Reviewing the physiological roles of the novel hormone-receptor pair INSL5-RXFP4: a protective energy sensor?

in Journal of Molecular Endocrinology
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  • 1 Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada
  • | 2 Department of Biology, The University of Winnipeg, Winnipeg, Manitoba, Canada
  • | 3 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada
  • | 4 Department of Biological Sciences, The University of Manitoba, Winnipeg, Manitoba, Canada

Correspondence should be addressed to S V Good: s.good@uwinnipeg.ca
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There is no common consensus on the physiological role of insulin-like peptide 5 (INSL5) and its cognate receptor, relaxin family peptide receptor 4 (RXFP4). The experimental data for INSL5–RXFP4 expression and function point to a potential role of the peptide hormone and receptor pair in linking energy availability, homeostasis, and inflammation. In this review, we summarize studies on the INSL5–RXFP4 system and propose that the current findings from diverse experimental settings point broadly to a role as a protective energy sensor (PES). Specifically, we review the evidence that (1) INSL5–RXFP4 could regulate immune response by decreasing the production of proinflammatory cytokines and may be involved in the stress response via the HPA axis; (2) INSL5–RXFP4 may signal through sensory neurons on the vagus nerve, transmitting signals to the CNS; and (3) INSL5–RXFP4 could have local autocrine/paracrine roles within the intestinal tract and immune cells. Further investigation and clarification of these proposed roles of INSL5–RXFP4 may prove a greater physiological relevance for the pair and add to existing evidence of INSL5–RXFP4 role as a PES.

Abstract

There is no common consensus on the physiological role of insulin-like peptide 5 (INSL5) and its cognate receptor, relaxin family peptide receptor 4 (RXFP4). The experimental data for INSL5–RXFP4 expression and function point to a potential role of the peptide hormone and receptor pair in linking energy availability, homeostasis, and inflammation. In this review, we summarize studies on the INSL5–RXFP4 system and propose that the current findings from diverse experimental settings point broadly to a role as a protective energy sensor (PES). Specifically, we review the evidence that (1) INSL5–RXFP4 could regulate immune response by decreasing the production of proinflammatory cytokines and may be involved in the stress response via the HPA axis; (2) INSL5–RXFP4 may signal through sensory neurons on the vagus nerve, transmitting signals to the CNS; and (3) INSL5–RXFP4 could have local autocrine/paracrine roles within the intestinal tract and immune cells. Further investigation and clarification of these proposed roles of INSL5–RXFP4 may prove a greater physiological relevance for the pair and add to existing evidence of INSL5–RXFP4 role as a PES.

Introduction: INSL5–RXFP4

The physiological role of hormone-receptor pair insulin-like peptide 5 (INSL5) and relaxin family peptide receptor 4 (RXFP4/GPCR42/GPR100) has been probed since their respective discoveries in 1999 (Conklin et al. 1999) and 2003 (Boels & Schaller 2003, Fredriksson et al. 2003, Liu et al. 2003a, b). The wide distribution of INSL5 includes its primary tissues of expression – L-cells of the distal colon, hypothalamus, kidney, thymus, and reproductive tissues (Conklin et al. 1999, Mashima et al. 2013, Thanasupawat et al. 2013, Grosse et al. 2014, Lee et al. 2016, Ang et al. 2018, Kocan et al. 2018, Vahkal et al. 2021), while the primary tissues of expression of RXFP4 are the colon, subtending vagus nerve efferents, cerebellum, reproductive tissues, and kidney (Liu et al. 2003b, 2005, “GTEx Portal” 2021, Vahkal et al. 2021). The overlap of expression patterns between the hormone-receptor pair indicates the potential function of INSL5 as a gut hormone (Grosse et al. 2014). RXFP4 has been established as the main receptor for INSL5 (Liu et al. 2005), although INSL5 is also a weak antagonist of RXFP3 (Liu et al. 2003a, 2005, Kocan et al. 2018), and relaxin-3/INSL7 has been shown to activate RXFP4 in human cell and mice models (Liu et al. 2003b).

INSL5 is produced and released from the L-cells of the colon and rectum. It is largely co-stored and co-released from a vesicular pool with two anorexigenic hormones – glucagon-like peptide (GLP)-1 and peptide YY (PYY) (Billing et al. 2018, Lewis et al. 2020). One difference between the anorexigenic hormones and INSL5 is the production and secretion site within the gastrointestinal tract. Both GLP-1 and PYY are produced and secreted throughout the gastrointestinal tract (GI), while the production and secretion of INSL5 are found in a gradient towards the colon and rectum (Gribble & Reimann 2016). This difference enhances the difficulty of understanding the physiological role(s) of INSL5. For example, it is unusual for nutrients to reach the distal gastrointestinal tract; thus, L-cells in the colon and rectum are more likely to be regulated by components found within the colonic lumen such as bile acids, short-chain fatty acids (SCFAs) and/or other luminal metabolites (Billing et al. 2018), plasma metabolite concentration(s) and/or the microbiota composition.

INSL5 has been reported to (1) modulate glucose homeostasis (Burnicka-Turek et al. 2012, Luo et al. 2015, Lee et al. 2016, Ang et al. 2017, 2018); (2) act as an orexigenic hormone (Grosse et al. 2014, Li et al. 2020); (3) play a role in both male and female fertility (Burnicka-Turek et al. 2012, Wagner et al. 2016, Yeganeh et al. 2017, Bicer et al. 2019); (4) be a marker of colorectal endocrine cells, neuroendocrine tumors, and breast cancer (Mashima et al. 2013, Thanasupawat et al. 2013, Lee et al. 2016, Sun et al. 2019); (5) regulate metabolic reprogramming and act as a potential diagnostic, prognostic, and therapeutic target of nasopharyngeal carcinoma (Li et al. 2020); (6) play a role in immune response (Vahkal et al. 2021); (7) stimulate colorectal propulsion (Diwakarla et al. 2020, Pustovit et al. 2021); and (8) based on evidence available from our previous studies and the work of others, we propose a novel function that INSL5-RXFP4 may modulate responses to the gut microbiome (Wichmann et al. 2013, Lee et al. 2016, Alnafea et al. 2019, Hecker et al. 2019, Vahkal et al. 2021). Some studies have suggested that since measurable effects of INSL5 are low within animal models, the physiological role of INSL5 may be less relevant than other gut hormones (Ang et al. 2018, Lewis et al. 2020); however, further research is necessary to fully understand the physiological role(s) of the pair in humans.

RXFP4 is found throughout the GI tract increasing towards the distal end, within immune organs and many immune cell subsets as well as on the vagus nerve that innervates the gut (Grosse et al. 2014; proteinatlas.org; Vahkal et al. 2021). Signalling via the vagus nerve can lead to changes in GI and pancreatic secretions, GI motility, regulation of food intake, and body weight. The abundance of receptors expressed on the vagus nerve responds dynamically to changes in nutritional status, indicating that it could be a nexus of control for maintaining physiological homeostasis (Raybould 2010). The vagal afferent neurons also regulate the cholinergic anti-inflammatory pathway – an immunological pathway that prevents excessive inflammation in response to injury or pathogens (Parekh et al. 2016). Given this, as well as evidence that the gene products of enteroendocrine cells (EECs) interact with vagal efferent neurons (Carabotti et al. 2015), the available experimental data for INSL5-RXFP4 expression and function together point to a potential role of the peptide hormone and receptor in linking metabolism and inflammation.

In this review, we summarize functional studies on the INSL5–RXFP4 system and present the available evidence from diverse experimental settings that point to their role as a PES via the microbiota–gut–brain axis. Given that INSL5–RXFP4 (1) is expressed in immune tissues and influences macrophage proliferation and cytokine levels (Box 1) (Vahkal et al. 2021), (2) can directly or indirectly regulate energy homeostasis (Tables 1, 2 and Box 2) (Burnicka-Turek et al. 2012, Grosse et al. 2014, Luo et al. 2015, Lee et al. 2016, Ang et al. 2017, 2018, Kocan et al. 2018, Li et al. 2020), (3) is associated with CRP, a general marker for inflammation in human clinical trials (Table 3) (Wagner et al. 2016, Bicer et al. 2019), and (4) is a marker of colorectal endocrine cells, neuroendocrine tumors, breast cancer, and nasopharyngeal carcinoma (Box 3) (Mashima et al. 2013, Thanasupawat et al. 2013, Lee et al. 2016, Sun et al. 2019, Li et al. 2020) collectively strengthens the hypothesis that INSL5 may influence communication via the microbiota–gut–brain axis to act as a PES (Fig. 1).

