An update on Galanin and Spexin and their potential for the treatment of type 2 diabetes and related metabolic disorders

Spexin (SPX) and galanin (GAL) are two neuropeptides widely expressed in the central nervous system as well as within peripheral tissues in humans and other species. SPX and GAL mediate their biological actions through binding and activation of galanin receptors (GALR), namely GALR1, GALR2 and GLAR3. GAL appears to trigger all three galanin receptors, whereas SPX interacts more specifically with GALR2 and GLAR3. Whilst the biological effects of GAL have been well-described over the years, in-depth knowledge of physiological action profile of SPX is still in its preliminary stages. However, it is recognised that both peptides play a significant role in modulating overall energy homeostasis, suggesting possible therapeutically exploitable


Introduction
Diabetes Mellitus (DM) is a major global epidemic, with around 10% of the adult population currently presenting with the disease equating to just over half a billion people globally (Sun et al. 2022).The underlying pathophysiology of the most prevalent form of the disease, namely type 2 diabetes mellitus (T2DM), is often a consequence of modern-day lifestyles and consumption of highly calorific meals combined with a physical inactivity (Zheng et al. 2018), leading to issues with obesity and associated dysfunctional metabolic control.As such, T2DM is characterised by the impaired insulin secretion from pancreatic beta-cells and reduced insulin sensitivity of peripheral tissues, that ultimately leads to overt hyperglycaemia (Galicia-Garcia et al. 2020).In a vicious circle type scenario, hyperglycaemia then results in excessive pancreatic beta-cell stimulation, beta-cell exhaustion and ultimately a reduction in total betacell mass (Galicia-Garcia et al. 2020).If left untreated, this can lead to hyperglycaemic-induced oxidative stress and inflammation, and subsequent longer-term damage to the micro-and macro-vasculature that culminates in characteristic T2DM disease complications (Harding et al. 2019).Although there is a proverbial arsenal of therapeutics to manage T2DM, many patients still fail to achieve adequate blood glucose control (Rohm et al. 2022), largely as a result of treatment failure over time linked to the degenerative nature of this disease.

J o u r n a l P r e -p r o o f
Obesity is recognised as an important independent risk factor for T2DM.Obesity refers to abnormal or excessive fat accumulation in the body, both within adipocytes as well as metabolically active tissues (Angelidi et al. 2022).Like T2DM, the prevalence of obesity has increased significantly during the last few decades, and it is now considered an epidemic of the 21 st Century (Wolosowicz et al. 2022).In that respect, it has been established that worldwide over 500 million adults currently present with obesity (Wolosowicz et al. 2022).Beyond T2DM, obesity is strongly linked to the increased prevalence of cardiovascular disease, stroke and cancer (Chavda et al. 2022).Although there have been a number of recently approved drugs for obesity, with some of these being linked to augmentation of the biological activity profile of the incretin peptide hormone glucagon-like peptide-1 (GLP-1) that is also employed for T2DM treatment (Chavda et al. 2022), there is still a scarcity of effective pharmacological interventions to effectively manage the disease.Indeed, the most successful approach for obesity is often lifestyle intervention including increased physical exercise and consumption of a healthy diet (Chavda et al. 2022), but long-term compliance and motivation can be a barrier to success.Thus, there is a real need for development of new safe and effective therapies for both obesity and related T2DM.Galanin (GAL) and spexin (SPX), neuropeptides with postulated benefits on energy homeostasis and the cardiovascular system, may have potential important therapeutic application for both obesity and T2DM, with this narrative review considering the use of both peptides in this regard.

GAL discovery
GAL is a biologically active neuropeptide first isolated from the porcine intestine in 1983 by Tatemoto and co-workers, who initially discovered that the peptide induced mild hyperglycaemia in mice (Tatemoto et al. 1983; Figure 2).The GAL family of peptides is now J o u r n a l P r e -p r o o f known to consist of GAL, galanin message-associated protein (GMAP), galanin-like peptide (GALP) and alarin (Jiang & Zheng 2022).GAL has a 29 amino acid primary sequence (30 amino acids in humans) and is distributed widely throughout the peripheral and central nervous systems (Table 1; Mohd Zahir et al. 2022).GAL is highly conserved across species (Table 1) and appears to be involved in a variety of physiological functions in humans including effects on the cardiovascular, respiratory, gastrointestinal, endocrine, reproductive and renal systems, as well as a role in inflammation and various other centrally mediated actions (Kastin 2013; Marcos & Coveñas 2021).

