GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22–51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22–51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22–51] into ApoE−/− mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22–51] antibody. GIP_HUMAN[22–51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22–51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22–51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22–51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.

exact native amino acid sequences in the peripheral circulation needs to be confirmed by mass spectrometry 20 . Despite long-term endeavours to elucidate the plasma proteome 11,21 , the comprehensive identification of plasma low-molecular weight native peptides has not been achieved until very recently 22 .
In a recent study, we established improved technology that improved the high-yield plasma extraction technique 22 , enabling the large-scale identification of plasma native peptides with mass spectrometry. We deposited the results into a newly generated human plasma native peptidomic database. We also synthesised peptides of identified sequences and validated their biological activity using cultured human cells. The present study aimed to use this plasma native peptidomic resource to identify a novel plasma native polypeptide hormone that may be involved in the pathophysiology of vascular diseases.

Results
In silico analysis of the mass spectrometry-identified human plasma native peptidomic resource. Our initial peptidomic sequencing data acquired from 189 analyses using liquid chromatography tandem mass spectrometry (LC-MS/MS) were searched against the SwissProt_2015_02.fasta database with two different data processing pipelines and search engines: (1) the Mascot Distiller (version 2.5.1.0, Matrix Science) deconvoluted the MS/MS spectra and performed an MS/MS ion search, and (2) the PEAKS Studio (version 7) used a PTM algorism and performed a database search based on a de novo sequencing. These analyses were conducted until the beginning of 2018 and after excluding peptides derived from the keratin protein family, they resulted in the identification of 18,552 polypeptide sequences with a peptide identification false discovery rate (FDR) of 1%. All these acquisition data were already deposited in the ProteomeXchange Consortium via the PRIDE 23 partner repository with the dataset identifier PXD003533. As described in the Methods section, we reanalysed these resources using the PEAKS Studio (version X), and a database search was performed against the SwissProt_2020_03.fasta with five variable PTMs in addition to the carbamidomethylation without using the PTM algorithm. This process resulted in the identification of 7,959 or 11,256 distinct native peptide sequences depending on FDRs of 0% or 1%, respectively 22 . We performed a bioinformatic analysis of this updated resource to select bioactive peptide candidates for chemical synthesis and functional validation using the criteria described in the Methods section. Synthesised peptides were tested for their high purity and liquid solubility using LC-MS/MS analysis and 135 peptides were confirmed to be appropriate for biological validation (Supplementary Table S1).
Searching for novel polypeptides that elicit cellular responses. Of the 135 synthetic peptides, we completed functional screening of 129 peptides in three types of cultured human cells, including the human monocytic leukaemia cells (THP1) induced to differentiate into macrophages, aortic endothelial cells (HAoECs), and aortic smooth muscle cells (HAoSMCs). We found that a 30-amino-acid peptide derived from GIP ( Fig. 1a,b), EKKEGHFSALPSLPVGSHAKVSSPQPRGPR (GIP_HUMAN , monoisotopic mass 3179.6951), caused an increase in intracellular free Ca 2+ levels ([Ca 2+ ] i ) in the THP-1-induced macrophages. The ability of GIP_HUMAN  to bind and elicit intracellular responses was examined, using the three types of cultured human cells. These cells were incubated with the peptide labelled with 5-carboxyfluorescein at the N-terminus (FAM-GIP_HUMAN ) to determine whether GIP_HUMAN  was bound to the intact cell surface. Confocal immunofluorescence microscopy revealed the presence of FAM-GIP_HUMAN  on the surface of the HAoECs (Fig. 2a) and THP1-derived macrophages (Fig. 2b), but not in the HAoSMCs. Of the remaining 128 peptides, two suprabasin-derived peptides elicited significant cellular responses in the HAoSMCs 22 . Three other peptides induced marginal increases in [Ca 2+ ] i in the HAoECs; however, their detailed biological activity has not subsequently been investigated.
Activation of NF-κB by GIP_HUMAN . The HAoECs and macrophage-differentiated THP1 cells were treated with GIP_HUMAN  for different periods and immunostained with an antibody against the p65 subunit of the NF-κB. GIP_HUMAN  increased the NF-κB immunoreactivity in the nuclei of both the HAoECs and THP1-derived macrophages within 60 min, indicating a nuclear translocation of the NF-κB (Fig. 2c,d). GIP_HUMAN  caused a rapid (within 5-15 min) and transient degradation of the IκB-α in the HAoECs and THP1-derived macrophages, which returned to baseline levels within 1 h (Fig. 2e,f). Next, we searched for genes upregulated by GIP_HUMAN  via NF-κB signalling in the HAoECs. GIP_ HUMAN  induced the expression of the matrix metalloproteinase-8 (MMP-8) gene in a time-and dosedependent manner (Fig. 2g,h). To determine the involvement of NF-κB signalling in the GIP_HUMAN -mediated MMP-8 gene expression, we pretreated the HAoECs with a proteasome inhibitor, MG132, and stimulated them over a time course with GIP_HUMAN . The inhibition of NF-κB by MG132 prevented the MMP-8 expression induced by GIP_HUMAN   (Fig. 2i). These results indicated the role of GIP_ HUMAN  in NF-κB activation in vascular endothelial cells and macrophages.