Box 1. INSL5–RXFP4 and immune system

Vahkal et al. 2021
  Insl5 expression in thymus and colon
   Highly correlated with Il-7 – marker of thymocyte development
  Rxfp4 expression in all immune organs, BMDM, and splenic DCs
   Regulated by immune-associated transcription factors (STATs and GATA)
Systemic effects (C57BL/6 mice, 70% CR INSL5 injection)
  Circulating immune mediators
   ↑ blood GLP-1
   ↑ blood GIP
   ↑ blood glucagon
   ↑ blood PYY
  Cytokines
   ↑ MIP-2
   ↑ M-CSF
   ↑ IL-15
   ↓ G-CSF
   ↓ IL-7
   ↓ IL-5 
   ↓ IL-27
   ↓ eotaxin
Mouse macrophage ANA-1 cells
   INSL5 co-treatment with LPS ⤓ cell growth
  Pretreatment 12 or 18 h with INSL5 then LPS treatment
   ↓ viable cells
   ↓ IL-1β
   ↓ IL-6
   ↓ TNFα

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit.

Table 1

Summary of INSL5–RXFP4 on metabolism in cell culture models.

Cell culture
Cell type and treatmentEffectReference
MIN6Luo et al. 2015
 INSL5⦸ insulin at low glucose

↑ insulin at high glucose
MIN6Ang et al. 2017
 INSL5 treatment⦸ insulin at low glucose

↓ insulin at high glucose
GLUTagLuo et al. 2015
 Increasing INSL5 incubation↑ GLP-1 at high glucose
GLUTagNo rxfp4 expression foundTrabelsi et al. 2015

Ang et al. 2018
NCI-H716 cellsAng et al. 2018
 Acute INSL5 treatment⦸ basal, carbachol, or insulin-stimulated GLP-1

⤓ forskolin-stimulated GLP-1
 Chronic INSL5 pre-treatment and INSL5 treatment⦸ basal secretion

Failed to ⤓ forskolin-stimulated GLP-1 release
 Chronic INSL5 pre-treatment↑ basal GLP-1

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit.

Table 2

Summary of INSL5-RXFP4 on metabolism in transgenic mice models.

Trangenic mice
Mouse strainEffectReference
Insl5−/− (129/Sv)↓ glucose tolerance w/age↓ pancreatic beta cell mass⚥ impaired fertilityBurnicka-Turek et al. 2012
 IPGTT⦸ GLP-1 levels
Insl5−/− (C57BL/6)⦸ glucose toleranceMashima et al. 2013
Insl5−/− (C57BL/6)↓ glycogen stores in liver↓ pyruvate toleranceLee et al. 2016
 IPGTT↓ glucose tolerance⦸ insulin levels
 ITT↑ insulin tolerance
Rxfp4−/−⦸ glucose intoleranceGrosse et al. 2014
db/db – IPGTT and i.p. INSL5 treatmentDose dependent ↓ blood glucoseLuo et al. 2015
C57BL/6 – IPGTT and i.p. INSL5 treatmentDose dependent ↓ blood glucose
C57BL/6 – OGTT and i.p. INSL5 treatment↑ insulin levels⦸ GLP-1 levels

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit.

Box 2. INSL5–RXFP4 on energy homeostasis

Grosse et al. 2014 (C57BL/6 Mice)
Plasma INSL5 (following overnight fast):
  Overnight CR: ↑ plasma INSL5, ↓ plasma INSL5 after refeed
  2-week 60% CR: ↑↑ plasma INSL5, ↓ plasma INSL5 after refeeding
  10-week 60% CR: ↑↑ plasma INSL5, ⦸ plasma INSL5 after refeed
Colonic mRNA:
  4-week 60% CR: ↑ Insl5 mRNA, ↑ Gcg mRNA
  4-week HFD: ⦸ Insl5 mRNA, ⦸ Gcg mRNA
Food Intake and body composition:
  WT: dose-dependent ↑ with INSL5 injections
rxfp−/−: ⦸ with INSL5 injections, ↓ mealtime to HFD, ↓ preference to HFD, ↓ fat mass at 11 weeks chow diet
Zaykov et al. 2019
WT mice: 1-week daily s.c. INSL5 administration: ⦸ food intake, body weight, or glucose intolerance
Diet-induced obese mice: 1-week daily s.c. INSL5 administration:↑ glucose tolerance at 5 days, ⦸ food intake or body weight
Lewis et al. 2020
Insl5 promoter-driven expression of Gq-coupled DREADD to activate colonic L-cells
  15-h fast: ↑ plasma GLP-1, PYY, and insulin, ⦸ blood glucose
  Fed mice: ↓ blood glucose
  IPGTT: ↑ glucose tolerance reversed with inhibition of GLP-1 receptor
  ↓ food intake abolished with Y2R antagonist
  ⤓ increase in RER, ↑ energy expenditure and activity rate
  ↑ food intake following Y2R antagonist and i.p. CNO
 2-week HFD and L-cell activation:
  ↑ glucose tolerance, ↓ food intake and body weight
Lewis et al. 2021
 Activation in all Rxpf4-expressing cells of a Gi-coupled DREADD (mimic INSL5-RXFP4 signalling)
  ⦸standard chow intake
  ↑ food intake of HFD and highly palatable liquid Ensure test meal (HPM)
  ↑ preference for HFD when given choice
  ⦸ambulatory activity, energy expenditure
 Activation of Gq-coupled DREADD in whole body and ventromedial hypothalamus Rxfp4-expressing cells
  ⦸ad libitum fed mice offered chow
  ↓ food intake when adapted to HFD or HPM
   95% body weight calorie restriction (whole body only)
    ↓ reduced motivation for the HPM
Vahkal et al. 2021
 Calorie restircted C57BL/6 mice
  i.p. INSL5 treatment: ↑ GLP-1, GIP, PP, PYY, and glucagon
 EMBL-EBI expression atlas revealed 5 experiments with ↓ Insl5 and Rxfp4 in association with metabolic disorders
Hecker et al. 2019
 INSL5–RXFP4 lost in obligate carnivores, present in obligate herbivores
  Diet composition, feeding patterns, and gut microbiome diversity

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit.

Table 3

Summary of INSL5 in human clinical trials.

Clinical trials
ParticipantConditionResult/effectReference
FemalesLean↑ circulating INSL5Wagner et al. 2016
Obese⦸ circulating INSL5

— serum INSL5 and CRP
MalesLean⦸ circulating INSL5

— serum INSL5 and testosterone

— serum INSL5 and estradiol
Obese⦸ circulating INSL5

— serum INSL5 and testosterone

— serum INSL5 and total cholesterol

— serum INSL5 and triglycerides

— serum INSL5 and cortisol
MalesPost bariatric surgery↓ circulating INSL5 regardless of diabetes status
FemalesControls+ INSL5 and BMI

+ INSL5 and waist circumference

+ INSL5 and fasting blood glucose

+ INSL5 and serum insulin

+ INSL5 and HOMA-IR

+ INSL5 and free androgen index

+ INSL5 and LH
Bicer et al. 2019
PCOS↑ serum INSL5↑↑ patients with insulin resistance+ serum INSL5 and BMI

+ serum INSL5 and waist circumference

+ serum INSL5 and fasting blood glucose

+ serum INSL5 and serum insulin

+ serum INSL5 and HOMA-IR

+ serum INSL5 and free androgen index

+ serum INSL5 and LH

+ serum INSL5 and hs-CRP
BMI ≥ 25 kg/m2↑ serum INSL5 regardless of PCOS status
Increased serum INSL5Higher odds of having PCOS

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit; (—) negative correlation; (+) positive correlation.

