Insulin-like peptide 5 (INSL5), a member of the insulin/relaxin superfamily, can activate the G-protein-coupled receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), but its precise biological functions are largely unknown. Recent studies suggest that INSL5/RXFP4 is involved in the control of food intake and glucose homoeostasis. We report in the present study that RXFP4 is present in the mouse insulinoma cell line MIN6 and INSL5 augments glucose-stimulated insulin secretion (GSIS) both in vitro and in vivo. RXFP4 is also expressed in the mouse intestinal L-cell line GLUTag and INSL5 is capable of potentiating glucose-dependent glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. We propose that the insulinotrophic effect of INSL5 is probably mediated through stimulation of insulin/GLP-1 secretion and the INSL5/RXFP4 system may be a potential therapeutic target for Type 2 diabetes.
Type 2 diabetes mellitus (T2DM) is an increasingly prevalent metabolic disorder across the world, with dramatic consequences in terms of human health and economic burden [1–4]. Its cause is linked to a combination of insulin resistance and pancreatic β-cell dysfunction . A large volume of literature has shown that several G-protein-coupled receptors (GPCRs) expressed in the pancreatic islets are involved in the regulation of β-cell function [6–8].
Insulin-like peptide 5 (INSL5), also known as relaxin/insulin-like factor 2 (RIF2), is a member of the insulin superfamily and was identified from the Expressed Sequence Tag Database in 1999. Its mRNA is primarily found in the rectum, colon and uterus . INSL5 is the endogenous ligand of the relaxin family peptide receptor RXFP4 (also known as GPCR142 or GPR100), a class A Gi/o-linked GPCR [10,11]. Although the precise biological functions of the INSL5/RXFP4 system still need to be fully elucidated, two published patent applications (WO2005/124361A2 and US20080269118) claimed that the phenotype of the RXFP4-knockout mice points to a role for INSL5 in both glucose and fat metabolism. A murine study found that RXFP4 is expressed in β-cells and Insl5−/− mice exhibited impaired glucose tolerance at an advanced age, resulting from reduced insulin secretion . Recently, Grosse et al.  identified INSL5 as a product of colonic enteroendocrine L-cells. Thus, the INSL5/RXFP4 system may be a potential therapeutic target for T2DM and detailed investigations into its physiological roles and mechanisms of action are obviously warranted.
In the present study, we report that INSL5 increases glucose-dependent insulin secretion in vitro and in vivo. In addition, RXFP4 mRNA is expressed in the mouse intestinal L-cell line GLUTag and INSL5 directly potentiates glucose-stimulated glucagon-like peptide-1 (GLP-1) release via this cell line. These results suggest that the insulinotrophic effect exerted by INSL5 may be mediated via two pathways in parallel: directly through RXFP4 in pancreatic β-cells and indirectly by promoting GLP-1 secretion.
Animal experimentation was conducted in accordance with the regulations adopted by the Animal Care and Use Committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Approval No.: 2013-SIMM-06). Male C57BL/6J and male db/db mice were housed at 22.7±0.8°C under a 12-h light–dark cycle. At 6–8 weeks of age, they were randomly assigned to acute treatments.
The INSL5, protein with a six-residue N-terminal extension of the A-chain (Mr=5632 Da, UniProt No. Q9Y5Q6), used in this study was prepared according to a procedure developed by Luo et al. .
Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 4.5 g/l of glucose, 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in 5% CO2. MIN6 cells were maintained in DMEM containing 15% FBS, 4.5 g/l of glucose, 55 μM 2-mercaptoethanol, 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in 5% CO2. GLUTag cells were maintained in DMEM containing 10% FBS, 1 g/l of glucose, 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in 5% CO2.
Receptor activation assays (at Tongji University), using HEK293 cells transiently transfected with human (NM_181885) or mouse (NM_181817) RXFP4 (pCMV/RXFP4), and a cAMP-response element (CRE)-controlled nanoluciferase reporter gene construct (pNL1.2/CRE), were performed as described by Wang et al. .
RNA extraction and real-time PCR
Cellular RNA was extracted using the reagent TRIzol (Life Technologies) according to the manufacturer's instructions. cDNA was prepared using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) and reverse transcription (RT)-PCR was performed using SYBR Green Master Mix (DRR820A; Takara Bio) on an ABI7300 system (Life Technologies). Changes in expression of mouse RXFP4 mRNA (forward 5′-CTGTCTTCTGCGTTTCCCCA-3′ and reverse 5′-GCTGTCCTGCCATTGTCGTG-3′; NM_181817) were calculated using differences in threshold cycle (CT) values compared with the housekeeping gene (β-actin: forward 5′-GAGACCTTCAACACCCCAGC-3′ and reverse 5′-ATGTCACGCACGATTTCCC-3′; NM_007393).
