Intestinal expression profiles and hepatic expression of LEAP2, ghrelin and their common receptor, GHSR, in humans

Liver-expressed antimicrobial peptide 2 (LEAP2) and ghrelin have reciprocal effects on their common receptor, the growth hormone secretagogue receptor (GHSR). Ghrelin is considered a gastric hormone and LEAP2 a liver-derived hormone and both have been proposed to be involved in the pathophysiology of obesity and type 2 diabetes (T2D). We investigated the mRNA expression of LEAP2, ghrelin and GHSR along the intestinal tract of individuals with and without TD2


Introduction
In recent years, liver-expressed antimicrobial peptide 2 (LEAP2) has gained interest due to its reciprocal relationship with the hunger hormone ghrelin [1].Ghrelin is considered a stomach-derived hormone and an agonist of the growth hormone secretagogue receptor (GHSR) stimulating appetite, increasing plasma levels of growth hormone and glucose, and reducing glucose-stimulated insulin secretion [2].Apart from the stomach, ghrelin is expressed in the intestine with higher expression levels in the proximal small intestine compared to distal small intestine and colon in humans [3,4].In rats, ghrelin tissue content was high in the gastric fundus with smaller contents in the small and large intestine [5,6] and ghrelin-containing cells were found throughout the gastrointestinal tract [7].Detailed gene expression of ghrelin along the intestinal tract in humans, however, remains poorly described.
LEAP2 is primarily considered a liver-derived hormone but has also been shown to be expressed in individual samples from the human jejunum, ileum, and colon with higher expression levels in jejunum compared to duodenum [8][9][10]; however, its expression profile along the human intestinal tract has not been described previously.In mice, LEAP2 mRNA expression was high in the jejunum with duodenal, iliac, and hepatic expression accounting for less than one-fifth of jejunal expression [1].In rats, however, hepatic LEAP2 exceeded that of the jejunum [11].In contrast to ghrelin, LEAP2 is an antagonist and inverse agonist of the GHSR [10,12] mediating attenuated ghrelin-induced food intake and growth hormone levels, as well as abolished blood glucose elevations when co-administered with ghrelin in rodents [1,11].In humans, exogenous LEAP2 infusion resulted in reduced ad libitum food intake and reduced postprandial glucose excursions [13].
GHSR is highly expressed in the human brain, especially the pituitary, with lower levels in the adrenal glands, pancreatic islet cells, spleen, and thyroid [3,4,14].In rodents, GHSR expression has mainly been investigated in the brain [15,16] but was also expressed in the rat stomach [17] and in the murine small intestine, but not in the liver [18].Despite ghrelin's appetite-promoting effect and the ghrelin-induced reductions in glucose-stimulated insulin secretion and glucose tolerance alluded to above, clear roles for ghrelin in the pathophysiology of obesity and type 2 diabetes (T2D) have not been established [19,20].Interestingly, increased levels of LEAP2 have been proposed as a marker of dysmetabolism in the context of obesity, T2D, and metabolic dysfunction-associated steatotic liver disease [21][22][23], but the underlying mechanisms are poorly understood.
Here, we sought to elucidate the potential roles of ghrelin and LEAP2 as well as their common receptor, GHSR, in the pathophysiology of T2D and obesity by evaluating their mRNA expression profiles along the entire intestinal tract in individuals with T2D and healthy matched controls as well as their hepatic expression in men with obesity and lean healthy men.

Regulatory approval
We obtained unpublished biopsy data from two studies approved by the Scientific-Ethical Committee / Research Ethics Committee of the Capital Region of Denmark (H-3-2010-115 and H-6-2014-097) and registered with the Danish Data Protection Agency.Clinicaltrials.gov(NCT03044860 and NCT02337660).The studies adhered to the latest revision of the Declaration of Helsinki and both oral and written consents were obtained from all participants before enrollment.

Participants
Gut mucosal biopsies collected by Rhee et al. [24] from 12 individuals with T2D (9 men) with a mean HbA1c of 48 mmol/mol, mean age of 51 years, and a mean BMI of 27 kg/m 2 and from 12 healthy age and body mass index (BMI)-matched controls (8 men) with a mean HbA1c of 34 mmol/mol, mean age of 50 years, and a mean BMI of 27 kg/m 2 were examined [24].The inclusion criteria for individuals with T2D were diagnosis of T2D at least 3 months prior to inclusion, diabetes management using lifestyle counseling alone or in combination with metformin and/or sulfonylurea, Caucasian ethnicity, age 25-70 years, negativity to glutamic acid decarboxylase 65 and islet cell autoantibodies, and informed oral and written consent.Key exclusion criteria included use of glucose-lowering drugs other than metformin and sulfonylurea, use of medicine that could not be withheld for 12 h, BMI greater than 35 kg/m 2 , and any condition that would contraindicate propofol sedation or double-balloon enteroscopy.For healthy controls, inclusion criteria were normal fasting plasma glucose and 75 g-oral glucose tolerance test, Caucasian ethnicity, and age 25-70 years, and exclusion criteria were identical to those for participants with T2D with the addition of no first-degree relatives with type 1 diabetes or T2D.Liver biopsies collected by Suppli et al. [25] were from 15 men with obesity (mean BMI 34 kg/m 2 ; mean age 36 years, mean HbA1c 31 mmol/mol) and from 15 lean men (mean BMI 23 kg/m 2 ; mean age 41 years, mean HbA1c 30 mmol/mol).Exclusion criteria included known liver disease, consumption of over 14 units of alcohol per week, HbA1c ≥ 42 mmol/mol, and first-degree relatives with diabetes.