Box 3. INSL5 as a disease marker

Thanasupawat et al. 2013
  INSL5 expressed by ECCs within colonic mucosa nearby colonocytes expressing RXFP4
  INSL5–RXFP4 expressed in human neuroendocrine/carcinoid tissues
Male C57BL/6 mice with chemically induced acute IBD:
  ⦸ number of INSL5-positive ECCs found at site of inflammation
Mashima et al. 2013
  INSL5 expressed in EECs along colorectum – increase towards s rectum
  RXFP4 expressed throughout digestive tract
  Neuroendocrine tumors co-expressed INSL5–RXFP4
  INSL5 not required for development of CgA-positive EECs
Cell proliferation:
  ⦸ proliferation of Caco-2 or COLO320DM cells
C57BL/6 mice chemically induced IBD:
  ⦸ severity or mucosal healing between WT and Insl5−/−
Li et al. 2020
  INSL5 elevated in nasopharyngeal carcinoma patients
  Higher plasma INSL5 associated with poor disease outcome
Cell proliferation:
  INSL5 overexpression ↑ proliferation of NPC cells
Metabolic reprogramming CNE2 overexpressed with INSL5:
  ↑ glycolysis, ↓ OXPHOS
  ↑ increase glucose uptake
  ↑ expression of glycolytic enzymes via RXFP4/STAT5 pathway
  ↑ activation of Akt, ERK1/2, and JAK1
   ⤓ JAK1 and ERK1/2 STAT5 inactivation
Strategy for NPC therapy:
 RXFP4 antibody diminished INSL5 oncogenic function
  INSL5 overexpression sensitizes NPC to glycolysis inhibitor
  Overexpression of INSL5 promoted chemoresistance → anti-INSL5 or RXFP4 supress tumor alone or renders NPC cells sensitive to chemotherapy
Skok et al. 2021
Microarray analysis using 7 datasets (4 UC and 3CD)
  INSL5 upregulated in UC (logFC = 3.22, P  < 0.001)
  INSL5 downregulated in CD (logFC = −1.26, P  = 0.015)
Experimental data (27 CD, 35 UC, and 22 normal) from routine ileocolonoscopies
 INSL5 downregulated in CD (logFC = −2.26, P  = 0.048)
  INSL5 downregulated in UC (logFC = −2.05, P  = 0.008)

(⦸) no effect; (↑) increase; (↓) decrease; (⤓) inhibit.

Figure 1
Figure 1

INSL5–RXFP4. A protective energy sensor in the microbiome–gut–brain axis. INSL5 and RXFP4 are expressed in the distal gut and in central immune tissues, while RXFP4 is additionally expressed in a range of peripheral immune tissues and cell types particularly those belonging to the innate immune system, nodose, and dorsal root ganglia as well the vagus nerve, ventromedial hypothalamus, and microglial cells in the brain. There is evidence that the ligand–receptor pair plays roles in glucose metabolism, colorectal propulsion, food-seeking behaviour, and, more recently, the immune system. We propose a synthetic view of these diverse roles that revolve around their role in the microbiome–gut–brain axis in an endocrine and/or paracrine manner. Levels of INSL5 expression in the distal gut are affected by the abundance of gut microbes and short-chain fatty acids, incubation of murine macrophages with INSL5 resulted in a predominantly anti-inflammatory LPS-induced cytokine response, serum levels of INSL5 are associated with those of C-reactive Protein, a general marker for inflammation, in human clinical trials, and INSL5-RXFP4 are markers of colorectal endocrine cells, neuroendocrine tumors, breast cancer, and nasopharyngeal carcinoma. Collectively, this points to a hypothesis that INSL5 may influence communication via the microbiome–gut–brain axis in an endocrine and/or paracrine manner to act as a PES. (Created using Biorender). A full colour version of this figure is available at https://doi.org/10.1530/JME-21-0241.

Citation: Journal of Molecular Endocrinology 69, 1; 10.1530/JME-21-0241

INSL5–RXFP4 signalling

Van der Westhuizen et al. (2012) and Kocan et al. (2018) published comprehensive reviews on the history and background of RXFP3 and RXFP4, including overviews of the downstream signalling pathway(s) discovered to date (van der Westhuizen et al. 2012, Kocan et al. 2018). Additional research has been published complementing and building upon the downstream signalling pathways of INSL5-RXFP4. Like RXFP3, RXFP4 is a G protein-coupled receptor (GCPR), coupled to inhibitory Gαi/o proteins, such that stimulation by INSL5 increases GTPγS binding and inhibits forskolin-stimulated cAMP accumulation (Liu et al. 2003b, 2005, Belgi et al. 2011, Ang et al. 2017, 2018, Kocan et al. 2018). INSL5 binding to RXFP4 can increase phosphorylation of ERK1/2, p38MAPK, Akt-Ser473, Akt-Thr308, JAK/STAT5 (Li et al. 2020), and S6 ribosomal protein (S6RP) (Ang et al. 2017, 2018, Kocan et al. 2018). Ang et al. (2017) demonstrated that RXFP4 activated by INSL5 undergoes classical GRK phosphorylation and β-arrestin-mediated receptor internalization followed by sequestration to early endosomal compartment (Ang et al. 2017). Chronic vs acute stimulation of RXFP4 by INSL5 within human NCI-H716 cells differentially impacts GLP-1 secretion. Acute incubation of NCI-H716 cells with INSL5 did not change GLP-1 levels in cells subjected to basal, carbachol, or insulin-stimulated GLP-1 secretion while forskolin-stimulated GLP-1 secretion was inhibited. On the other hand, chronic pre-treatment of cells with INSL5 did not inhibit forskolin-stimulated GLP-1 secretion but did result in increased basal GLP-1 secretion (Ang et al. 2018). The significance of these results is discussed further in section ‘INSL5-RXFP4 on metabolism in cell culture models’. The authors speculate that the difference in acute vs chronic exposure of NCI-H716 cells to INSL5 may be heterologous sensitization of adenylyl cyclase by Gi/o-coupled receptors (Brust et al. 2015). For example, other known Gi/o-coupled receptors such as the δ-opioid, µ-opioid, and dopamine D2 receptors share this paradoxical change in cAMP production after acute or chronic agonist stimulation (Ang et al. 2018).

INSL5–RXFP4 and immune system

Summarized in Box 1.

INSL5–RXFP4 impact on immune cells

Recently, Vahkal et al. (2021) explored the hypothesis that INSL5–RXFP4 have roles in the central (thymus) and/or peripheral (spleen, lymph node, blood, and intestine) immune system. Insl5 transcripts were detected in thymus and colon. Using in silico analyses, high levels of Insl5 expression were found in specific subpopulations of thymocytes but low expression in other immune tissues and cell subsets. Promoter analysis revealed that INSL5–RXFP4 are regulated by a host of immune and metabolism-associated transcription factors as well as cortisol (glucocorticoid response element), indicating that it could respond to immune system mediators and stress. Rxfp4 expression was detected in all murine immune organs and in diverse immune cell subsets in the innate immune system, particularly bone-marrow-derived macrophages (BMDM) and splenic dendritic cells. Splenic dendritic cells can relay information between the gut lumen and immune tissues (spleen and lymph node) to help initiate an appropriate (i.e. pro-inflammatory and anti-inflammatory) immune response (Sathe & Shortman 2008, Wu et al. 2018). Consistent with the broad expression of Rxfp4, i.p. injection of INSL5 resulted in altered systemic levels of cytokines and chemokines (IL-5, IL-7, M-CSF, IL-15, IL-27, and MIP-2). In a mouse BMDM cell line (ANA-1), incubation with INSL5 resulted in impeded cell growth, a transient elevation of IL-15 and a sustained reduction in IL-1β, IL-6, and TNFα, all pro-inflammatory immune mediators (Vahkal et al. 2021).

IL-6 is elevated in gut inflammatory diseases and is secreted in response to microbial proteins (Cauvi et al. 2017). Tessaro et al. (2017) found that the stimulation of BMDM with insulin and LPS resulted in increased IL-6 and TNFα via the PI3K and ERK1/2 pathways in diabetic but not in non-diabetic mice. However, insulin and LPS downregulated IL-6 and TNFα within alveolar and peritoneal macrophages. Vahkal et al. (2021) found that INSL5 may downregulate IL-6, IL-1β, and TNFα in BMDMs from non-diabetic C57BL/6 mice. This suggests that both insulin and INSL5, which share similar three-dimensional structures but signal via different classes of receptors, can influence immune cells.

INSL5–RXFP4 on the microbiota–gut–brain axis

The intestinal microbes communicate with the gut and brain via the microbiota–gut–brain axis utilizing multiple pathways including neural (vagus nerve and/or spinal cord), endocrine (HPA), immune (cytokines), and metabolic systems. Some pathway components may reach the brain through the blood and circumventricular organs (Bonaz et al. 2018). The vagus nerve is a key component of the parasympathetic nervous system due to its role in interoceptive awareness (Strigo & Craig 2016) and contains 80% afferent and 20% efferent fibres (Agostoni et al. 1957). The vagal afferent fibres do not cross the epithetical layer and, thus, they are not in direct contact with the gut luminal bacteria (Wang & Powley 2007). As a result, a part of the EECs ability to detect the bacterial content and by-products which regulate gastrointestinal motility, secretion, and food intake is due to their indirect effect on vagal afferent fibres (Bonaz et al. 2018). In addition to their role in energy balance, metabolism, and ability to modulate function in different tissues, microbial metabolites such as SCFAs may have a direct effect on afferent terminals and/or may exert central effects through G-protein coupled receptors (Canfora et al. 2015). The expression and functional profiles of INSL5 and RXFP4, the latter of which has been detected on vagal afferents (Grosse et al. 2014), indicate that they could be involved in the microbiota–gut–brain axis.