GAL synthesis, secretion and distribution
GAL is encoded by a gene on the 11Q13.3human chromosome and is processed from a 123 amino acid precursor pro-peptide known as preprogalanin (Figure 1A; Dunning & Taborsky 1988).GAL expression, as demonstrated in bovine chromaffin cells, is regulated by three distinct signal transduction systems that includes depolarisation-stimulated Ca 2+ influx, activation of protein kinase C (PKC) as well as protein kinase A (PKA), which together differentially upregulate and downregulate the expression of several other neuropeptides, resulting in different patterns of expression between GAL and other neuropeptides (Rokaeus et al. 1990).GAL is distributed widely throughout the brain in mammals but is most abundant in the hypothalamus and pituitary (Lang et al. 2007).As such, GAL mRNA is detectable in various regions of the brain in non-mammalian vertebrates that are known to impact energy regulation (Mohd Zahir et al. 2022), being co-expressed alongside various neurotransmitters such as acetylcholine, serotonin, glutamate, GABA, noradrenaline, dopamine, enkephalin, neuropeptide Y (NPY), substance P, vasopressin, calcitonin gene-regulated peptide (CGRP) and gonadotropin-releasing hormone (GRH) (Webling et al. 2012).As well as been located centrally, GAL is also evidenced within the periphery including the gastrointestinal tract and J o u r n a l P r e -p r o o f endocrine system (Gesmundo et al. 2017), where the full bioactivity profile is now beginning to be resolved, as described below.Studies to date reveal that the secretion of GAL is modulated by several factors within the bloodstream including oestrogens, thyroid hormones, corticosteroids, growth hormone-releasing hormone, dopamine, somatostatin and thyrotropinreleasing hormone (TRH) (Invitti et al. 1999).

GAL receptors and signalling
GAL receptors (GALRs) are responsible for mediating the functions of GAL, as well as the more recently discovered phylogenetically related peptide SPX (Kim et al. 2014).GALRs are G-protein coupled receptors (GPCRs) with three different GLARs identified to date, namely GALR1, GALR2 and GLAR3 (Mohd Zahir et al. 2022).All three receptors are expressed in the periphery and centrally.For example, GALR1 and GALR2 mRNA are present in the similar brain regions including the hippocampus, hypothalamus and cortex, as well as within the gastrointestinal tract (Jiang & Zheng 2022).Accordingly, GALR3 has been evidenced in peripheral tissue and the central nervous system (Yu et al. 2020).Interestingly, Barreto et al. conducted in situ hybridisation studies with an aim of localising pancreatic GALRs, with GALR1 and GALR2 detected in endocrine pancreatic islets only, and GALR3 located in both endocrine and exocrine pancreatic tissue (Barreto et al. 2011).In terms of cell signalling, GALR1 and GALR3 interact with the Gi-class of GPCR to induce inhibitory effects at the cellular level (Bai et al. 2018).Specifically, GALR1 modulates the G protein-coupled inwardlyrectifying potassium channel (GIRK) and stimulates mitogen-activated protein kinase (MAPK) activity (Lang et al. 2015).In terms of GALR3, signalling interactions are more poorly defined.
However, because GALR3 acts via Gi/o-type GPCR, resulting in activation of G proteinregulated inwardly rectifying potassium channels (GIRK), it seems likely that GALR3 signalling pathways are at least partially similar to that of GALR1 and GALR2 (Smith et al.J o u r n a l P r e -p r o o f 1998).Fascinatingly however, GALR2 can couple with different G-protein classes, including Gs, Gq/11 and Gi/o to mediate both stimulatory and inhibitory downstream effects (Cohen et al. 2020).The best characterised GALR2 signalling pathway involves phospholipase C activation to induce Ca 2+ release from intracellular stores (Lang et al. 2007).Of interest, GALR2 signalling has also been linked to promotion of cell survival, associated with activation of phosphoinositide 3-kinases (PI3Ks) leading to inhibition of caspase activity (Brzozowska & Całka 2021).