Effect of GIP_HUMAN[22-51] infusions on atherosclerotic lesion progression.
A sequence comparison of mouse and human proGIP proteins revealed that the N-terminal sequence of GIP_HUMAN  is highly homologous to the corresponding mouse GIP N-terminally extended putative peptide that was located immediately downstream of the signal peptide. Therefore, the in vivo effect of a continuous infusion of GIP_   (red horizontal line) and other cleaved products of proGIP identified with an FDR of 0% (blue horizontal lines) or 1% (yellow horizontal lines) by our peptidomic analysis. The predicted signal peptide of the preproprotein is shown in yellow letters and authentic GIP peptide in green letters, respectively. (b) Annotated MS/MS fragmentation spectra for plasma GIP_HUMAN  filtered by peptidomic analysis and comparisons with those of the corresponding synthetic GIP_HUMAN  peptides. MS/MS spectra with sequence assignments of fragment ions corresponding to synthetic GIP_HUMAN  "EKKEGHFSALPSLPVGSHAKVSSPQPRGPR" with a m/z 530.9565 (z = 6; upper panel) are compared with those of endogenous peptides (lower panel) to confirm the putative identification. The lower panel shows the MS/MS spectrum obtained by LC-MS/MS analysis in Fig. 5b with a retention time of 10.82 min. MS/MS spectra were deconvoluted into singly charged ions from the observed spectra and peaks were assigned theoretical m/z values for fragment ions. The annotations of the identified matched N-terminal-containing ions are shown in blue and the C-terminal-containing ions in red. The m/z differences between theoretical and observed values for most assigned peaks were less than 0.01 Da.   on atherosclerotic plaque formation in apolipoprotein E-deficient (ApoE −/− ) mice was investigated. Two osmotic mini pumps were implanted subcutaneously and loaded with saline alone, saline-dissolved GIP_HUMAN , or anti-GIP_HUMAN  IgG. The continuous infusion commenced at 17 weeks of age and lasted until 21 weeks of age in all four groups of mice that were assigned to be infused with (1) saline and saline, (2) GIP_HUMAN  and saline, (3) GIP_HUMAN  and anti-GIP_HUMAN  IgG or (4) saline and anti-GIP_HUMAN  IgG. The daily food intake during the 4-week infusion period did not differ significantly between any two groups of mice (vehicle group: 3.57 ± 0.19 g/day; GIP_HUMAN  group: 3.51 ± 0.16 g/day; GIP_HUMAN  plus antibody group: 3.48 ± 0.19 g/day; antibody only group: 3.48 ± 0.22 g/day, n.s.). There were no significant differences in body weight among the four groups at any time points ( Supplementary Fig. S1), nor were there any differences in serum chemistry measurements at the end of the 4-week infusion, except for an elevation of serum GIP levels in the GIP_HUMAN  plus anti-GIP_ HUMAN  IgG group (Supplementary Table S2). After 4 weeks of infusion, the atherosclerotic lesions were evaluated according to the oil red O-stained aortic surface area ( Fig. 3a-d), the aortic root area (Fig. 3e-h), and the anti-MOMA-2 antibody-stained aortic root area ( Fig. 3i-l). GIP_HUMAN  increased the atherosclerotic lesion areas and atheromatous plaque formation significantly when compared with that of the vehicle infusions ( Fig. 3m-o), whereas the combined infusions with the neutralising antibody inhibited the proatherosclerotic effects caused by GIP_HUMAN   (Fig. 3c,g,k).

