientific community.Following the identification of relevant literature, each publication underwent a meticulous review process.The gathered information was then synthesized and critically evaluated to highlight the current understanding of insulin signaling in humans and corals, identify gaps in knowledge, and provide a comprehensive view of potential future research directions.

Systems biology of INS in humans: INS-related signal transduction cascades
The complexity of the INS-related signal transduction pathways is depicted in Fig. 2, and the proteins involved are listed in Table 1.Proteins are separated by pathway involvement based on whether the pathways are initiated by GCG, GLP-1, INS, or SST.Note that GLP-2 is omitted from Fig. 2 due to our gaps in knowledge of how it interfaces with the action of the other hormones and downstream signaling pathways.Full protein names, UniProt entry names, PDAM ID, E-value, P-value, sequence identity, similarity and references regarding biological function are provided and all protein isoforms of a related gene are listed in Tables 1 and 2 are discussed together.A total of 75 proteins (excluding isoform counting) are involved across the three pathways in humans (GCG and GLP-1 receptor binding events are treated as one pathway for simplicity).Of these, 17 proteins function exclusively within the GCG/GLP-1 signaling pathway, while 36 are exclusive to canonical INS signaling, and 10 are exclusive to the SST pathway.Eleven proteins are involved in two    of the pathways, while only one protein is ubiquitous to all three pathways AKT (RAC-a/β/γ serine/threonine-protein kinase).Inhibitory signaling is found in GCG and INS pathways, while only stimulatory signaling is maintained in the SST pathway.

Introduction to corals
Corals are colonial marine invertebrates (cnidarians) that depend on a symbiotic relationship with dinoflagellate algae of the family Symbiodiniaceae (LaJeunesse et al., 2018).The algae harvest light and synthesize nutrients in exchange for shelter and nitrogen sources (Putnam et al., 2017).Coral reefs cover only 0.1% of the ocean floor but are home to the largest density of animals on earth, rivaling rain forest habitats in species diversity (LaJeunesse et al., 2018).The symbiosis, which was originally thought to be restricted to algae, is now known to extend to a much more complex community than anticipated with thousands of bacteria, bacteriophages, viruses, and fungi, in addition to endosymbiotic algae (Bourne et al., 2009).The entirety of the organism community in a coral is referred to as a holobiont, while the individual cnidarian host animals forming the colonies are called polyps.
The holobiont is characterized by balanced host-microbe molecular interactions.The complexity of these interactions in relation to stress and disease resistance, and recovery grow with every new study as questions arise regarding what molecules are responsible for symbiosis establishment and partner coexistence (Ainsworth & Gates, 2016;Kelly et al., 2021).These inter-partner exchanges are still poorly understood, and this is a particularly severe gap in our knowledge, since it is at the heart of the worldwide phenomenon of coral reef bleaching which refers to the breakdown of symbiosis (particularly the cnidarian host and endosymbiotic algae) due to thermal stress and high irradiance, including that brought about by global climate change.A recent study assessed 100 worldwide locations and found that the annual risk of coral bleaching has increased from an expected 8% of locations in the early 1980s to 31% in 2016 (Hughes et al., 2018;IPCC, 2022).Human impacts on coral reef ecosystems threaten fisheries and tourism, industries valued at hundreds of billions of dollars annually (Putnam et al., 2017).We are in urgent need of innovative solutions to increase corals' resiliency to anthropogenic activities and facilitate their survival.
Climate change driven coral bleaching has now been recognized as the leading cause of the worldwide decline of coral reef cover and, overall, the biggest threat to reef-building coral survival (Hughes et al., 2017).Mass bleaching events have increased both in frequency and severity since the first recorded event in the 1980s (Oliver, Berkelmans & Eakin, 2018) and show no signs of reprieve as ocean warming gets compounded with traditional climate patterns such as the El Niño-Southern Oscillation (McPhaden, Zebiak & Glantz, 2006).Coral bleaching is the common term used to describe dysbiosis in the coral holobiont, specifically, the breakdown of symbiosis (xenophagy and/or expulsion) between the cnidarian host and the dinoflagellate endosymbionts (i.e., dinoflagellates provide most of the coral tissue pigmentation and as dysbiosis progresses, the tissue becomes transparent, thereby revealing the white calcium carbonate skeleton) (Suggett & Smith, 2020).While the full signaling cascade leading to dysbiosis is still poorly defined, we know it leads to damage to cell membranes, lipids, proteins and DNA via nitro-oxidative stress (i.e., the accumulation of free radicals, reactive oxygen species and reactive nitrogen species), a failing antioxidant machinery (e.g., catalase, ascorbate peroxidase, superoxide dismutase) and the organisms' innate immune response (Weis, 2008;Lesser, 2011;Suggett & Smith, 2020).
In the context of coral bleaching induced by heat stress, a study involving the tropical sea anemone Aiptasia pallida identified over 500 up-regulated genes, categorized into Cluster I linked to immunity and apoptosis and Cluster II related to protein folding, with potential regulators influenced by transcription factors NFκB and HSF1.A total of 337 genes in symbiotic anemones exhibited declining expression levels before visible bleaching, suggesting their involvement in algal symbiosis loss (Cleves et al., 2020).These findings hint at potential interactions of these genes with the INS signaling pathway, considering known roles of INS signaling in apoptosis and immune responses (Yuyama et al., 2018).Furthermore, experiments inducing ERK activity in corals via UV radiation and thermal stress (Courtial et al., 2017) and heat-shock experiments on Aiptasia (Sloan & Sawyer, 2016) contribute to our understanding of ERK and AKT phosphorylation and MAPK activities in these organisms, potentially implicating the INS signaling pathway in coral bleaching.