GAL structure
The N-terminal 16 amino acid sequence of GAL, GAL(1-16), is considered important for receptor binding and activation (Fathi et al. 1997;Sobrido-Cameán et al. 2019).As such, this N-terminal region of GAL is highly conserved among species (Table 1), with peptides that have a similar structure to GAL and bind to GALRs, such as in GALP and SPX, displaying similarly prominent levels of N-terminal amino acid sequence conservation (Jiang & Zheng 2022).
Interestingly, the C-terminal region of GAL is less well conserved and is thought to be more important for protection against proteolytic attack rather than receptor binding or recognition (Table 1; Webling et al. 2012).It is predicted that GAL forms a horseshoe-like conformational shape, where the N-terminal region is organized in an alpha-helix form, followed by a betabend around the Pro 13 residue, with a more uncertain configuration of the C-terminal region (Schrank et al. 2013).Studies with GAL and GAL(1-16) suggest that phosphoramidon sensitive zinc-metalloprotease are primarily responsible for the degradation of GAL within cerebrospinal fluid (Bedecs et al. 1995), whereas endopeptidases seem to be responsible for GAL degradation within the circulation (Land et al. 1991).However, the exact degradation profile of GAL in plasma has yet to be confirmed.

GAL biological actions
The most well described physiological role of GAL relates to its effects on appetite control and energy regulation in rodents (Fang et al. 2012; Figure 2).Direct administration of GAL into the paraventricular nucleus (PVN) of rats elicits a preferential increase in fat and carbohydrate consumption, with no effect on protein ingestion (Tempel et al. 1988).As such, lipid intake increases GAL levels in the paraventricular hypothalamic nucleus (PVH) in a dose-dependent fashion, suggesting GAL signalling could play a role in the onset of obesity (Marcos & Coveñas 2021).These GAL mediated effects are thought to be related specifically to activation of GALR1 (Fang et al. 2012;Marcos & Coveñas 2021).In terms of direct effects of GAL on the endocrine system, at the level of the pancreatic islet GAL appears to function in a comparable manner as noradrenergic modulation of islet hormone secretion in both rodents and humans (Dunning & Taborsky Jr 1988).In keeping with this, GAL reduces glucose-induced insulin secretion in isolated rat and pig islets (Guo et al. 2011) as well as augmenting glucagon secretion (Gregersen et al. 1991;Gesmundo et al. 2017; Figure 2).These findings have been confirmed in the in vivo environment in obese hyperglycaemic mice (Ahren et al. 1986), although there is still debate as to whether this effect fully translates to humans.Conversely, GAL exerts beneficial effects on insulin sensitivity (Figure 2) with GAL levels thought to decline during the onset of T2DM (Fang et al. 2020).Thus, intracerebroventricular (ICV) administration of GAL in animals with obesity, insulin resistance and T2DM, leads to increased GLUT4 expression with related beneficial effects on insulin sensitivity (Guo et al. 2021).To reinforce this concept, both M35 and M617, recognised are GALR1 agonists (Lundström et al. 2005), also augment GLUT4 expression in muscle and adipose tissues in T2DM rats (Guo et al. 2011).In addition, a GALR2 agonist, M1145 (Runesson et al. 2009), was reported to improve glycaemic control via restoration of insulin and leptin sensitivity in diet-induced obese (DIO) mice (Fang et al. 2018a).Overall, studies would indicate that GAL can alleviate insulin J o u r n a l P r e -p r o o f resistance in myocytes, hepatocytes and adipocytes (Fang et al. 2012), suggesting potential therapeutically exploitable actions (Figure 2).
Interestingly, although M35 is characterised as a GALR1 agonist (Lundström et al. 2005), in the presence of GAL is appears to inhibit GAL-mediated biological actions, suggesting that it may be a GALR1 partial agonist.Despite this, M35 has been demonstrated to inhibit glucose utilisation and oxidation in mice (Idelevich et al. 2018).The appetite suppressive actions of GAL are blocked by galantide in goldfish (Unniappan et al. 2004).In addition, M40 and galantide decrease fat intake when injected directly into the hypothalamus in rodents, in contrast to native GAL (Zorrilla et al. 2007).Interestingly, M40 fails to antagonise GAL-induced inhibition of glucose-induced insulin release from mouse pancreatic islets (Dunning & Taborsky Jr 1988) but is thought to displace GAL from receptor binding sites in the hippocampus, hypothalamus and spinal cord (Bartifai et al. 1993), suggesting possible tissuespecific effects of this antagonist.Non-peptide agonists of GALR1 and GALR2, such as galmic and galnon, have been described and intriguingly shown to stimulate insulin secretion from isolated rat pancreatic islets (Bartfai et al. 2004;Quynh et al. 2005).