Scientific Reports
To investigate whether chronic infusions of GIP_HUMAN  stimulated the production and/or release of bioactive substances, serum samples withdrawn from the ApoE −/− mice after 4 weeks of infusions with or without GIP_HUMAN  or anti-GIP_HUMAN  IgG, were applied to protein antibody arrays that detected 111 mouse cytokines and chemokines (Fig. 4a). The representative blots generated by these analyses showed appreciable regulation by GIP_HUMAN  and anti-GIP_HUMAN  IgG (Fig. 4b-e). The relative signal intensities of a variety of protein levels induced by GIP_HUMAN  or anti-GIP_HUMAN  IgG versus the vehicle-treated control experiments were quantified (Fig. 4f,g). The infusions of GIP_HUMAN  increased the secretions of many proinflammatory and proatherosclerotic proteins, including angiopoietin-2, serum amyloid P (SAP), CXC chemokine ligand 16 (CXCL16), proprotein convertase subtilisin kexin type 9 (PCSK9), fetuin A and MMP-3, whereas the anti-GIP_HUMAN  IgG reduced the serum levels of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), fetuin A, PCSK9,   for 60 min, and immunocytochemical staining was performed using an NF-κB p65 subunit antibody to detect its nuclear translocation. (e, f) Degradation of IκB-α. HAoECs (e) or THP1-derived macrophages (f) were stimulated with 10 -7 M GIP_HUMAN  for the indicated times and subjected to western blot analysis using the anti-IκB-α antibody to assess the time-course of IκB-α degradation. The panels show the cropped blots and the full-length blots are presented in the Supplementary Information. (g-i) Upregulation of MMP8 gene expression by GIP_HUMAN . HAoECs were incubated with 10 -7 M GIP_HUMAN  for the indicated times (g) or with indicated doses for 48 h (h), and MMP8 mRNA and β-actin levels were quantified. Data represent the fold changes (mean ± S.E.M) of MMP8 mRNA copies relative to β-actin mRNA (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle. (i) HAoECs pre-treated with or without MG132 (500 nM) for 30 min were incubated with 10 -7 M GIP_HUMAN  and MG132 (100 nM) for 24 h and MMP8 and β-actin mRNA levels were quantified. **p < 0.01, ***p < 0.001, compared with vehicle. The relative mRNA levels are shown as fold changes (mean ± S.E.M; n = 5). www.nature.com/scientificreports/ CXCL16, C-reactive protein and SAP. These results indicated that GIP_HUMAN  was an endogenous inducer of various proinflammatory and proatherosclerotic proteins.

Presence of GIP_HUMAN[22-51] in human plasma and organs.
To verify that GIP_HUMAN  was reproducibly detected according to the identified amino acid sequence in human plasma and to determine its accurate plasma levels, we synthesised a stable isotope-labelled GIP_HUMAN  and spiked the human plasma samples with a dilution series of this peptide prior to extraction to generate and extrapolate the extracted ion chromatogram (XIC) intensities using an ultra-high resolution mass liquid chromatography mass spectrometry method. The plasma concentration of GIP_HUMAN  extrapolated from the XICs generated by the respective endogenous peptides and the reference peptides was ~ 0.6 nM (    , a healthy human tissue microarray was stained using a specific polyclonal anti-GIP_HUMAN  IgG. Immunohistochemistry with IgG purified from preimmune rabbit serum did not detect any signal ( Fig. 6a-l). Conversely, immunoreactive GIP_ HUMAN  was abundant in the cerebellum, liver, stomach, kidney, heart, small intestine, and colon, and less in the cerebral cortex, tonsils, lymph nodes, and testes ( Fig. 6a'-l'), and undetected in lung, pituitary, spleen, pancreas, adrenal gland, spine, thyroid, bone marrow, fallopian tube, skin, thymus, uterus, bladder, ovary and prostate. GIP_HUMAN [22] is biosynthesised theoretically, concomitantly with GIP from their common precursor protein, and GIP is expressed abundantly in a limited number of organs, such as the small intestine and duodenum. Therefore, we investigated whether GIP and GIP_HUMAN  peptides were co-localised www.nature.com/scientificreports/ within cells by co-staining the two peptides. Confocal immunofluorescence microscopy revealed a distinct GIP and GIP_HUMAN  expression within each type of tissue ( Fig. 6a"-l"). These results indicate differential intracellular production and/or localisation of the GIP_HUMAN  and the GIP peptides.