Glucose regulation in corals: an opportunity for understanding INS action in non-model organisms
The symbiotic algae provide as much as 90% of the energy corals consume by light harvesting and photosynthesis (Gierz, Forêt & Leggat, 2017).Thus, corals must be able to measure and regulate nutrient balance (Cunning et al., 2017).Given the crucial role of INS signaling for this task in other organisms, we here hypothesize that INS signaling may also exist in corals, although this hypothesis is purely theoretical and remains to be experimentally validated.Support for this hypothesis comes from transcriptomic studies (Yuyama et al., 2018).A comparison between the expression of INS signaling related genes in the presence and absence of the symbiotic algae strongly suggests that INS signaling is induced at the transcriptomic level in response to algal density in the tissue.A likely interpretation of this finding is that corals need to respond to the sugars produced by the algae through light harvesting and perhaps too much sugar could have detrimental effects on corals, similar to the diabetic response through aberrant INS signaling in humans.The symbiotic interaction between algae and coral involves algae entering the host, and the facilitation of energy and metabolite exchange.Algae utilize seawater substrates to synthesize a spectrum of organic compounds, effectively transferring vital nutrients, including amino acids, small peptides, sugars, carbohydrates, and lipids, to coral cells with glucose being a major metabolite transferred in this exchange as demonstrated by Burriesci and colleagues (Burriesci, Raab & Pringle, 2012).It is also possible that the mechanism for bleaching (loss of symbiotic algae from the coral holobiont) involves an imbalance in nutrient regulation and possible involvement of the INS signaling pathway.This raises an interesting speculation: could corals have diabetes, and could insulin resistance be related to the bleaching that is threatening coral species survival?While corals of course do not have Langerhans islets nor blood, the diabetes analogy at the molecular level may stimulate new ways of thinking about coral and human health (see below).
Indeed, there is evidence for INS signaling in corals at the molecular level.First, remote homology detection using HHblits have identified homologues for human IR and INSR in corals (Roger et al., 2022).HHblits is a so-called Hidden Markov Model (HMM)-based alignment approach developed by Remmert et al. (2011).Unlike traditional HMM profiles, in HHblits, both query and template are HMMs.The search for homologues is through an HMM-HMM alignment and the query HMM is generated by using amino acid distributions which makes this method extremely sensitive.It has been shown that, in many instances, HHblits successfully outperforms the identification and alignment of remote homologues, as compared to the traditional profile HMM approach, such as HMMER3 (Remmert et al., 2011).Given the 700 million years of evolution between corals and humans, this enhanced sensitivity of HHblits is instrumental to the comparison between corals and humans.We have already described the sequence alignments of the ligand-receptor pair, INS with INSR for human and for coral (Roger et al., 2022).In both cases, the alignments were identified with high confidence and cover a large fraction of the sequences: 1,164 out of 1,382 amino acids in the case of INSR and 101 out of 110 in the case of INS.The comparison of the sequences of human insulin (UniProt ID P01308) and Pocillopora damicornis (pdam) protein pdam_00006633, and the extracellular domain of the human INSR (UniProt ID P06213) and pdam_00013976 are shown in Tables 3A and  3B, respectively.
The finding of a human INS homologue in Pocillopora damicornis has prompted us to test the effect of human INS on corals experimentally (Roger et al., 2022).An average 20% reduction in viability at 100 µg/mL INS concentration was observed in line with its proteotoxicity in other systems (Rege et al., 2020).Due to the importance of INS administration in diabetes, its folding and stability has been studied extensively (Weiss & Lawrence, 2018;Liu et al., 2018).High concentrations of salts are known to promote INS aggregation and misfolding (Grudzielanek et al., 2007;Chatani et al., 2014), and the use of seawater in our experiments may induce similar effects, which may be the cause for the observed cytotoxicity.
As shown in Fig. 2, not only INS and INSR, but a total of 75 proteins (excluding isoform counting) are involved across the three INS related pathways in humans.Application of the non-model organism pipeline described above (Kumar et al., 2023) reveals that the majority of downstream signaling proteins, namely 67 of the 75 human proteins, are likely conserved in Pocillopora damicornis.In Fig. 2, all human proteins shown in black have a predicted Pocillopora damicornis homologue, while those shown in red do not.Crosstalk between the SST, CGC and INS pathways is mediated by several proteins that are common to two or even all three pathways.We were not able to identify suitable Pocillopora damicornis homologues for eight proteins: GLP-1, GLP-1R, CGC, CGCR, SST, SSTR, BAD, and CALM, as judged by their poor e-values as well as low percent alignments of amino acids.It is important to realize that GLP-1R, CGCR and SSTR are all GPCR's and thus it is difficult to differentiate GPCR variation within organisms as compared to across organisms.This complication has been discussed in detail, and it was proposed that the GPCR repertoire of Pocillopora damicornis is 151 as compared to 825 in human (Kumar et al., 2023).The results of the remote homology search can be accessed through supplementary file S4 of that article, where all three human GPCR sequences (CGCR, SSTR and GLP-1R) are never ranked first for any of the coral GCPR candidates.The most closely related GPCR is pdam_00008152-RA, which is more similar to the GLP-2 receptor than those three.Thus, it may be possible that GLP-2 is a more ancient modulation of the INS pathway than the GLP-1, CGC and SST pathways (Amato, Baldassano & Mulè, 2016).This finding suggests that the lack of interest in GLP-2 in previous human studies reviewed above is perhaps unjustified.It is important to note that these new evidences are computational only and await future experimental validation.While SST, GLP-1 and GCG are the ligands initiating their respective signaling pathways through their respective GPCR's, BAD is located at the effector end of the main INS pathway, indicating that this pathway is mostly functional.Similarly, CALM functions only to stimulate PYGL in the GCG signaling cascade, and TSC1/2 in INS signaling.Importantly, there is a clear homologue of both INS and the INSR present.