GMAP and GALP
GMAP is a 59 amino acid residue peptide (60 amino acids in humans) that serves as a GAL precursor (Rökaeus & Brownstein 1986).GMAP has been reported to possess biological effects distinct from that of GAL, which include antimicrobial actions as part of the innate immune system (Webling et al. 2012).GMAP also effects nociception via the spinal cord in rats, and it J o u r n a l P r e -p r o o f is likely that GMAP exerts this and other actions independent of GALRs (Xu et al. 1995), but to date no further actions of GMAP have been acknowledged.There is an overall lack of mechanistic knowledge in relation to these ascribed actions of GMAP (Šipkova et al. 2017).
GALP is a 60 amino acid peptide, with residues 9-21 of sharing 100% sequence homology with GAL(1-13) (Robinson et al. 2006).GALP was isolated from the porcine hypothalamus in 1999 and found to bind to GALR1 and GALR2, with greater affinity than GAL itself (Ohtaki et al. 1999).However, there is an indication that GALP may interact with other receptors, and that its putative role in the regulation of metabolism and energy homeostasis may not involve GALRs (Lawrence & Fraley 2011).GALP has been shown to reduce body weight and food intake in both obese-diabetic ob/ob and DIO mice following intranasal administration (Kageyama et al. 2016), but the underlying mechanism(s) remain unclear.

Spexin (SPX)
4.1 SPX discovery SPX, also known as neuropeptide Q (NPQ), is a 14 amino acid peptide hormone that signals through GAL membrane bound receptors to exert its physiological effects (Table 1; Türkel et al. 2022a).SPX was first recognised in 2007 through a bioinformatic approach, using evolutionary probabilistic models based on the genome database in different vertebrates (Mirabeau et al. 2007).Subsequently, in situ hybridization (ISH) studies confirmed detection of SPX mRNA within the submucosal layer of the oesophagus and stomach (Sonmez et al. 2009).Elevated levels of SPX have also been detected in the liver, brain, ovaries, hypothalamus and thyroid in both mammalian and non-mammalian species (Sonmez et al. 2009;Türkel et al. 2022a).In 2014, SPX was confirmed as a member of GAL/kisspeptin (KISS) peptide family of peptides, where a second form of SPX, namely SPX2, was discovered in non-mammalian species (Kim et al. 2014).SPX2 represents a mature peptide with a somewhat similar amino J o u r n a l P r e -p r o o f acid sequence to SPX and a further amidation at the C terminus (Lv et al. 2019).However, to date most focus on SPX biology relates to the actions of SPX, rather than SPX2, given that SPX2 is not detected in humans.