Lee et al. (2016) showed that Swiss Webster mice that lacked a microbiota and were germ-free (GF) or conventional/wildtype Swiss Webster mice treated with antibiotics exhibited a significant increase in Insl5 expression. Previous research from their lab showed that colonic L-cells deprived of energy from either caloric restriction (CR) or removal/disruption of the microbiota exhibited increased expression of Gcg (and thus GLP-1 and GLP-2), similar to that reported by Grosse et al. (2014) (Wichmann et al. 2013). Wichmann et al. (2013) proposed that the action of colonic L-cell GLP-1 may be similar in times of caloric excess and restriction. An increase in GLP-1 from either a response to nutrients (as seen higher in the GI tract) or energy deprivation in the colon appears to slow intestinal transit to increase nutrient absorption as well as to regulate bacterial colonization via transit time. On the other hand, during times of caloric excess (non-energy-deprived colonic L-cells), colonic GLP-1 is suppressed, thereby increasing the rate of intestinal transit and preventing bacterial overgrowth (Stephen et al. 1987, Wichmann et al. 2013). This pattern is reminiscent of the changes found in expression of Insl5 in the colon. Cells within the distal GI tract deprived of energy either via CR or disruption of the gut microbiota exhibit increased Insl5 expression which is reduced following refeeding (Grosse et al. 2014, Lee et al. 2016). However, in mice fed a high-fat diet (HFD), Insl5 expression remained low regardless of microbial status. Together, these results indicate the critical influence that energy intake, whether at the cellular level (cells within the distal GI tract), microbiome, or organism level, can have on the expression and plasma levels of INSL5. In this context, it is worth mentioning that the recently developed and commonly used analogue, INSL5-A13, has been found to stimulate colorectal propulsion, thus offering a potential pharmacological agent for constipation as well as bacterial diarrhoea by the inhibition of RXFP4 via INSL5-A13NR (Diwakarla et al. 2020, Pustovit et al. 2021). Both studies utilized exogenous RXFP4 stimulation, either via direct RXFP4 stimulation or SCFA addition. Endogenous INSL5 release, either during times of CR (Grosse et al. 2014, Lee et al. 2016) or by alternative microbiota manipulation (Lee et al. 2016), may have opposing effects on colonic propulsion, dependent upon starting energy status.

Arora et al. (2018) investigated how the gut microbiota regulates the transcriptome of L-cells within the ileum and colon by studying microbial colonization of GF GLU-Venus mice, which express yellow fluorescent protein (YFP) under control of the proglucagon promoter. They found high expression of Gcg, Pyy, cholecystokinin, secretin, and neurotensin in proglucagon-expressing YFP-positive cells in the ileum and colon, whereas gastric inhibitory peptide (Gip) and Insl5 were only expressed at high levels in the ileum and colon, respectively. In contrast to previous findings, the expression of these hormones was the same in GF and conventionally raised GLU-Venus mice; however, the authors suggest assay saturation as a cause (Arora et al. 2018).

Another critical route of communication from the gut to brain is immune signalling mediated by cytokines (El Aidy et al. 2015). Cytokines produced in the gut can travel via the bloodstream to the brain. Increasing evidence suggests that cytokines can signal across the blood–brain barrier to influence specific brain areas. For example, IL-1 and IL-6 activate the hypothalamic–pituitary–adrenal (HPA) axis resulting in the release of cortisol. Dysregulation of the stress response via the HPA axis can have a significant impact on the microbiota–gut–brain axis, both of which have been shown to be implicated in many disease states (Dinan & Cryan 2017). Thus, given the evidence discussed in section ‘INSL5–RXFP4 impact on immune cells’, specifically, that INSL5 may downregulate IL-6, IL-1β, and TNFα in BMDM’s from non-diabetic C57BL/6 mice and both INSL5 and RXFP4 are regulated by multiple STATs in humans and mice (Vahkal et al. 2021), provide evidence that INSL5-RXFP4 may signal indirectly through the HPA axis.

INSL5–RXFP4 and metabolism

INSL5–RXFP4 on metabolism in cell culture models

A summary of INSL5’s impact on metabolism in cell culture models is presented in Table 1. Previous findings of impaired glucose tolerance, abnormal islet morphology in aging Insl5−/− mice, and the expression of RXFP4 found in mouse pancreatic islets (Burnicka-Turek et al. 2012) collectively suggest that INSL5 may have a direct influence on insulin secretion. Luo et al. (2015) utilized a mouse insulinoma cell line, MIN6, and showed that the addition of INSL5 had no impact at 2.8 mM glucose but elicited a significant increase in insulin secretion at 16.8 mM glucose. Similar results were seen with 100 nM GLP-1 treatment at both glucose concentrations, leading the authors to conclude that the insulinotropic effect of INSL5 on mouse pancreatic islets is glucose-dependent (Luo et al. 2015). Thus, in times of glucose excess, INSL5 appeared to increase insulin secretion in MIN6 cells and isolated mouse islets (Luo et al. 2015). In contrast, Ang et al. (2017) found that MIN6 cells treated with INSL5 (0.1–200nM) did not have altered insulin secretion at low glucose levels (0 mM), while at high glucose (10 mM), INSL5 slightly inhibited insulin secretion (Ang et al. 2017). β-islet cell GPCRs that are Gαs or Gαq-coupled have been known to increase insulin secretion, while Gαi/o-coupled receptors, such as RXFP4, would be expected to reduce insulin secretion (Ahrén 2009, Kocan et al. 2018).

Further probing the relationship between INSL5 and GLP-1, Luo et al. (2015) used GLUTag cells, a murine intestinal L-cell line, in 16.8 mM glucose and found that with increasing incubation levels of INSL5, there was an increase in GLP-1, complementing their findings that INSL5 may have insulinotropic effects (Luo et al. 2015) under conditions of caloric excess. In contrast, both Trabelsi et al. (2015) and Ang et al. (2018) found no Rxfp4 expression in GLUTag cells. These differences may be attributed to culture methods and/or conditions which could influence transcriptomic variability between laboratories (Frattini et al. 2015, Trabelsi et al. 2015, Ang et al. 2018).

Ang et al. (2018) utilized NCI-H716 cells (common human model of enteroendocrine L-cells) and found that INSL5 treatment inhibited GLP-1 release in response to forskolin, an adenylyl cyclase activator, but had no impact on basal GLP-1 release or carbachol or insulin-stimulated GLP-1 secretion. However, chronic INSL5 pre-treatment of NCI-H716 cells resulted in a significant increase in basal GLP-1 secretion and abolished the inhibition of forskolin-stimulated GLP-1 secretion. It was proposed that INSL5–RXFP4 act as a regulatory mechanism for GLP-1 secretion (Ang et al. 2018). During the fasted state and/or at the end of feeding when the action of GLP-1 is no longer required, INSL5 inhibits secretion (as shown by the inhibition of forskolin-stimulated GLP-1 secretion). These results complement those of Grosse et al. (2014) who found that INSL5 levels peak during the fasted state and lower with refeeding. However, there are still many unanswered questions. GLP-1 release within L-cells is regulated by multiple mechanisms. In addition, the factors controlling INSL5 secretion are not clear. Billing et al. (2018) concluded that within colonic L-cells, INSL5 is largely co-stored with both PYY and GLP-1 and upon L-cell stimulation, all three hormones are co-secreted in vesicular pools, but the conditions driving differential expression of the hormones along the GI-tract remain unclear (Billing et al. 2018).

The results from Ang et al. (2018) indicate that in the presence of nutrients, chronic INSL5 stimulation may increase GLP-1 secretion by heterologous sensitization of adenylyl cyclase and/or the activation of ERK1/2 and Akt signalling pathways downstream of RXFP4 (Ang et al. 2018). Acute INSL5 treatment, however, inhibits GLP-1 secretion by coupling to Gi/o proteins and decreasing cAMP. Together, these results provide important insight into future studies of INSL5, specifically to further understand the downstream pathways and crosstalk within the INSL5–RXFP4 system under various conditions of energy availability (Ang et al. 2018).