Outlook: coral pharmacology
In this review, we have pointed out numerous possible analogies between human and coral INS biology.The enormous pharmacological importance of treating INS resistance in diabetes makes it tempting to speculate that we can translate what we know about human INS pharmacology to corals, coining a new field of "coral pharmacology" which opens the door to thinking about drug discovery and treatments for corals.While at present we do not know if and how we can deliver medicines to corals in the vast ocean in practical terms, ideas include coating surfaces on which coral larvae settle, or feeding corals or dispersing such compounds into the ocean in proximity to coral reefs.This is not unthinkable given that there are many known examples of small molecules secreted into the ocean used in communication between different inhabitants of a reef, e.g., for attracting fish to anemones (Kamio & Derby, 2017;Saha et al., 2019;Kamio, Yambe & Fusetani, 2022;Morgan et al., 2022) or to mediate biological interactions with surfaces during settling of coral larvae (Petersen et al., 2023).Nanocarriers could also assist with this purpose (Roger et al., 2023).
Given the fluid nature of the ocean environment, such small molecules can be dispersed easily and thus the environmental impact of treatment of corals with small molecule "coral drugs" will need to be carefully addressed.Nonetheless, the idea of "coral pharmacology" may open new avenues to think about how to tackle the coral bleaching crisis.How might coral drug discovery look like?The high quality of sequence alignments of INS and INSR with respective coral homologues shown in Table 3 provides an opportunity to exploit the large amount of INSR structural data that has become available recently (McKern et al., 2006;Menting et al., 2013;Gutmann et al., 2018;Weis et al., 2018), especially due to the advances in cryoelectron microscopy (Uchikawa et al., 2019).Shown in Fig. 3 are homology models for the various domains in human used to predict coral INSR using the sequences shown in Table 3B.These models open the door to the first step of future exploration of potential drug targets in coral, exemplified here by the INSR as a drug target, extrapolated from its role as a human drug target (Kumar, Vizgaudis & Klein-Seetharaman, 2021).The concept of "coral pharmacology" aims to develop pharmacological approaches towards potentially treating corals who have been harmed by human activities.Using membrane receptors as a proof of concept, we developed a pipeline for establishing the functional similarities between human and coral membrane receptor signaling systems (Kumar et al., 2023).This pipeline extends to the INS-INSR pair and its related signaling pathway (Fig. 2).Given the role of INS signaling for regulation of nutrient concentration in humans, we surmise that the coral homologues will likely carry out a similar function in corals.This suggests that early metazoans such as corals use the INS system despite their simple organization.This may have major implications for coral bleaching and the communication across cnidarian host and symbiotic algae.Transcriptomic analysis has revealed that INS signaling is clearly affected by the establishment of symbiosis between cnidarian host animals and algal symbionts (Yuyama et al., 2018).Given that one of the major benefits of symbiosis is the delivery of sugars obtained through photosynthesis of the algae to the host, we can expect that the role of INS signaling is analogous in corals to that in humans, despite their evolutionary distance.It is tempting to speculate that under high light conditions, when the algae synthesize excess sugars, that the cnidarian host may experience INS resistance, a hypothesis that remains to be validated experimentally.By inference, pharmacological treatment of INS resistance may allow coral rescue.We have already shown that human INS does have an effect on corals (see above), and in fact is cytotoxic (Roger et al., 2022).The presence of receptors such as IR (described here) and GPCR (described in Kumar et al., 2023) suggests that many other functions of corals could potentially be targeted by pharmacological means to help prevent their extinction predicted under current climate trajectories.