SPX synthesis and secretion
As noted above, SPX belongs to the GAL/KISS family of peptides and is encoded by the C12ORF39 gene located on chromosome 12 (Mirabeau et al. 2007).Figure 1B highlights how SPX is initially synthesised from a 116 amino acid precursor peptide (Lv et al. 2019), with the highly conserved 14 amino acid form of SPX noted in both mammalian and non-mammalian species (Türkel et al. 2022a).Strong evolutionary conservation would imply that SPX exerts important biological actions (Table 1; Darakci & Bozkurt 2022).As such, SPX has a range of pleiotropic metabolic effects including an increase of glucose and lipid metabolism as well as modulation of insulin secretion and energy homeostasis (Figure 3; Yu et al. 2022).SPX secretion appears to be restricted to metabolically active tissues and organs such as adipose tissue, liver, stomach and the pancreas in mammals (Lv et al. 2019).In this regard, although SPX is defined as and adipokine, SPX immunoreactivity has been observed across various tissues in humans (Lv et al. 2019), with expression and secretion actually revealed to be downregulated within omental and subcutaneous fat in obese individuals (Walewski et al. 2014).
Exercise is the primary physiological secretory stimulus for SPX, as evidence in both rodents and humans (Turkey et al. 2022b).In addition, hormones such as adrenocorticotropic hormone, insulin, glucagon and oestradiol are known to modulate SPX secretion in rodents, supporting the idea that SPX is responsive to input from the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal circuits (Wang et al. 2020).Notably, previous studies document that SPX is co-localised with insulin in pancreatic beta-cell secretory vesicles of mice, implying exocytosis as the primary method of secretion, similar to many other peptide J o u r n a l P r e -p r o o f hormones (Gowdu & Dayanand 2021).In the porcine pancreas, SPX secretion is increased after a short-term 90 min incubation with glucose but decreased after 24 hours glucose exposure (Sassek et al. 2018a), implying that glucose augments SPX secretion from pancreatic islets but that insulin may inhibit this.Accordingly, glucose ingestion has been demonstrated to increase the expression and secretion of SPX from the stomach of fish, mediated by GLUT4, whereas subsequent glucose-induced insulin secretion then inhibits SPX release (Ma et al. 2017).

SPX structure
The three-dimensional structure of SPX was first confirmed in goldfish.While the first 4 Nterminal amino acids (Asn-Trp-Thr-Pro) form a random coil-like structure at the N-terminus, the amino acids from position 5-14 create an alpha-helical motif that is common in many peptides (Wong et al. 2013).The same study also revealed that Lys 11 of SPX plays a critical role in receptor activation [Wong et al. 2013).Using a combination of molecular dynamic simulations and nuclear magnetic resonance (NMR) analysis, a beta-turn-helix-beta-turn (βαβ) conformity for human SPX has been demonstrated, relating to interactions between amino acid residues Asn 1 and Ala 13 (Darakci & Bozkurt 2022).Furthermore, cyclic analogues of SPX with a disulphide bond between Asn 1 and Ala 13 are still capable of activating GALR2 and GALR3, and N-terminal acetylation can increase peptide potency (Huang 2017).

SPX biological actions
SPX is known to inhibit feeding with an elevation of SPX expression observed following nutrient ingestion (Figure 3; Darakci & Bozkurt 2022).The inhibitory effect of SPX on food intake is regulated centrally by either suppressing orexigenic factors such as neuropeptide Y (NPY), agouti-related peptide (AgRP) and apelin or increasing anorexigenic factors such as proopiomelanocortin (POMC), cocaine-and amphetamine-regulated transcript (CART), J o u r n a l P r e -p r o o f melanin-concentrating hormone (MCH), cholecystokinin (CCK), nucleobindin-2 (NUCB-2) or peptide YY (PYY) in the nuclei of the hypothalamus, as evidenced in piscine models (Wong et al. 2013;Ma et al. 2018).In addition, nutrient intake augments SPX secretion from the stomach, which may contribute this satiety signal through either activation of vagal afferents or direct passage of SPX through the blood brain barrier (Yu et al. 2022).
SPX appears to be involved in the up-regulation of exercise related insulin action in liver, adipose tissue and skeletal muscle (Figure 3; Yu et al. 2022;Türkel et al. 2022b;Fang et al. 2022).In agreement, SPX was previously shown to induce weight loss in DIO mice, with related improvements in HbA1c, glucose tolerance and insulin resistance observed (Kolodziejski et al. 2021).This would suggest that SPX can improve metabolic status in T2DM through direct improvements on insulin action (Figure 3).SPX also increases lipolysis while suppressing lipogenesis and hepatic fat accumulation in adipose tissue through activating GALR2 and GALR3 in DIO mice (Figure 3; Yu et al. 2022).It follows that SPX may be able to exert metabolic benefits in diseases such as non-alcoholic fatty liver (NAFLD), that are often linked with obesity and insulin resistance (Hazlehurst et al. 2016).In keeping with this, SPX has been revealed to reverse hepatic and hepatocellular steatosis in DIO mice (Wang et al. 2022).In addition, treatment with 50 μg/kg/day SPX for 21 days in DIO mice improved liver function through positive effects on the regulation of carbohydrate and lipid metabolism leading to reduced levels of pro-inflammatory cytokines such as IL-6 and TNF-α (Figure 3; Kolodziejski et al. 2021).At the level of the pancreatic islet, akin to the actions of GAL (Sun et al. 2023) SPX has been shown to inhibit glucose-induced insulin secretion both in vitro and in vivo (Figure 3; Sassek et al. 2018b).Thus, benefits of GAL and SPX in T2DM could be partly linked to induction of beta-cell rest that is known to improve enduring glycaemic control under situations of metabolic stress (Tanday et al. 2022), as well as improvements of insulin action.Furthermore, in obese-diabetic patients, improvements of metabolic indicators such as J o u r n a l P r e -p r o o f waist circumference and BMI lead to increased serum SPX concentrations (Gu et al. 2022).In keeping with this, circulating SPX levels appear to be directly correlated with a decline in pancreatic beta-cell function in a Chinese population of T2DM subjects (Dai et al. 2023).Other reported, but less well characterised, functions of SPX in rodents include inhibition of adrenocortical cell proliferation, stimulation of bowel movements and up-regulation of hyperoxia (Walewski et al. 2014;Pruszynska-Oszmalek et al. 2020).