At least three studies, Grosse et al. (2014), Luo et al. (2015), and Ang et al. (2018) provide evidence that there is a relationship between INSL5 and GLP-1, but the mechanism(s) underlying their association requires further research. Studies indicate that the influence of INSL5 on metabolism is differentially affected by acute vs chronically sustained INSL5 levels, BMI, gender, disease state, and diet composition. Under conditions of high energy availability, both colonic INSL5 and GLP-1 appear to work in concert to increase insulin secretion, thus, protecting the body from prolonged nutrient excess. When deprived of nutrients either by CR or depletion of the gut microbiota, serum levels of INSL5 increase, potentially to promote an increase energy availability through gluconeogenesis, act as an orexigenic hormone, and/or favour beneficial microbial colonization, that is short-chain fatty acid producers, which in turn would aid in increasing energy availability.

If INSL5 increases GLP-1 secretion following chronic exposure as suggested in vitro, this could help explain the interaction of INSL5 with metabolic syndrome and insulin resistance (Ang et al. 2017, 2018). Increased GLP-1 under times of nutrient excess within the upper GI tract slows intestinal transit, increases absorption, promotes adipogenesis, and increases insulin secretion. Chronic elevation of INSL5, which may contribute to a chronic increase in GLP-1 secretion, could lead to sustained insulin secretion (Luo et al. 2015). Although the assays for INSL5 remain to be fully reliable (Kay et al. 2017), Bicer et al. (2019) proposed that PCOS patients develop higher levels of circulating INSL5, which in turn may contribute to the development of insulin resistance and metabolic syndrome (Bicer et al. 2019).

INSL5–RXFP4 on metabolism in transgenic mice

INSL5’s effect on glucose levels has been probed by several studies using a mouse model (summarized in Table 2). Burnicka-Turek et al. (2012) first reported on blood glucose levels in an Insl5–/– knockout mice on a 129/Sv background. They observed no significant body weight or composition differences between Insl5–/–and WT mice, but glucose tolerance in Insl5–/– mice worsened with age. They attributed the reduced insulin secretion and increased blood glucose of older cohorts to reduced pancreatic beta-cell mass as Insl5–/– mice had 1.6-fold smaller mean islet area compared to WT and no differences in GLP-1 levels before or after an i.p. glucose tolerance test (IPGTT) (Burnicka-Turek et al. 2012). These results are contrary to those found in cell culture models (Luo et al. 2015, Ang et al. 2018), perhaps due to in vitro vs in vivo differences and/or mode of nutrient administration.

Lee et al. (2016) used Insl5–/– mice on a C57BL/6 background and performed glucose tolerance tests to assess the impact of INSL5 on in vivo glucose homeostasis. Glucose tolerance following IPGTT was impaired in the Insl5–/– mice and the mice had improved insulin tolerance (Lee et al. 2016), leading the authors to hypothesize that the Insl5–/– mice had exhibited counter-insulin responses (glucagon, catecholamines, etc.). They also found a small difference in the pyruvate tolerance test between WT and Insl5–/– mice indicating a potential reduction in hepatic gluconeogenesis, since reduced glycogen stores were found in the livers of Insl5–/– mice. It is possible that reduced hepatic gluconeogenesis in combination with reduced glycogen levels and glycogenolysis may explain the delay in counter-regulatory responses and therefore result in improved insulin sensitivity (Lee et al. 2016). Lee and colleagues proposed that INSL5 is a low-energy sensor and regulator of hepatic glucose production under times of CR that helps mediate increases in energy availability. In particular, they proposed that colonic L-cells respond to decreased energy by increasing GLP-1 to slow intestinal transit and increase absorption (Wichmann et al. 2013) and secrete INSL5 to increase hepatic glucose production (Lee et al. 2016). It remains to be determined whether INSL5 can directly impact hepatic glucose production in humans.

In Rxfp4–/– mice, no glucose intolerance was observed (Grosse et al. 2014), although a C57Bl/6 Rxpf4–/– available from Jackson laboratory is catalogued as having an elevated fasting glucose phenotype (http://www.informatics.jax.org/allele/key/844491). In WT mice, Burnicka-Turek et al. (2012) demonstrated the expression of RXPF4 in pancreas, whereas no RXFP4 was previously reported in human whole pancreatic islets (Eizirik et al. 2012). Our analyses of human RNA expression database (gtexportal.org) and the protein atlas (proteinatlas.org) indicate that RXFP4 is expressed in human pancreas (median transcripts per million (TPM) 0.045), pancreatic endocrine, and exocrine glandular cells, albeit at similarly low levels (~0.1TPM) (Nica et al. 2013).

Luo et al. (2015) utilized C57BL/6 and diabetic db/db mice to investigate the effect of INSL5 on blood glucose levels and plasma levels of insulin and active GLP-1. They found that blood glucose in both WT and diabetic mice decreased significantly in a dose-dependent manner following i.p. injection of INSL5. Plasma insulin levels after an oral glucose tolerance test were significantly higher 5 and 10 min after i.p. injection, while levels of active GLP-1 were not significantly different between mice injected with INSL5 or control (Luo et al. 2015). Thus, INSL5 may lower blood glucose levels by increasing insulin secretion either directly or indirectly.

INSL5–RXFP4 on energy homeostasis

Summarized in Box 2. Grosse et al. (2014) reported that INSL5 was upregulated under conditions of CR and that INSL5 is produced in the colon by enteroendocrine L-cells, near RXFP4-positive enteric vagal nerve endings. Levels of INSL5 were suppressed following refeeding in C57BL/6J mice given ad libitum chow following an overnight fast. CR (60% of ad libitum) mice exhibited a significant increase in fasting INSL5 which lowered after refeeding. In HFD (45% fat) fed mice, no significant difference in INSL5 concentrations were found during the fasted state or a HFD refeed. Interestingly, after a prolonged CR of 10 weeks, INSL5 levels remained high after refeeding, suggesting a difference in INSL5 response following acute and chronic fasting (Grosse et al. 2014), indicating the potential role INSL5-RXFP4 may have in influencing long-term energy imbalance.

An analysis of enteroendocrine hormone gene expression in the jejunum, ileum, and transverse colon from mice fed HFD or CR for 4 weeks revealed that levels of Insl5 were elevated in the colon of CR mice. Gcg expression was also increased in the CR group. Thus, dietary interventions can impact mRNA levels of both Insl5 and Gcg within the colon (Grosse et al. 2014). Injection of exogenous INSL5 increased food intake in a dose-dependent manner, but Rxfp4−/− mice had ablated orexigenic behaviour, exhibited shorter mealtimes, and lost the preference for an HFD. Rxfp4−/− mice also exhibited no difference in glucose metabolism or insulin levels but had lower fat mass after a chow diet for an 11-week period (Grosse et al. 2014). These results indicate that at times of CR, or low energy state within the colon, Insl5 levels increase which may contribute to changes in energy intake. INSL5 may not only have orexigenic effects but may also increase the preference for energy-dense foods (i.e. HFD), as suggested by the meal differences of Rxfp4−/− mice (Grosse et al. 2014).

There have been differing results reported on the role of INSL5 on appetite and other known appetite regulators (specifically leptin and ghrelin); these may play a larger and more significant role in energy intake. Zaykov et al. (2019) chemically synthesized INSL5 and examined its orexigenic properties and impact on glucose homeostasis. They found no significant difference in food intake, body weight, or glucose tolerance in healthy, lean mice exposed to chronic INSL5 administration (1-week period of daily s.c. injections at 10, 50, 300 nmol/kg). Within diet-induced obese mice, acute INSL5 treatment failed to significantly improve glucose tolerance, but chronic treatment slightly improved glucose tolerance, although no dose–response relationship was found (Zaykov et al. 2019). These results are contrary to many of the previous findings of INSL5 (Burnicka-Turek et al. 2012, Grosse et al. 2014, Luo et al. 2015, Lee et al. 2016, Ang et al. 2017, 2018). The lack of an effect of INSL5 on glucose metabolism in these studies may be attributed to the stability of the INSL5 peptide; specifically, the Zaykov et al. (2019) study employed a novel peptide that exhibited high integrity ex vivo, but its stability in vivo was not demonstrated (Zaykov et al. 2019).

A major question is why and how colonic L-cells respond to energy levels since they do not normally experience large fluctuations in nutrient exposure. The data presented by Grosse et al. (2014) suggest that L-cells in the colon may signal long-term measures of energy balance and are not stimulated by nutrient levels. Given that there was no effect of food intake on intracerebroventricular INSL5 and that the expression of Rxfp4 was found within the enteric plexus and nodose ganglia and not within the hypothalamus suggest that the orexigenic properties of INSL5 may be initiated peripherally and transmitted by afferent sensory nerves to the CNS (Grosse et al. 2014).