Figure 1
Figure 1 The role of insulin signaling in glucose regulation.(Left) Regulation of blood glucose in humans.The rise in the blood glucose level releases INS from the pancreas into the bloodstream.This INS stimulates the liver to convert blood glucose into glycogen for storage and SST secreted inhibits GCG secretion.When blood glucose level is low, pancreas release GCG, which causes the liver to turn stored glycogen back into glucose and release it into the bloodstream.SST in this case inhibits INS secretion.(Right) Schematic of Islet of Langerhans architecture.Created with BioRender.com.Full-size  DOI: 10.7717/peerj.16804/fig-1

Figure 2
Figure 2 Overview of insulin related signaling pathways.Conservation of INS-related signaling pathways.Proteins (and their associated isoforms as detailed in Table 1) are represented by their gene name.Proteins exclusive to the GCG signaling pathway are colored in blue and proteins exclusive to INS signaling are colored in orange, while those of the SST signaling pathway are colored pink; proteins involved in two or more pathways are shaded light green.Created with BioRender.com.Full-size  DOI: 10.7717/peerj.16804/fig-2

Table 1
Proteins related to human insulin signaling.Full list of proteins involved in INS and INS-related signaling pathways (i.e., GCG and SST signaling), detailing associated pathway, full name, UniProt entry name, and references to biological function.

Table 1 (
continued ) Table 2 Remote homology detection of candidate insulin signaling related proteins in Pocillopora damicornis.Full list of proteins involved in INS and INS-related signaling pathways (i.e., GCG and SST signaling), matched to Pdam ID with number of residues overlayed (Cols), P-value, E-value, matched sequence length, probability, query template length and percentage (%) identity retrieved from hhblits.

Table 3
Comparison of the sequences of human insulin and coral insulin.Sequence comparison of human and Pocillopora damicornis insulin and insulin receptor sequences.A. Comparison of the sequences of human and coral INS.B. Matching residues in human INSR (visible in the 6pxv structure) with corresponding residues in coral INSR.
Note:* Indicates missing sequence in the structure.