Discussion
We described the identification of a novel peptide hormone, GIP_HUMAN , using native peptide resources generated by plasma peptidomics. We completed functional screening of 129 synthetic peptides selected in silico using cultured human macrophages and vascular cells. Since GIP_HUMAN  activated NF-κB signalling in the HAoECs and the macrophages, its proatherogenic activity was investigated using ApoE −/− animal models. GIP_HUMAN  accelerated the development of atherosclerotic lesions without affecting body weight and    www.nature.com/scientificreports/ food intake, whereas combined infusions of the anti-GIP_HUMAN  antibody inhibited the atheromatous plaque formation and macrophage accumulation induced by GIP_HUMAN . GIP_HUMAN  upregulated the serum levels of many important proatherosclerotic and proinflammatory proteins that are transcriptionally upregulated by NF-κB, whereas the neutralising endogenous GIP_HUMAN  with the anti-GIP_HUMAN  antibody suppressed these levels. Thus, GIP_HUMAN  revealed a novel endogenous proatherogenic peptide that activates NF-κB signalling. GIP_HUMAN  is located immediately downstream of the signal peptide of the preproGIP and is theoretically biosynthesised simultaneously upon generation of the mature GIP from its common precursor, proGIP. Signal peptides of preproproteins are cleaved cotranslationally 25,26 , whereas the prohormone convertase (PC) 1/3 flanks the C-terminus of GIP_HUMAN  to generate both GIP_HUMAN  and GIP from proGIP 5 . The endogenously processed GIP_HUMAN  has a C-terminal PC consensus motif that can be removed by carboxypeptidase E, which is a common feature of most neuropeptides and peptide hormones 27 . However, the LC-MS/MS analysis did not detect GIP_HUMAN , which does not contain a C-terminal arginine residue. The absence of GIP_HUMAN  in our repetitive LC-MS/MS analysis was not likely to be caused by a reduced ionisation efficiency due to the loss of the arginine because GIP_HUMAN  has four positively charged residues, that is, three lysines and one arginine. In addition to GIP_HUMAN , our current LC-MS/MS analysis identified GIP_HUMAN  and GIP_HUMAN [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] in human plasma with the highest confidence probability of an FDR of 0%. These two plasma peptides did not exert any appreciable biological activity when compared with that of GIP_HUMAN , suggesting that the effect of GIP_ HUMAN  required its N-terminal sequences.
The potential implications of GIP_HUMAN  in the pathophysiology of human diseases is supported by its presence in the human peripheral circulation. One of the major obstacles in the study of plasma peptides is the difficulties associated with their precise quantification. Our LC-MS/MS measurements by high resolution Orbitrap MS used plasma pre-spiked with synthetic stable-isotope labelled peptides to exclude any interference associated with the plasma extraction 28 . Further, we used the sums of the XIC areas of the three ion precursors generated by the endogenous and synthetic peptides to ensure accuracy in the quantification 29,30 (Fig. 5). This yielded a human plasma concentration of approximately 0.6 nM. The levels were higher than those of the major pituitary, pancreatic and vasoactive peptide hormones and comparable with other hormones, such as cortisol, and some growth factors.
Systemic immunohistochemical analysis using a normal human tissue microarray revealed an abundant expression of GIP_HUMAN  in several digestive organs and kidneys. Conversely, GIP is synthesised throughout the small intestine, mainly in the duodenum and proximal jejunum in the enteroendocrine K cells 31,32 , and is secreted in response to nutrient ingestion, especially glucose or fat 33,34 . Our confocal immunofluorescent microscopy results showed the concomitant expression of GIP and GIP_HUMAN  at tissue levels; however, these two peptides were not colocalised at the cellular level, despite both peptides being, in principle, biosynthesised simultaneously from the proGIP. GIP_HUMAN  is considered to act on its own unidentified receptor distinct form the GIP receptor, because GIP and GIP_HUMAN  exert totally distinct responses in both cultured cells and animal models. In a previous study using ApoE -/mice, GIP induced antiatherogenic effects by activating cAMP via the GIP receptor 35 , while in the current study, GIP_HUMAN  caused NF-κB activation and contrasting proatheroslcerotic effects. Several classical bioactive peptides are derived from the same precursor proteins as a result of tissue-specific posttranslational processing mechanisms and they show distinct biological activities and systemic expression profiles. For example, the prohormone convertase 2 expressed in the pancreatic α-cells converts proglucagon into glucagon, while prohormone convertase 1/3 in small intestinal enteroendocrine L-cells processes proglucagon into GLP-1 36 . Other examples include proopiomelanocortin-derived peptides in the anterior pituitary 37,38 and salusin-α/salusin-β in systemic tissues 15 . GIP/ GIP_HUMAN  is another pair that shows distinct biosynthetic mechanisms and biological activities.
Identification of plasma bioactive peptides and disease biomarkers is challenging because these peptides usually exist at trace levels, while plasma contains a considerable dynamic range of high-molecular-weight proteins. This study demonstrated that the use of our plasma native peptide library identified using our improved peptidomics technology, enabled an easier identification of a novel bioactive peptide than conventional methods. Synthetised peptides of the identified sequences can be tested for biological responses using experiments that detect the desired activity. These peptides can also be tested to identify ligands of orphan GPCRs. The nonbiased peptidomic information generates a human "orphan ligand" library available for the discovery of novel bioactive peptides and disease biomarkers applicable to various scientific fields. Our results will facilitate the ultimate application of peptidomics techniques in biomedical fields and further promote innovations in the health science industry.