Probing the role of the L-cells in the distal colon on energy levels Lewis et al. (2020) found positive metabolic outcomes following the stimulation of murine colonic L-cells using an Insl5 promotor-driven Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) system. Intraperitoneal treatment with clozapine N-oxide (CNO), a ligand for DREAD-Dq, in fasted mice resulted in a significant increase of GLP-1 (2.7-fold) and PYY (3.3-fold) in plasma compared to mice receiving vehicle stimulation. INSL5 levels were measurable in vitro but undetectable in plasma. Mice receiving L-cell stimulation exhibited improved glucose tolerance following IPGTT but reduced food intake; inhibition of the GLP-1 receptor reversed glucose tolerance but did not restore food intake. This demonstrates that direct murine colonic L-cell stimulation improves glucose tolerance via GLP-1, but its inhibitory effects on food intake are presumably mediated via a GLP-1 independent pathway (Lewis et al. 2020).

The inhibition of food intake after distal colon L-cell stimulation in healthy mice is an important insight. Satiety signals originating from L-cells may be mediated by increased expression of PYY following L-cell stimulation; PYY acting via its’ cognate receptor, neuropeptide Y2 receptor (Y2R), is known to be anorexigenic. Supporting this link, CNO administration following pre-treatment with a selective blood–brain barrier penetrating small molecule antagonist of the Y2R, JNJ-31020028, resulted in a significant increase in food intake, potentially explained via the previously reported orexigenic action of INSL5-RXFP4. Given that INSL5 is co-released with PYY and GLP-1, Lewis et al. (2020) suggest that the orexigenic activity of INSL5 may be insignificant as the anorexigenic action of PYY appeared to override the orexigenic effect of INSL5-RXFP4. Although reasonable, the conditions favouring physiological action of INSL5 within and across species as well as in compromised metabolic states (Li et al. 2020), dietary manipulations (Grosse et al. 2014, Lee et al. 2016), and/or shifts in the microbiome (Lee et al. 2016) warrant further investigation. Perhaps, under most physiological conditions, the orexigenic effects of INSL5 are masked by dominant satiety signals such as those mediated through PYY, cholecystokinin, amylin, GLP-1, and other nutrient and distension signals (Wichmann et al. 2013, Dinan & Cryan 2017, Bonaz et al. 2018).

In a follow-up study by Lewis et al. (2021), the authors detected Rxpf4 expression within the CNS specifically within nuclei which have previously been implicated in food intake control. To mimic the INSL5–RXFP4 signalling, Gi-coupled DREADD (Di) was utilized and resulted in an increase in highly palatable food intake. Activation of Gq-coupled DREADD (Dq), however, like their previous study resulted in reduced intake of highly palatable meals. Similar results were found when the ventromedial hypothalamic Rxfp4-expressing neurons were selectively targeted. In addition, the ventromedial hypothalamic population was further characterized by single-cell RNA sequencing and projection mapping; together the authors propose RXFP4 as a potential target for manipulation of food preference. This study is not without limitations, although the proposition and demonstration that INSL5–RXFP4 may function via the CNS and modulate food preference rather than overall energy homeostasis are interesting findings (Lewis et al. 2021), and consistent with genome wide association study (GWAS) results that indicate that genetic variants in or neighbouring RXFP4 influence BMI in humans (c.f. GWAS catalogue, six studies have identified genetic variants linked to RXFP4 that influence BMI, https://www.ebi.ac.uk/gwas/genes/RXFP4).

Despite observations and epidemiological data linking intestinal inflammation, neuropathophysiology, and behavioural effects, the functional pathways which affect this complex relationship are not well understood. Several hypotheses have been proposed including; imbalance of microbes eliciting pro- and anti-inflammatory responses; barrier dysfunction leading to systemic inflammation; influence of microbial amyloids on host amyloidosis; and age-related defects in immune function (see review by Fung 2020). Given the previously proposed actions of INSL5–RXFP4, the potential role of INSL5–RXFP4 in mediating behavioural responses to changes in the gut microbiome-immune response requires further study.

INSL5 has been shown to have insulinotropic effects (Burnicka-Turek et al. 2012, Luo et al. 2015) but may also decrease insulin secretion (Lee et al. 2016, Ang et al. 2017, 2018). A possible resolution to these apparently contradictory results is that INSL5–RXFP4 may serve as a protective energy sensor and modulate the physiological response of the organism depending on the overall state of the system (healthy or unhealthy) and/or energy availability. For example, Vahkal et al. (2021) i.p. injected calorie-restricted C57/Bl6 mice with INSL5 and found higher levels of serum GLP-1, GIP, PP and PYY 12, and 24 h post-injection (Vahkal et al. 2021). This is in agreement with previous results indicating the role that INSL5 may play in glucose metabolism and hepatic glucose production (Burnicka-Turek et al. 2012, Luo et al. 2015, Lee et al. 2016) as well as complementing those by Luo et al. (2015) who reported an increase in GLP-1 secretion using a murine L-cell model GLUTag cell line by stimulating ERK1/2 phosphorylation. In addition, the location of expression of RXFP4, throughout the GI tract as well as in a number of mucin 2 (MUC2)-expressing, goblet-like cell human colorectal carcinoma cell lines (Mouradov et al. 2014, Ang et al. 2018) together supports the hypothesis that multiple gut hormones, including INSL5, may interact in an autocrine/paracrine manner (Mashima et al. 2013, Thanasupawat et al. 2013, Ang et al. 2018, Billing et al. 2018) and play a role in energy homeostasis.

Another clue for the hypothesis that INSL5–RXFP4 play a role in energy homeostasis is the repeated loss of INSL5–RXFP4 found in obligate carnivores compared to obligate herbivores (Hecker et al. 2019). Three aspects of dietary specialization can explain the loss of INSL5–RXFP4 in carnivores – dietary composition, feeding patterns, and gut microbiome diversity (Hecker et al. 2019). Carnivores feed at irregular intervals compared to herbivores given accessibility and nutrient density. Their diets are rich in protein and fat, whereas herbivore diets are rich in carbohydrates. Thus, the multiple independent losses of INSL5–RXFP4 in carnivorous lineages suggest that it contributes significantly to carbohydrate digestion, potentially via a role as an orexigenic hormone, a blood glucose regulator (Hecker et al. 2019), or potentially via digestion of SCFA (such as butyrate) which is a byproduct of microbial digestion of fibre that plays roles in appetite and blood glucose regulation.

Ben-Dor et al. (2021) utilized multidisciplinary methods to reconstruct the human trophic level during the Pleistocene (2,580,000–11,700 years ago) which form the basis of many explanations regarding human evolution, behaviour, and culture. In contrast to most of the reconstructions to date that state the trophic level of 20th century hunter gatherers is flexible (regarding plant and animal-sourced foods), Ben-Dor et al. (2021) reviewed evidence of biological, ecological, and behavioural systems and their impact on the human trophic level. They identified 25 sources of evidence that together illustrate the trophic level of the Homo lineage that likely led to modern humans, evolved from a low base to high carnivorous position during the Pleistocene. This critical evolutionary data, specifically, how dietary intake affects INSL5–RXFP4 and the loss of the pair in obligate carnivores, complement the previously discussed reports in cell culture and animal models on INSL5–RXFP4 and energy homeostasis. The average human diet, within the last century and foreseeable future, is not obligate carnivorous. Thus, the role of the hormone–receptor pair may be even more relevant and/or modifiable with dietary intake.

INSL5–RXFP4 in human clinical trials

Summarized in Table 3. Wagner et al. (2016) investigated associations between circulating levels of INSL5 and metabolic and hormonal variables within lean and obese men and women, as well as INSL5 levels after bariatric surgery. They observed gender-specific differences in circulating INSL5, with highest levels in lean women (between 30 and 35 ng/mL) (Wagner et al. 2016). Their data indicate that the physiological serum concentrations of INSL5 may be significantly higher than those reported for other gut peptides (Wagner et al. 2016) and could point to a more systemic role for INSL5–RXFP4 than other gut peptides. Wagner et al. (2016), Bicer et al. (2019) and Li et al. (2020) report different ranges of circulating INSL5 using three different assays. Clinical data should be reviewed cautiously for both comparison groups – experimental and control – due to lack of validation of INSL5 assays (Kay et al. 2017).