Activation of extracellular-signal-regulated kinase (ERK) in HEK293 cells after treatment with 100 nM INSL5 was determined according to the procedure of Nanamori et al. . In brief, cells were cultured in 12-well plates and serum starved for 2 h before INSL5 stimulation in DMEM containing 0.5% fatty acid-free BSA. Total cell extracts were prepared and analysed by SDS/PAGE and Western blotting using anti-ERK1/2 antibody at 1:1000 dilution and anti-phospho-ERK1/2 antibody at 1:2000 dilution (Cell Signaling Technology).
The relaxin family peptide receptor 4 (RXFP4) and the G-protein Gα16 were co-transfected into HEK293 cells, followed by analysis of the intracellular Ca2+ concentration [Ca2+]i. Cells were seeded at a density of 3×104 cells/well on 96-well plates in the medium 24 h before assaying. Mobilization of Ca2+ was monitored essentially as described by Zhang et al. .
MIN6 insulinoma cells were plated in 96-well plates (5×105 cells per well) for assay of insulin release. On day 1 of the experiment, the culture medium was aspirated and cells were washed twice with Hepes-balanced Krebs/Ringer bicarbonate buffer (KRBB) containing 0.5% fatty acid-free BSA (KRBB buffer), and then starved at 37°C for 2 h in KRBB containing 2.8 mM glucose. After starvation, cells were incubated with INSL5 for 2 h at 37°C in KRBB buffer containing 2.8 or 16.8 mM glucose. Supernatant insulin levels were determined using an HTRF insulin assay kit (Cisbio Assays) and EnVision Multilabel Plate Reader (PerkinElmer) according to the manufacturers’ instructions.
An assay of insulin release in pancreatic islets isolated from male C57BL/6 mice (approximately 25 g body weight) was carried out as described by Li et al. . Briefly, pancreases were digested by collagenase V [0.8 mg/ml in Hanks Balanced Salt solution (HBSS); Sigma] and the islets were separated by hand picking under a microscope. Batches of islets were incubated in RPMI-1640 (Life Technologies) supplemented with 10% FBS at 5% CO2 and 37°C for 24 h. Thereafter, groups of five islets each were placed in incubation wells. After a 60-min preincubation in Hepes-buffered Krebs/Ringer buffer (pH 7.4) containing 0.5% BSA, islets were incubated with various concentrations of INSL5 at 5% CO2 and 37°C for 2 h. Supernatants were collected for insulin determination as described above.
GLUTag cells were plated in 24-well plates (2×105 cells per well) and culture medium was replaced with DMEM (containing 2.8 mM glucose) supplemented with 10% FBS 24 h before analysis of GLP-1 release. On the day of the experiment, cells were washed twice with PBS and incubated with INSL5 at the desired concentrations in serum-free DMEM and 16.8 mM glucose for 2 h at 37°C and 5% CO2. The supernatants were collected by centrifugation at 3000 g for 3 min. GLP-1 in the supernatant was detected by an HTRF GLP-1 assay kit (Cisbio Assays).
An intraperitoneal glucose tolerance test (IPGTT) was carried out in male C57BL/6 and db/db mice. Animals were fasted overnight and given either PBS (vehicle) or INSL5 (n=12 per treatment group) at various doses intraperitoneally. AR231453 (positive control, 20 mg/kg body weight; Abcam) was orally administrated 30 min before a glucose challenge [19,20]. An intraperitoneal D-glucose bolus was then delivered (2 g/kg body weight), plasma glucose levels were determined at desired time points over a 2-h period using blood collected from the tail vein, and plasma glucose levels were determined with a glucose meter.
In a separate experiment, male C57BL/6 mice were fasted overnight and their plasma insulin and active GLP-1 levels measured after a single intraperitoneal dose of INSL5 or PBS (vehicle) together with an oral load of D-glucose (2 g/kg body weight). Blood samples were collected from the retro-orbital vein and rapidly mixed with a DPP-IV inhibitor (DPP4-010, Millipore). Plasma samples were obtained via centrifugation at 3000 g for 15 min for insulin and active GLP-1 determination using ELISA kits (Millipore, EZRMI-13K and EGLP-35K).