Experimental procedures
Mucosal samples were collected along the entire length of the intestinal tract using anterograde and retrograde double-balloon enteroscopy on separate days as described by Rhee et al. [24].In short, enteroscopies were performed under propofol sedation with an EN-450 T5 enteroscope (Fujinon Inc., Saitama City, Saitama, Japan).Prior to each enteroscopy, individuals with T2D paused any antidiabetic medications for one week.Individuals with and without T2D fasted 6 h before the enteroscopic procedures.During the anterograde enteroscopy, the enteroscope's maximal insertion depth was marked submucosally with ink once further advancement was not feasible.Mucosal biopsies were then, upon scope retraction, obtained at 30 cm intervals throughout the ileum and the jejunum as well as at Treitz ligament and in the duodenum.During the subsequent retrograde double-balloon enteroscopy, the objective was to reach the abovementioned ink mark, and during retraction of the scope, biopsies were obtained every 30 cm of the small intestine and in the coecum, in the ascending, transverse, descending, and sigmoid colon, and in the rectum.Region 1 and 2 represented duodenum as the Treitz ligament marks the duodenojejunal transition.Region 10 represented the ileocecal transition The small intestine between the Treitz ligament and the ileocecal transition was segmented into 7 equal parts, designated as regions 3-9 representing jejunum and ileum.A precise distinction of the jejunoileal transition was not possible.Ultrasound-guided transcutaneous liver biopsies were sampled from men with and without obesity after a 10-h overnight fast as previously described by Suppli et al. [25].Until fasting all individuals maintained their normal diet.An illustration of the study setup can be found in Fig. 1.

Gene expression analysis
For both mucosal and liver biopsies, tissue homogenization, total RNA isolation and mRNA purification and quantification, generation of cDNA libraries and sequencing of these were performed as previously described by Jorsal et al. and Suppli et al., respectively [25,26].Gene expression is reported as reads per kilobase of transcript per million mapped reads (RPKM), and RPKM values over 1 were considered to reflect robust expression, while the threshold for detectability was set at an RPKM of 0.1.Thresholds were based on the principle of excluding lower expressed genes to minimize analytical noise and enhance the accuracy of detecting meaningful gene expression patterns.Smaller detection limits tend to generate large and misleading fold changes during analysis, as elucidated by Sheng et al. [27].The approach aligns with established practices in the field, but to date, no consensus has been reached on what RPKM threshold to use for gene detection.

Statistical analysis
The dataset was summarized using counts or mean values with corresponding ranges.Data visualization included the use of box and whisker plots and scatter plots.The analysis of the mucosal samples aimed to discern differences in LEAP2, ghrelin and obestatin prepropeptide (GHRL), and GHSR mRNA expression levels between individuals with T2D and those without, as well as across various regions of the small and large intestines.We employed a linear mixed-effects model that considered group and location as fixed effects and the interaction between these two factors, along with an unstructured covariance pattern to accommodate repeated measures within individual participants.For the analysis of GHRL mRNA expression, the analyses did not converge due to missing data at the ileocecal region.Hence, this region was removed from the analysis.For comparisons between men with and without obesity, paired t tests were utilized.Only tissue biopsies in which more than 50 % of participants exhibited nonnegligible expression levels (>0.1 RPKM) were included in the statistical analyses, and the liver, small intestine, and large intestine, respectively, were considered independently.This was to ensure, that only information on statistical significance was provided for regions where genes are more reliably expressed, thus avoiding potentially skewed A. Englund et al. interpretations of our data.Due to non-normal distributions, all data were log-transformed and expressed as relative median differences with 95 % confidence intervals (Cis).Site-specific differences between individuals with T2D and healthy controls were reported, as were intersite variations, using the duodenum and rectum as reference points for the small and large intestines, respectively.Adjustment for multiple comparisons was performed using the Benjamini-Hochberg method to control the false discovery rate.A corrected p value below 0.05 was deemed indicative of statistical significance.Analyses were conducted using SAS software (SAS Studio version 3.8), and figures were generated using R (Version 2022.07.2+576).

Expression of LEAP2 in the intestines and the liver
Both in individuals with and without T2D, mucosal LEAP2 mRNA levels increased from the duodenum through the small intestine until dropping just proximal of the ileocecal valve (Fig. 2A; Tab.S1).In the large intestines of both groups, LEAP2 was expressed in uniformly low levels (Fig. 2A; Tab.S1).No significant differences were observed between the two groups across the sampled intestinal regions (Tab.S3).In the liver, no significant differences in LEAP2 mRNA expression were observed between men with and without obesity (Fig. 2B; Tab.S4).