Several studies, including that of Wagner et al. (2016), indicate that INSL5–RXFP4 may interact with male steroid hormones and/or influence male fertility. RXFP4 expression is observed in the gonads of mice (Burnicka-Turek et al. 2012) and humans (https://www.gtexportal.org/home/gene/RXFP4); notably, RXFP4 expression was found in the neck and middle portion of human sperm and is hypothesized to play a role in sperm motility by enhancing mitochondrial function by attenuation of reactive oxygen species (ROS) (Yeganeh et al. 2017). These results complement those of Burnicka-Turek et al. (2012) who found that male Insl5−/−mice had impaired fertility, in particular low sperm motility in male and a disrupted oestrus cycle in female mice. Wagner et al. (2016) found that serum levels of INSL5 were negatively correlated with serum testosterone and blood lipids in obese men prior to but not after bariatric surgery. In contrast, serum INSL5 levels had no significant correlations with metabolic or hormonal values within obese or lean women. However, in obese women, INSL5 levels were found to be negatively correlated with CRP, potentially indicating a negative role of low-grade inflammation in INSL5 biosynthesis.

Bicer et al. (2019) investigated the relationship between INSL5 and diverse metabolic and hormonal parameters in women with polycystic ovarian syndrome (PCOS). They performed a randomized, control trial on 82 women with PCOS and 82 controls and measured a broad range of anthropometric traits and metabolic parameters including the homeostasis model assessment of insulin resistance (HOMA-IR) and used a commercially available ELISA to measure serum INSL5. They found that individuals with the highest levels of INSL5 (26.49–48.46 ng/mL), a range elevated from controls compared to circulating levels observed by Wagner et al. (2016), had significantly higher odds (OR = 2.26, 95% CI = 1.87–2.73) of having PCOS compared to those with lower circulating levels (8.57–19.57 ng/mL). They calculated that INSL5 levels greater than 25.10 ng/mL were an optimal cut-off value of INSL5 for predicting PCOS, albeit with moderate sensitivity (67%) and specificity (69%). In addition to the association between INSL5 and PCOS, they observed an independent association between INSL5 and HOMA-IR (Bicer et al. 2019). The authors state that increased levels of INSL5 may contribute to the development of insulin resistance within PCOS subjects, but the higher levels of circulating INSL5 were not significantly correlated with lipid profiles, a 2-h oral glucose tolerance test, or HbAlc A1C (a long-term measure of blood glucose). Further evidence for a relationship between INSL5 and IR is given by their finding that circulating INSL5 levels were significantly higher in overweight compared to lean women in both the control and treatment groups.

In contrast to the results of Wagner et al. (2016), Bicer et al. (2019) found a positive correlation between INSL5 and high sensitivity C-reactive protein (hs-CRP) in PCOS patients. Subject differences used in these studies may help explain these results. Obese females did not appear to have large differences in metabolic changes within the Wagner et al. (2016) study, whereas participants in Bicer et al. (2019) were insulin resistant. Despite the strong association of elevated INSL5 in women with PCOS, no associations between circulating INSL5 and ovarian follicular number or volume were found, thus it was concluded that INSL5 did not have a direct effect on the development of ovarian follicles in women with PCOS (Bicer et al. 2019). Taken together, these studies infer that BMI, gender, and disease states and/or diet composition may influence serum levels of INSL5 that may interact with hormonal, metabolic, and inflammatory parameters. This clinical data should be reviewed cautiously as the comparison groups and INSL5 assays employed are neither constant between studies nor accepted to be fully reliable (Kay et al. 2017).

INSL5 as a disease marker

Summarized in Box 3. A variety of studies have implicated INSL5 in disease states. Thanasupawat et al. (2013) identified INSL5 as a novel marker of an EEC population within the mucosa of the colon. Immunoreactive INSL5 was expressed by EECs located within the colonic mucosa, while RXFP4 was found expressed in colonocytes. Both INSL5 and RXFP4 were detected in human neuroendocrine/carcinoid tissues. These results provide much of the foundation for the potential autocrine/paracrine action of INSL5–RXFP4 within the colon as well as the presence of INSL5 and RXFP4 in carcinoid tumours (Thanasupawat et al. 2013). Within animal models of colitis (Oshima et al. 1999, O’Hara et al. 2004) and Crohn’s disease (Bishop et al. 1987), EECs levels can increase. Using two mouse models of inflammatory bowel disease (IBD), Thanasupawat et al. (2013) demonstrated that acute IBD did not result in a change in the number of INSL5-positive EECs at the inflammatory sites, suggesting that the number of INSL5 immunopositive EECs within the mucosal layer did not change during the acute phase of inflammation (Thanasupawat et al. 2013). Additionally, no difference was found between chemically induced colitis severity and mucosal healing in Insl5−/−vs WT mice (Mashima et al. 2013).

If INSL5 and/or RXFP4 play roles in metabolic or intestinal disease, associations between INSL5 and/or RXFP4 expression in healthy compared to diseased individuals could be observed. Vahkal et al. (2021) retrieved RNA-seq data from the EMBL-EBI expression atlas and identified 26 and 25 experiments that identified significant changes in Insl5 and Rxfp4, respectively. Twenty-two of these studies highlighted significant changes in gene expression associated with various cancers, while five found decreased expression of Insl5 or Rxfp4 in association with metabolic disorders, including Crohn’s disease and ulcerative colitis (Vahkal et al. 2021).

Skok et al. (2021) recently provided an expression profile of cytokines in ulcerative colitis (UC) and Crohn’s disease (CD) using a bioinformatics approach with experimental validation of expression of the selected genes. Seven microarray datasets (4 UC and 3 CD) were obtained from GEO. Active UC samples were selected and included in the analysis for a total of 47 UC, 226 CD, and 247 normal tissues. The authors identified 201 significant differentially expressed cytokine-encoding genes within UC and 36 in CD samples. A total of 28 genes were differentially expressed common to both UC and CD compared to normal tissue. Ten of these DEGs were selected for further study based on an inverse regulation between the UC and CD groups. One of these was INSL5 which exhibited the largest difference in logFC among UC and CD (delta logFC = 4.49) and was upregulated in UC and downregulated in CD. The ten DEGs were validated using quantitative real-time PCR in 27 samples of CD, 35 samples of UC, and 22 samples of normal mucosa collected from patients during routine ileocolonoscopies. The results did not confirm the difference in expression between CD and UC but found that INSL5 was downregulated in both CD and UC when compared to control tissues and significantly so in UC (logFC = −2.26, P  = 0.048 and logFC = −2.05, P  = 0.008, respectively). Given the expression location of INSL5 within the colon, it is reasonable that INSL5 may be a significant marker of UC and/or play a role in colonic inflammation (Skok et al. 2021). The results of this study in addition to the RNA-seq data analysed by Vahkal et al. (2021) support INSL5 as a PES. Chronic intestinal inflammation within IBD is often and broadly attributed to the failure of anti-inflammatory mechanisms to suppress pro-inflammatory immune responses (Yeshi et al. 2020). Given the data surrounding the physiological roles of INSL5, specifically the location as well as the anti-inflammatory role found by Vahkal et al. (2021), the downregulation of INSL5 within IBD supports our proposed peptide hormone as a PES.

Given the co-location of INSL5 and RXPF4 in the colorectal epithelium, it is conceivable that the hormone−receptor pair plays a role in rectal neuroendocrine tumors (NETs). Mashima et al. (2013) found that all examined NETs expressed both INSL5 and RXFP4, but INSL5 was not found to be necessary for the development of chromogranin A-positive EECs or the general structure of the colonic epithelium (Mashima et al. 2013). Notably, other members of the relaxin family have been shown to mediate growth and metastasis of human cancers (Klonisch et al. 2007, Feng et al. 2010, Hombach-Klonisch et al. 2010, Vinall et al. 2011, Li et al. 2020). Consistently, elevated INSL5 levels could contribute to the pathology of colorectal NETs via the mammalian/mechanistic target of rapamycin (mTOR) pathway (Klonisch et al. 2007, Feng et al. 2010, Hombach-Klonisch et al. 2010, Vinall et al. 2011, Mashima et al. 2013). In patients with advanced pancreatic NETs, inhibition of the mTOR pathway with everolimus and/or Sunitinib has been shown to prolong progression-free survival (Raymond et al. 2011, Yao et al. 2011), indicating that INSL5 and its downstream pathways may have an autocrine/paracrine role in the colorectal epithelium and be a unique marker of colorectal EECs and rectal neuroendocrine tumors (Mashima et al. 2013).