SPX receptor and signalling
SPX binds specifically to GALR2 and GALR3, but not to GALR1, corresponding to primary effects on modulation of insulin action and feeding behaviour (Fang et al. 2018b;Lee et al. 2020).SPX is believed to regulate energy homeostasis through interaction with hypothalamic circuits linked to augmentation of leptin and melanocortin 4 (MC4) receptor signalling, whilst diminishing neuropeptide Y5 (NPYR5) and ghrelin receptor signalling (Wong et al. 2021).
Thus, intranasal administration of a SPX-based GALR2 agonist effectively reduced food intake and body weight in obese mice (Yun et al. 2019).SPX has also been shown to inhibit lipogenesis and glucose absorption in murine 3T3-L1 as well as human adipocytes, through activating both GALR2 and GALR3 (Kolodziejski et al. 2018).Specifically, SPX activation of GALR2 and GALR3 results in the triggering of the PI3K/PKB and cyclic adenosine 3,5monophosphate (cAMP)/PKA cell signalling pathways in most tissues (Yu et al. 2022).
J o u r n a l P r e -p r o o f

SPX metabolism
Studies reveal that modifications of the N-terminal amino acid of SPX, Asn 1 , via substitutions of L-Asn 1 to D-Asn 1 , addition of fluorenyl methoxycarbonyl (Fmoc), acetylation or polyethylene glycosylation, increase the enzymatic stability of the peptide when compared to native SPX (Reyes-Alcaraz et al. 2016).In addition, substitution of L-Gln 14 for D-Gln 14 also enhanced stability of the peptide in plasma (Lee et al. 2020).Thus, although the exact degradation profile of SPX is unknown, cleavage of N-and C-terminal residues by serum proteases seems the most likely process in this regard, but further confirmation is required.Furthermore, L-Ala 4 for D-Ala 4 substitution improved enzymatic stability of the peptide, indicating other potentially important enzyme cleavage sites beyond the N-and C-termini (Reyes-Alcaraz et al. 2016).Together it is clear that further investigation of SPX mediated degradation within the bloodstream is warranted.