Quantitative data are presented as means±S.E.M. from at least three independent experiments. Data were analysed using GraphPad Prism 5 software. Non-linear regression analysis was performed to generate dose–response curves and EC50 values. Two-tailed Student's t-tests were performed to analyse the data and P<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
INSL5 is equally efficacious to activate human and mouse RXFP4s
INSL5 bears an extended N-terminus of the A-chain which possesses full activity compared with the chemically synthesized peptide, as shown previously . To compare the activation pattern between species, we used transfected HEK293 cells expressing human or mouse RXFP4s (Figure 1A) to detect the inhibition of forskolin-stimulated cAMP production and induction of calcium mobilization by INSL5 respectively. Although the mouse RXFP4 gene shows a lower overall conservation (74% identity at the amino acid level) compared with the human gene , INSL5 was capable of suppressing cAMP accumulation (Figure 1B) and inducing cellular calcium influx (Figure 1C) in these two cell preparations, in a concentration-dependent manner and with similar efficacies (for cAMP accumulation, the negative logarithm of the EC50, pEC50=9.20±0.07 and 9.27±0.08 respectively; for calcium mobilization, pEC50=8.23±0.06 and 8.10±0.05 respectively).
INSL5 activates RXFP4-mediated signalling pathway
It is recognized that activation of extracellular ERK1 and ERK2 results from agonist stimulation of many GPCRs and plays an important role in numerous cellular functions . In the present study, we examined the effect of INSL5 on RXFP4 signalling with an ERK1/2 phosphorylation assay. HEK293 cells expressing either human or mouse RXFP4 responded positively to INSL5 stimulation in a time-dependent manner. In contrast, in HEK293 cells transfected with an empty vector (control), only the protein kinase C (PKC) activator PMA (Sigma) induced ERK1/2 phosphorylation (Figure 1D, left panel). On the other hand, after 2 h of treatment with 20 μM MEK (mitogen-activated protein kinase/ERK kinase) inhibitor PD98059, a (Sigma), the pERK1/2 expression level was down-regulated (Figure 1D, right-hand panel). These results indicate that INSL5 activates the RXFP4-mediated ERK signalling pathway.
INSL5 augments glucose-dependent insulin secretion in vitro
As reported earlier, INSL5 may have an effect on insulin secretion, on the basis of: (i) the initial observation of impaired glucose tolerance and exhibited abnormal islet morphology in aging Insl5−/− mice; and (ii) the existence of RXFP4 in mouse islets . Following this clue, we investigated the question of whether the INSL5/RXFP4 system has a direct impact on glucose homoeostasis. We first examined the expression of RXFP4 mRNA in mouse pancreas (results not shown) and insulinoma MIN6 cells using RT-PCR techniques: the size of the resultant PCR product (indicated by an arrow) corresponded to the expected size for RXFP4 (167 bp, Figure 2A). We then found that INSL5-induced ERK activation in MIN6 cells (Figure 2B) was similar to that seen in RXFP4-expressing HEK293 cells (see Figure 1D). Next, we studied the effect of INSL5 on insulin secretion. MIN6 cells were exposed to low or high glucose concentrations with or without INSL5 for 2 h to measure insulin content in the supernatant. As shown in Figure 2C, INSL5 had a limited effect on insulin secretion at low glucose levels but caused a significant increase at high ones (P<0.05), suggesting that INSL5 augmented glucose-stimulated insulin secretion (GSIS) from MIN6 cells.
Effects of INSL5 on insulin secretion in vitro
On the basis of the above data and the presence of RXFP4 in the islets of Langerhans [12,13], we used isolated mouse islets to examined the effect of INSL5 on GSIS. At high glucose concentrations (16.8 mM), INSL5 enhanced insulin release in mouse islets in a manner similar to GLP-1 (Figure 2D), whereas it had no impact on islets incubated in 2.8 mM glucose. The insulinotrophic effect of INSL5 on pancreatic islets is therefore glucose dependent.
INSL5 stimulates GLP-1 release by GLUTag cells
As INSL5 mRNA is primarily found in the rectum, colon and uterus, and INSL5 has recently been claimed as a peptidic hormone secreted by L-cells, better known for their secretion of GLP-1 and peptide YY [13,22], we were keen to learn whether there is a relationship between INSL5 and GLP-1. Thus, we investigated the RXFP4 mRNA in GLUTag cells using real-time PCR techniques: the size of the resultant PCR product (indicated by an arrow in the Figure) corresponded to the expected size for RXFP4 (167 bp, Figure 3A), similar to that shown in MIN6 cells (see Figure 2A). This was followed by the detection of INSL5-induced ERK activation in GLUTag cells (Figure 3B), an outcome similar to that seen in both RXFP4-expressing HEK293 and MIN6 cells (see Figures 1D and 2B). When GLUTag cells were treated with various concentrations of INSL5 in a medium containing 16.8 mM glucose for 2 h, the GLP-1 contents in the supernatant were significantly increased from the basal level and the phenomenon was dose dependent (Figure 3C), implying that INSL5 is able to stimulate GLP-1 secretion in GLUTag cells.