Our annotation of the identified peptide, GIP_HUMAN , may lead to a misunderstanding of the amino acid position as the authentic active GIP peptide is often called GIP(1-42) to distinguish it from its inactive form GIP , indicating that this peptide consists of 42 amino acids. However, we have proposed the use of the UniProt entry names with their amino acid positions, such as GIP_HUMAN , rather than assigning new names to each new bioactive peptide discovered, because the current peptidome has already revealed the presence of a large number of fragmented peptides of many classical proteins/peptides, which may facilitate the discovery of multiple new bioactive peptides 22 and biomarkers derived from a single proprotein. Classical bioactive peptides and biomarkers can also use such aliases in the event that the discovery of their brotherhood peptides can cause confusion. Authentic active and inactive incretin hormones, GIP(1-42) and GIP , can be called GIP_HUMAN  and GIP_HUMAN[54-93], respectively, to compare their amino acid positions in the entire preproGIP protein.
In conclusion, this was the first study to discover a novel proatherosclerotic peptide by using the native peptide resources identified by the human plasma peptidome. This outcome was made possible by our improved www.nature.com/scientificreports/ technology that efficiently enriched plasma low-molecular-weight, low-abundance native peptides, enabling their identification by LC-MS/MS analysis. Human plasma peptidomics holds significant promise as a powerful revolutionary tool for identifying endogenous bioactive peptides and peptide biomarkers that may detect, diagnose, and treat diseases.

Database searching of LC-MS/MS acquired peptidomic resource. LC-MS/MS acquisition data
obtained up until the beginning of 2018 were initially searched against the SwissProt_2015_02.fasta database (selected for Homo sapiens; 20,199 entries) using two different data processing pipelines and search engines, Mascot (Matrix Science, London, UK) and PEAKS Studio (Bioinformatics Solutions, Waterloo, Canada) 22 . In the Mascot workflow, raw MS and MS/MS data files were processed using Mascot Distiller (version 2.5.1.0, Matrix Science). Data processing included peak picking, de-isotoping and charge deconvolution of fragment ions. The resulting peak list files were searched using the Mascot search engine (version 2.4.1). In the PEAKS Studio workflow, the PEAKS Studio (version 7.0) was used to perform peak picking, de-isotoping, charge deconvolution of fragment ions and a de novo peptide sequencing-based database search from the MS and MS/MS spectra of peptides. The PTM algorithm of the PEAKS Studio was applied to identify variable modifications and substitutions [39][40][41] . The FDR was set as 1%. All the peptidomic sequencing data were deposited into the Pro-teomeXchange Consortium via the PRIDE 42 partner repository with the dataset identifier PXD003533. In our initial analyses of the peptides deposited in the peptidomic database, we used 485 built-in modifications in the PTM search of the PEAKS, which hampered the PTM accuracy. Furthermore, a native peptide database search was performed using the Mascot MS/MS ion search, in which peptides longer than 30 amino acids may show high scores despite having poor MS/MS spectra. Therefore, we reanalysed the entire peptide database using the SwissProt_2020_03.fasta database (selected for Homo sapiens; 20,365 entries) with the PEAKS database search algorithm (Bioinformatics Solutions, Waterloo, Canada). The PEAKS Studio (version X) performed peak picking, de-isotoping, charge deconvolution of fragment ions and a de novo peptide sequencing-based database search from the MS and MS/MS spectra of peptides. The search parameters were: enzyme, no enzyme; fixed modification, carbamidomethyl (C, only for samples with reductive alkylation); variable modifications, acetyl (N-term, K), amidated (C-term), pyro-glu from Q (Q), oxidation (M), carbamidomethyl (N-terminal, only for samples with reductive alkylation); peptide ion mass tolerance, 6 ppm; fragment ion mass tolerance, 0.02 Da. The FDR was set at 1%. The results of the analysis using this updated database and the PEAKS search algorithm were used for the subsequent exploration of the bioactive peptides.