GAL and SPX: opportunities for the treatment of T2DM and obesity
It seems apparent that both GAL and SPX have untapped therapeutic promise for obesity and obesity-driven forms of diabetes such as T2DM (Mills et al. 2021).The peptides induce direct effects on to appetite and overall energy homeostasis, making them desirable targets for clinical application.Interestingly, from the current literature it seems that SPX may have more potential in this regard as a treatment for obesity, possibly owing to the GALR selectivity of this peptide (Modh Zahir et al. 2022).On the other hand, GAL stimulates increased fat intake and may be better suited to the treatment of eating disorders such as anorexia and bulimia (Baranowska et al. 2001).However, compounds that antagonise the actions of GAL could yet find therapeutic applicability for diseases such as obesity.In contrast to GAL, SPX suppresses appetite and can reduce body weight in obese rodents (Mills et al. 2021).Indeed, circulating fatty acids may act J o u r n a l P r e -p r o o f as a trigger for SPX secretion to induce satiety (Modh Zahir et al. 2022).Such effects of GAL and SPX are predominantly mediated centrally through direct modulation of hypothalamic circuits (Marcos & Coveñas 2021).Notably, SPX is documented to inhibit adipogenesis and stimulate lipolysis in both human and murine adipocytes (Kolodziejski et al. 2018), suggesting direct benefits in obesity.In good agreement with this, once daily SPX administration (25 μg/kg body weight) for 30 days in DIO mice lead to reductions of body weight, with subsequent improvements in insulin action and overall metabolism (Kolodziejski et al. 2021), similar to previous observations (Walewski et al. 2014).In keeping with this, GAL and SPX have previously been shown to improve peripheral insulin action and up-regulate GLUT4 membrane expression, resulting in positive effects on glucose tolerance (Gu et al. 2019).Interestingly, at the level of the pancreatic beta-cell, SPX inhibits glucose-stimulated insulin secretion (GSIS) (Sassek et al. 2018a).This inhibitory effect of GAL and SPX on GSIS may at first appear counterintuitive for T2DM treatment.However, there are well documented benefits of inducing beta-cell rest in diabetes (Tanday et al. 2022), with such agents shown to promote beta-cell health and prevent beta-cell apoptosis that is characteristic of all forms of diabetes.Indeed, SPX-induced enhancement of pancreatic islet cell viability and proliferation would fully support this notion (Sassek et al. 2018a), with the presence of SPX in islet beta-cells suggesting possible important autocrine and paracrine interactions within and between endocrine islet cells (Sassek et al. 2018b).Therefore, the impact of GAL and SPX in this setting requires further detailed study.

Conclusion and future developments
Recent literature clearly documents that GAL and SPX and exert numerous biological actions both within the CNS and periphery (Figures 2&3).However, the relative importance of each of these peptide mediated effects in terms of overall physiology and control of metabolism is still J o u r n a l P r e -p r o o f largely unknown, but their impact on the modulation of energy homeostasis appears to be particularly significant (Figure 2&3).Of the two peptides, GAL is the most extensively investigated to date given its earlier discovery, although potential anti-obesity actions of SPX may see interest in this molecule increase exponentially over the coming years.Development of pharmacological and/or genetic methods to determine the precise actions of GAL and SPX at individual GALRs, such as characterisation and use of specific GALR antagonists or CRISPR-Cas9 mediated knockdown of GALRs (Eskova et al. 2020), may help to uncover the underlying mechanisms of such benefits.Thus, there are still unanswered questions regarding the pharmacology and cellular mechanisms that coordinate GAL and SPX biological actions.
Nevertheless, owing to direct positive effects on energy homeostasis and insulin sensitivity, GAL and SPX related peptides have exciting potential for the future treatment of obesity and associated metabolic disorders such as T2DM.

Highlights
• Galanin (GAL) and spexin (SPX) are neuropeptides that bind to GALR1, GALR2 and GLAR3 galanin receptors • GAL activates all galanin receptors, whereas SPX interacts with GALR2 and GLAR3 • GAL and SPX appear to have beta-cell resting actions at the level of the endocrine pancreas J o u r n a l P r e -p r o o f Yun S, Reyes-Alcaraz A, Lee YN, Yong HJ, Choi J, Ham BJ, Sohn JW, Kim DH, Son GH, Kim H & Kwon SG 2019 Spexin-based galanin receptor type 2 agonist for comorbid mood disorders and abnormal body weight.Frontiers in Neuroscience 13 391.Zheng Y, Ley SH & Hu FB 2018 Global aetiology and epidemiology of type 2 diabetes mellitus and its complications.Nature Reviews Endocrinology 14 88-98.Zorrilla EP, Brennan M, Sabino V, Lu X & Bartfai T 2007 Galanin type 1 receptor knockout mice show altered responses to high-fat diet and glucose challenge.Physiology & Behavior 91 479-485.

Figure 3 .
Figure 3. Biological actions of SPX within brain, muscle, pancreas, liver and adipose tissue.

Table and figure legendsTable 1 .
Amino acid sequences of SPX, and GAL including species variations.Amino acids are abbreviated using standard single letter amino acid codes