Due to the increased energy requirements for growth and division, cancer cells often shift to the strict use of glucose via glycolysis, known as aerobic glycolysis or the Warburg effect (Hsu & Sabatini 2008). Given the evidence that INSL5 plays roles in glucose homeostasis and is proposed to be a marker of neuroendocrine tumors (Burnicka-Turek et al. 2012, Luo et al. 2015, Lee et al. 2016, Ang et al. 2018), Li et al. (2020) examined the hypothesis that INSL5 plays a role in metabolic reprogramming in individuals with nasopharyngeal carcinoma (NPC). Following extensive experimental analyses, they find support for the hypothesis that INSL5 is a biomarker for NPC and that it exerts this effect through a direct role in metabolic reprogramming of NPC cancer cells to increase rates of glycolysis by activating the RXFP4/STAT5 axis (Li et al. 2020).

Nuclear translocation and activation of STAT5 are a proposed mechanism by which INSL5 promotes gene expression of glycolytic enzymes (hexokinase 2, transporter Glut3, and phosphofructokinase 1) and results in NPC metabolic reprogram (Li et al. 2020). The authors also demonstrated that INSL5 increased the phosphorylation of Akt, ERK1/2, and JAK1, and in cells treated with JAK1 and ERK1/2 inhibitors, STAT5 activation was reversed. Thus, JAK and ERK1/2 activation downstream of RXFP4 may lead to STAT5 signalling (Li et al. 2020). Extensive research on STAT5 and its activation in various cancers has been conducted (Cotarla et al. 2004), wherein growth factors, JAKs, SRC family kinases, and other tyrosine kinases can activate STAT5 (Levy & Darnell 2002).

Concluding remarks

The pathophysiology of many chronic diseases, including gastrointestinal, neurodegenerative, and mood disorders, involves disturbances in the microbiota−gut−brain axis (Bonaz & Bernstein 2013, Cenit et al. 2017). In addition, dysregulation of the stress response via the HPA axis can have a significant impact on the microbiota−gut−brain axis (Dinan & Cryan 2017). The close relationship between metabolism and the immune system is supported by mounting evidence that obesity-related low-grade inflammation leads to a variety of metabolic syndromes (Parekh et al. 2016). Increased levels of inflammation in the gut, caused by a multitude of factors, are associated with a host of autoimmune gastrointestinal disorders (Izcue et al. 2009, Round & Mazmanian 2009, Campbell 2014). Intriguingly, at least two other gut hormones, ghrelin and GLP-1, are showing promise as disease therapies for such diseases: GLP-1 agonists are being used as a treatment for type 2 diabetes (Barrera et al. 2011); ghrelin is a treatment for gastrointestinal disorders such as Celiac disease, and IBD because of its anti-inflammatory effects (Prodam & Filigheddu 2014). Many of the summarized results presented here suggest that INSL5 may play similar roles either via autocrine/paracrine and/or endocrine signalling, wherein systemic signalling could be possible via the vagus nerve.

It remains to be validated whether INSL5−RXFP4 has an impact on human metabolism. Liu & Lovenberg (2008) discussed the differences between the rodent and human RXFP4 gene. It is a pseudogene in rats (which are carnivores) and has a 74% identity at the amino acid level including differences in C-terminus in mice compared to humans. These findings reiterate the importance of using other mammalian species which share higher sequence identity with humans (97% monkey, 85% cow, and 87% pig) for future functional studies (Liu & Lovenberg 2008). In addition, it is widely accepted that RXFP4 is mainly activated by INSL5. However, as mentioned, relaxin-3/INSL7 is also capable of activating RXFP4 (Liu et al. 2003a). Thus, the difference observed within Insl5−/− knockout mice may be due to compensatory effect of RXFP4 activation. As seen in other relaxin-family knockout models, the inability to identify altered phenotype in knockout studies has been attributed to the compensation by other INSL factors (Mashima et al. 2013).

In summary, the physiological role(s) of INSL5–RXFP4 remain to be fully elaborated. Given the available evidence, it is conceivable that the signalling pathways of INSL5–RXFP4 and the way in which they act as a protective energy sensor are multi-layered but centred around modulating the microbiota–gut–brain axis. Research focused on the conflicting results surrounding insulin secretion, whether INSL5 can directly impact hepatic glucose production in humans and perhaps most significantly the potential relationship and interaction with other gut hormones, specifically GLP-1, may prove to provide important insights into the role of INSL5–RXFP4. Further investigation and clarification of these proposed roles of INSL5–RXFP4 may prove a greater physiological relevance for the pair and add to existing evidence of INSL5–RXFP4 role as a protective energy sensor (Fig. 2).

Figure 2
Figure 2

Further proposed actions of INSL5–RXFP4 as a protective energy sensor (PES). (A) INSL5 may activate RXFP4 on the vagus nerve (VN) which in turn may have behavioural impacts (orexigenic, intake of calorie-dense foods). (B) INSL5 may stimulate nearby cells expressing RXFP4 in a paracrine manner thereby impacting local cell metabolism. (C) INSL5 may stimulate RXFP4 located on dendritic cells, impacting immune cell function (pro/anti-inflammatory signals) and/or cell differentiation. (D) INSL5 may stimulate RXFP4 located on EECs and regulate GLP-1 secretion, thus having an indirect impact on insulin levels. (E) INSL5 may inhibit macrophage proliferation, thus reducing the immune response. (F) INSL5 may act in an endocrine fashion, for example, activate RXFP4 receptors in the pancreas, thus directly impacting insulin secretion. Together, these proposed actions reveal the possibility of INSL5–RXFP4 acting as a protective energy sensor (created using Biorender). A full colour version of this figure is available at https://doi.org/10.1530/JME-21-0241.

Citation: Journal of Molecular Endocrinology 69, 1; 10.1530/JME-21-0241

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This work was supported by the the Natureal Sciences and Engineering Research Council (NSERC) Discovery Grant (Grant number 06203 to S V G).

Acknowledgement

The authors would like to thank Ian McNicol for his comments on an earlier version of this manuscript.

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    INSL5–RXFP4. A protective energy sensor in the microbiome–gut–brain axis. INSL5 and RXFP4 are expressed in the distal gut and in central immune tissues, while RXFP4 is additionally expressed in a range of peripheral immune tissues and cell types particularly those belonging to the innate immune system, nodose, and dorsal root ganglia as well the vagus nerve, ventromedial hypothalamus, and microglial cells in the brain. There is evidence that the ligand–receptor pair plays roles in glucose metabolism, colorectal propulsion, food-seeking behaviour, and, more recently, the immune system. We propose a synthetic view of these diverse roles that revolve around their role in the microbiome–gut–brain axis in an endocrine and/or paracrine manner. Levels of INSL5 expression in the distal gut are affected by the abundance of gut microbes and short-chain fatty acids, incubation of murine macrophages with INSL5 resulted in a predominantly anti-inflammatory LPS-induced cytokine response, serum levels of INSL5 are associated with those of C-reactive Protein, a general marker for inflammation, in human clinical trials, and INSL5-RXFP4 are markers of colorectal endocrine cells, neuroendocrine tumors, breast cancer, and nasopharyngeal carcinoma. Collectively, this points to a hypothesis that INSL5 may influence communication via the microbiome–gut–brain axis in an endocrine and/or paracrine manner to act as a PES. (Created using Biorender). A full colour version of this figure is available at https://doi.org/10.1530/JME-21-0241.

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    Further proposed actions of INSL5–RXFP4 as a protective energy sensor (PES). (A) INSL5 may activate RXFP4 on the vagus nerve (VN) which in turn may have behavioural impacts (orexigenic, intake of calorie-dense foods). (B) INSL5 may stimulate nearby cells expressing RXFP4 in a paracrine manner thereby impacting local cell metabolism. (C) INSL5 may stimulate RXFP4 located on dendritic cells, impacting immune cell function (pro/anti-inflammatory signals) and/or cell differentiation. (D) INSL5 may stimulate RXFP4 located on EECs and regulate GLP-1 secretion, thus having an indirect impact on insulin levels. (E) INSL5 may inhibit macrophage proliferation, thus reducing the immune response. (F) INSL5 may act in an endocrine fashion, for example, activate RXFP4 receptors in the pancreas, thus directly impacting insulin secretion. Together, these proposed actions reveal the possibility of INSL5–RXFP4 acting as a protective energy sensor (created using Biorender). A full colour version of this figure is available at https://doi.org/10.1530/JME-21-0241.

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