Selection of peptides for chemical synthesis and dissolution of synthetic peptides. The pep-
tides for synthesis and the biological function analysis were selected in silico based on: (1) being uniquely assigned to a precursor protein family; (2) being assigned a gene name or a gene name of a protein family; (3) being encoded by secretory proteins as defined by the SwissProt keywords; (4) having potential extracellular release predicted by PSORT, a bioinformatics tool used for the prediction of protein localisation sites in cells 43 ; (5) being identified with an FDR of 1%; (6) having amino acid lengths of 5-38 residues; (7) having no substitutions or modifications except for C-terminal amidation; and (8) having no cysteine residues. For the initial round of chemical synthesis, 146 sequences were selected and synthesised (Scrum Inc., Tokyo, Japan), reconstituted to 2-10 × 10 −6 M with 10% acetonitrile/0.1% trifluoroacetic acid and then subjected to an LC-MS/MS analysis to confirm the purity and solubility.
Production and purification of peptides antibodies. The specific polyclonal antibody against GIP_ HUMAN  was raised and purified as described previously 20,44 , with the following modifications: two micrograms of [Cys 0 ]-PEG-EKKEGHFSALPSLPVGSHAKVSSPQPRGPR were chemically synthesised and approximately half was pre-treated with a protein crosslinking and fixation reagent, mixed with the remaining respective 1 μg of untreated peptide, coupled to a maleimide-activated mariculture keyhole limpet hemocyanin (Pierce) and immunised on days 1, 15, 29, 43 and 57 into Japanese white rabbits. Blood was collected prior to the first injection and on days 36, 50 and 64 post-injection. The antibody titer was determined using ELISA. The polyclonal antisera were purified using a Melon Gel IgG Spin Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), which removed the non-relevant proteins often present in high abundance 20 .
Cell culturing. The human aortic endothelial cells (HAoECs) and human aortic smooth muscle cells (HAoSMCs) were purchased from Promocell (Heidelberg, Germany) and cultured using the appropriate medium and supplements recommended by the supplier. Successive experiments were performed with passage of four to six cultures. The THP1 was obtained from the Riken Cell Bank (Ibaraki, Japan). The THP1 cells were grown in a RPMI1640 medium containing 10% foetal bovine serum for 2-3 days and then incubated with 500 ng/mL phorbol-12-myristate-13-acetate for 24 h to facilitate the differentiation into macrophages. growing HAoECs were incubated for 5-60 min after the addition of 10 -6 M FAM-labelled GIP_HUMAN . The cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. The nuclei were counterstained using DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and the fluorescence signals were photographed using an LSM710 confocal microscope (Carl Zeiss, Jena, Germany), as described previously 46,47 . Immunocytochemistry staining. To assess the NF-κB nuclear translocation, the THP1-derived macrophages and HAoECs grown in a 4-well Slide&Chamber (Watson Bio Lab, Tokyo, Japan) for 2-3 days were washed twice with PBS and incubated with GIP_HUMAN  (10 -7 M) for 60 min. The cells were fixed with 4% paraformaldehyde for 15 min, blocked with Blocking One (Nacalai Tesque Inc., Kyoto, Japan), incubated for 60 min with an antibody against p65 (1:500, Santa Cruz Biotechnology, Inc., CA, USA) at room temperature and incubated for 30 min with the biotinylated goat anti-rabbit antibody (1:3000, Vector Laboratories Inc., Burlingame, CA, USA). The avidin-biotin-peroxidase complex was formed by using the Vectastain ABC Kit (Vector Laboratories Inc.) and visualized by using the DAB peroxidase substrate kit (Vector Laboratories Inc.).

Determination of the intracellular free Ca 2+ concentration [Ca
Immunoblotting. Western blotting was performed as described previously 46,48 . The subconfluent HAoECs and THP1-derived macrophages grown in 6-well plates were incubated with GIP_HUMAN  for the indicated times, washed twice with ice-cold PBS, solubilised in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (1:100, Thermo Fisher Scientific), sonicated, and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected and the protein concentrations were measured. The extracted protein samples were loaded onto 4%-20% gradient polyacrylamide gels (Mini-PROTEAN TGX Gels, Bio-Rad Laboratories, Irvine, CA, USA) and transferred to PVDF membranes (Trans-Blot Turbo Mini PVDF Transfer Packs, Bio-Rad Laboratories). After blocking with Blocking One (Nacalai Tesque Inc.), the membranes were incubated with the anti-IκB-α antibody at a 1:3000 dilution (ab32518, Abcam, Cambridge, MA, USA) overnight at 4 °C, washed extensively, and then incubated with the peroxidase-conjugated goat anti-rabbit secondary antibody at 1:5000 dilution (Bio-Rad Laboratories) for 1 h at room temperature. The protein bands were detected using ECL Prime (GE Healthcare, Tokyo, Japan). The signals of each blot were visualised and analysed quantitatively using ImageQuant LAS 4000 (GE Healthcare) 47 .

Real-time RT-PCR.