Effects of INSL5 on GLP-1 release in GLUTag cells
It is known that β-cell proliferation and insulin secretion are stimulated by intrinsic and extrinsic growth factors and hormones . Several lines of evidence revealed that intestinally produced GLP-1 regulates insulin secretion [8,19,20]. These are consistent with our finding that the mouse intestinal L-cell line GLUTag expresses RXFP4 and responds to INSL5 stimulation by secreting GLP-1. This observation is of particular importance in terms of both a potential autocrine mechanism and a newly found incretin role for INSL5.
INSL5 enhances GSIS and improves glucose tolerance in vivo
A glucose tolerance test is a conventional measure for insulin resistance and β-cell function . The insulinotrophic action of INSL5 observed in vitro was substantiated by our acute experiments in vivo. To evaluate a potential role of INSL5 in regulating glucose homoeostasis, IPGTT studies were performed in both male C57BL/6 and db/db mice after overnight fasting. It was found that the recombinant peptide INSL5 significantly and dose dependently reduced blood glucose concentrations in normal animals at 15, 30 and 60 min after a glucose load, compared with the vehicle control (Figure 4A), resulting in an overall reduction in the area under the curve (AUC0–120) for blood glucose (Figure 4B). Such a hypoglycaemic effect of INSL5 was more pronounced in diabetic mice: both the magnitude of glucose decline in every time point examined (Figure 4C) and the reduction of the absolute AUC0–120 relative to each dose (Figure 4D: −14% compared with −6% at 10 μg/kg body weight, −20% compared with −16% at 30 μg/kg body weight and −29% compared with −16% at 90 μg/kg body weight respectively) were larger than those seen in the control group, consistent with the view that INSL5 is a regulatory peptide and might be involved in glucose metabolism.
Acute glycaemic regulating effects of INSL5 in vivo
Plasma insulin and active GLP-1 levels were subsequently measured in overnight-fasted male C57BL/6 mice receiving intraperitoneal INSL5 or vehicle treatment together with an oral glucose challenge. As shown in Figure 5(A), animals treated with 270 μg/kg body weight of INSL5 had markedly elevated insulin levels 5 and 10 min after the procedure (P<0.05 and P<0.01, respectively), whereas simultaneous measurements of active GLP-1 did not show any statistically significant differences (Figure 5B). These observations not only confirm the direct insulinotrophic effect of INSL5 but also point to a potentially complex interaction of INSL5, insulin and GLP-1 in vivo. Examination of total GLP-1 in such an experimental setting is in progress and may help better understanding of the regulatory role of these hormones.
Plasma insulin and active GLP-1 levels in male C57BL/6 mice after a single intraperitoneal injection of INSL5 (270 μg/kg body weight)
In summary, it appears that INSL5 may exert its insulinotrophic action in two modes: directly through RXFP4 in pancreatic β-cells and indirectly by promoting incretin (GLP-1) secretion. Obviously, further studies in this direction are urgently required to substantiate these preliminary findings.
Dulbecco’s modified Eagle’s medium
glucose-stimulated insulin secretion
Human embryonic kidney 293
insulin-like peptide 5
intraperitoneal glucose tolerance test
Krebs/Ringer bicarbonate buffer
mitogen-activated protein kinase/ERK kinase
reverse transcription PCR
relaxin/insulin-like family peptide receptor 4
Type 2 diabetes mellitus
Xiao Luo, Zhan-Yun Guo and Ming-Wei Wang conceived and designed the experiments. Xiao Luo, Ting Li, Yue Zhu, Yunbin Dai, Jianwei Zhao performed the experiments. Xiao Luo and Ming-Wei Wang analysed the data. Zhan-Yun Guo contributed materials. Xiao Luo and Ming-Wei Wang wrote the paper.
We are indebted to Xiaoying Li for valuable discussions, to Shuyong Zhang and Xinyi Wang for technical assistance, and to Institut de Recherches Servier (Suresnes, France) for the GLUTag cell line.
This work was partially supported by grants from the National Health and Family Planning Commission of China [grant numbers 2012ZX09304-011, 2013ZX09401003-005, 2013ZX09507-001 and 2013ZX09507-002], Shanghai Science and Technology Development Fund [grant number 13DZ2290300] and the Thousand Talents Program in China.