Total RNA was harvested using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the HAoECs grown in 6-well plates treated with or without GIP_HUMAN , reverse transcribed with the PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan) and quantified using a C1000 Touch Thermal Cycler and CFX Real-Time PCR Detection Systems (Bio-Rad Laboratories) and the Kapa Syber Fast qPCR Master Mix (Kapa Bio Systems Inc., Cape Town, South Africa) as described previously 47 , but with the following modifications: after the reverse transcription, the reaction mixtures were denatured at 94 °C for 3 min followed by 40 cycles of PCR at 94 °C for 10 s and 60 °C for 30 s. The data were expressed as Ct values and used to determine ΔCt values. The human MMP8 mRNA was amplified using synthetic oligomers (MMP8: forward primer 5′-tctgcaaggttatcccaagg-3′, reverse primer 5′-gctccatgaattgtctttggt-3′; β-actin: forward primer 5′-attggcaatgagcggttc-3′, reverse primer 5′-ggatgccacaggactcca-3′) synthesised by Eurofins Genomics (Tokyo, Japan). The relative MMP8 gene expression level versus the reference β-actin expression level as the housekeeping gene was calculated using the 2 −ΔΔCT method equipped in software packages for PCR Detection Systems 49 .
In vivo analysis of atherosclerosis lesion development. Twenty-four ApoE −/− 9-week-old mice purchased from Sankyo Labo Service (Tokyo, Japan) were maintained under controlled temperatures (22-25 °C) with free access to standard rodent chow (Labo MR Stock, NOSAN, Yokohama, Japan) for 4 weeks. At 13 weeks of age, the mice were fed the High-Fat-Diet (D12451, Oriental Yeast Co., Ltd., Tokyo, Japan) containing 45% fat. At 17 weeks of age, two micro-osmotic pumps (Alzet Ⓡ Model 1002, Durect Corp., Cupertino, CA, USA) were implanted subcutaneously into the dorsum of mice that were anaesthetised with isoflurane in order to chronically infuse GIP_HUMAN  and/or its neutralising antibody. The pumps were replaced after 2 weeks. The mice were assigned into the following four groups with two pumps loaded with: 1) saline only (vehicle); 2) GIP_HUMAN  and saline; 3) saline and anti-GIP_HUMAN  IgG; and 4) GIP_HUMAN  and anti-GIP_HUMAN  IgG. The pumps containing GIP_HUMAN  or anti-GIP_HUMAN  IgG delivered 0.6 nmol/kg/h or 1.4 μg/kg/h of peptide or antibody dissolved in saline, respectively. After 4 weeks, mice were anaesthetised with isoflurane and, after blood collection from the descending vena cava, transcardially perfused with PBS and then with 4% paraformaldehyde 35 . The entire length of the aorta was carefully dissected from the root to the abdominal area, and cross sections of the aortic roots and the longitudinally exposed lumen surface were stained with oil red O for evaluation of the atherosclerotic plaques 35  www.nature.com/scientificreports/ Antibody array analysis. Two-hundred-microliter serum samples obtained from mice infused with or without GIP_HUMAN  or anti-GIP_HUMAN  IgG for 4 weeks were applied to the Mouse XL Cytokine Kit (ARY028, R&D Systems, Minneapolis, MN, USA) and assayed according to the manufacturer's instructions. Cytokine array signals were detected using the ImageQuant LAS 4000 (GE Healthcare) 47 and quantified using ImageJ software (http:// rsb. info. nih. gov/ ij/ 50 . Values from duplicate spots were averaged and the relative signals obtained from the serum of mice treated with GIP_HUMAN  or anti-GIP_HUMAN  IgG were compared with the control serum samples that were obtained from vehicle-treated mice. Peptide extraction. Blood samples were collected from healthy volunteers into vacutainers containing Na 2 -EDTA and immediately separated in a refrigerated centrifuge at 1,000 g for 20 min. Aliquots were immediately flash-frozen in liquid nitrogen and stored at − 80 °C until processing. The following stable isotope-labelled peptide was synthesised by Scrum Inc. using L-phenylalanine-N-9-fluorenylmethoxycarbonyl ( 13 C 9 , 98%; 15 N, 98%): SI-GIP_HUMAN , EKKEGHFSALPSLPVGSHAKVSSPQPRGPR, with the underlined residue containing a stable isotope. The stable isotope-labelled peptide was spiked into human plasma at the final concentration of 100 pM, enriched using the modified differential solubilisation method, as described previously 28

Measurements of plasma peptide levels using stable isotope-labelled peptides. Peptide extracts
were analysed using a quadrupole Orbitrap benchtop mass spectrometer, Q-Exactive (Thermo Fisher Scientific), equipped with an EASY-nLC 1000 system (Thermo Fisher Scientific). The peptides were injected directly onto an analytical column (C18, particle diameter 3 µm, 75 μm i.d. × 125 mm, Nano HPLC Capillary Column, Nikkyo Technos, Tokyo, Japan) and then separated with a gradient composed of solvent A (0.1% FA) and solvent B (0.1% FA and 90% ACN) (0-20 min, 5-32% B; 20-26 min, 32-55% B; 26-27 min, 55-95% B; 27-30 min, 95% B) at a flow rate of 300 nL/min. MS spectra were collected over an m/z range of 350 to 900 at a resolution of 70,000 at 200 m/z to set an automatic gain control (AGC) target of 1 × 10 6 . The 12 most-intense ions with charge states of 2 + to 6 + that exceeded an intensity of 1.3 × 10 5 were fragmented in data-dependent mode via collision-induced dissociation with a normalised collision energy of 27%. Tandem mass spectra were acquired on the Orbitrap mass analyser with a mass resolution of 35,000 at 200 m/z to set an AGC target of 2 × 10 5 . The dynamic exclusion time was set to 30 s. The plasma concentrations were extrapolated from the XICs generated using the respective endogenous peptides and the corresponding spiked stable isotope-labelled peptides 30,52 .

Immunohistochemistry.
A human healthy tissue microarray, MNO341 (US Biomax Inc., Rockville, MD, USA), which containsed biospecimens of 33 tissue types mostly from surgical resectiosn, was used to examine the systemic distribution of each peptide expression. The tissue preparations were deparaffinised, rehydrated and autoclaved in pH 7.0 antigen retrieval solution (Histo VT One, Nacalai Tesque Inc.) at 121 °C for 10 min. The preparations were blocked with 0.3% hydrogen peroxidase in methanol for 20 min at room temperature to block endogenous peroxidase, followed by treatment with the Avidin Biotin Blocking Kit (Nichirei Bioscience Inc., Tokyo, Japan) and incubated with Blocking One (Nacalai Tesque Inc.) for 30 min to block the non-specific sites. The sections were incubated for 90 min at room temperature with either the anti-GIP_HUMAN  IgG or the control non-immune IgG purified from the preimmune serum (1:500 dilution). After washing three times in PBS, the biotinylated goat anti-rabbit IgG antibody (1:500, Vector Laboratories Inc.) was incubated for 60 min at room temperature. Antibody binding was visualised using the avidin-biotin-complex peroxidase method with the Vectastain ABC kit (Vector Laboratories Inc.) and a DAB peroxidase substrate kit (Vector Laboratories Inc.) 53 . The tissue preparations were stained with Meyer's hematoxylin (Muto Pure Chemicals, Tokyo, Japan). For the comparison of the exact expression sites of GIP_HUMAN  and the mature GIP using dual immunofluorescence staining, we followed the same procedures as described for normal immunohistochemical staining with tissue array, MNO341, except that we used two primary antibodies, the rabbit anti-GIP_ HUMAN  IgG (1:1000) and the mouse anti-human GIP (1:500, R&D Systems) simultaneously, and two secondary antibodies, the goat anti-rabbit-Alexa Fluor 488-conjugated antibody (1:500, Abcam) and the goat anti-mouse-Alexa Fluor 594-conjugated antibody (1:1000, Abcam). The images were acquired using an LSM710 confocal microscope (Carl Zeiss).
Statistical analysis. The data were presented as means ± SEM and were analysed using GraphPad Prism software 5.02 (GraphPad Software Inc., San Diego, CA, USA). The data between the two groups were compared using the two-tailed unpaired Student's t-test or Welch's t-test, and data involving three or more groups were analysed using a one-way analysis of variance followed by a Bonferroni's post hoc test. Statistical significance was set at P < 0.05.

Scientific Reports
| (2021) 11:14470 | https://doi.org/10.1038/s41598-021-93862-w www.nature.com/scientificreports/ Ethics approval and consent to participate. All the animal experimental procedures were approved by the Animal Experimentation and Ethics Committee of the Showa University School of Medicine (permission number: 09062). Procedures were performed in compliance with the ARRIVE guidelines and with guidelines for animal experiments by the Showa University School of Medicine. The study protocol using human blood samples was approved by the Ethics Committee of Kitasato University Hospital/School of Medicine (C19-245). All the study methods were undertaken in accordance with the relevant guidelines and regulations of this organisation as well as the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan. Written informed consent was obtained from all the healthy volunteers who provided blood samples.