Expression of ghrelin in the intestines and the liver
Levels of ghrelin mRNA in small intestinal mucosa were highest in the duodenum and decreased throughout the small intestine for both T2D and healthy controls, with uniformly low levels along the large intestine (Fig. 2C; Tab.S5, S6).Significantly higher ghrelin mRNA levels were found in the small intestinal segment 7 and 8 (p adj = 0.02 and 0.02) of individuals with T2D compared to healthy controls (Tab.S7).The liver expression of ghrelin was non-detectable or negligible with only a few samples having more than 0.1 RPKM (Fig. 2D).

Expression of GHSR in the Intestine and Liver
The mRNA expression of the GHSR was minimal or non-detectable throughout the intestinal tract for both individuals with and without T2D as well as in the liver of men with and without obesity (Figs.2E and  2F).
A. Englund et al.

Fig. 2. Expression levels of LEAP2
, ghrelin and GHSR in intestine and liver.mRNA expression of liver-expressed antimicrobial peptide 2 (LEAP2) (A, B), ghrelin (GHRL) (C, D) and their common receptor, growth hormone secretagogue receptor (GHSR) (E, F), in mucosal biopsies sampled throughout the small intestine (white background) and the large intestine (grey background) (A, C, E) in 12 individuals with type 2 diabetes (blue) and in 12 age and body mass index-matched healthy controls (grey), and in transcutaneous liver biopsies (B, D, F) from 15 men with obesity (green dots), n = 15) and 15 lean controls (grey dots, n = 15).Dots are individual data points, boxes represent inter-quartile ranges and whiskers extend from the 25th percentile to the smallest value within 1.5 the interquartile range below it and from the 75th percentile to the largest value within 1.5 times the interquartile range above it (encompassing data points not deemed outliers).Asterisks (left (*/) for healthy controls, right (/*) for T2D) show statistically significant difference between location and reference (duodenum in small intestine and rectum in large intestine).Abbreviations: Asc., ascending; Trans., transverse; Desc., descending; RPKM, reads per kilobase of transcript per million mapped reads.intestine, increasing from the duodenum throughout the small intestine before dropping at the terminal ileum, and uniformly low expression levels throughout the large intestine, in both individuals with T2D and controls; 2) robust and similar mRNA expression of LEAP2 in the liver of men with and without obesity; 3) robust ghrelin mRNA expression levels in the duodenum gradually decreasing throughout the small intestine with uniformly low expression through the large intestine in both individuals with T2D and healthy controls; 4) negligible or non-detectable mRNA expression of ghrelin in the liver; and 5) no significant expression of GHSR in the intestines or in the liver in individuals with or without T2D/obesity.
The present study extends findings from Howard et al. [9] and Hagemann et al. [10], showing LEAP2 expression in the liver, ileum, and colon from disease-free donors [9], by comparing intestinal and hepatic LEAP2 mRNA expression in individuals with T2D and men with obesity, respectively, with matched healthy controls, and providing unprecedented granularity of the LEAP2 expression profile along the intestines in man.Similar to Krause et al., we find higher jejunal LEAP2 expression compared to the duodenum with low expression levels in the colon.Iliac expression levels, however, were not investigated [8].Intestinal LEAP2 expression levels in mice were somewhat comparable to our findings with higher expression in the jejunum compared to the duodenum, but showed low iliac expression levels and was absent in the colon [1].In rats there was no difference in duodenal and jejunal LEAP2 expression levels and no expression was found in the colon [11].Our findings of profound small intestinal LEAP2 expression in combination with a recent study showing higher LEAP2 plasma concentrations following oral glucose administration compared to intravenous argues, that LEAP2 is not only expressed but is also secreted from the human intestine [28].Plasma levels of LEAP2 have been reported to be higher in individuals with T2D compared to healthy controls [22].In individuals with obesity and prediabetes, fasting levels of LEAP2 were positively associated with HbA1c, fasting plasma glucose and insulin while negatively associated with insulin sensitivity [29].Also, plasma levels of LEAP2 have been shown to positively correlate with BMI, and Mani et al. [21] and Holm et al. [30] reported elevated LEAP2 levels in individuals with severe obesity (BMI ≥ 40 kg/m 2 ) compared to lean individuals.The mechanisms underlying elevated LEAP2 levels in T2D and obesity and their association with glycaemia and circulating insulin levels remain unknown.Unfortunately, measurement of LEAP2 plasma levels was not possible in the present study, but our results do not support exaggerated intestinal or hepatic LEAP2 gene expression as an explanation of any increased plasma LEAP2 concentrations in T2D and obesity.
As alluded to above, ghrelin is considered primarily a stomachderived hormone with some findings suggesting expression in the small intestine and the liver as well.Gnanapavan et al. found a slightly higher ghrelin expression in the jejunum compared to the duodenum before reaching a nadir in the ileum and increased in the colon [3].Ueberberg et.al found higher small intestinal ghrelin expression, though not investigated in specific segments, compared to the colon [4].Rats showed an expression profile with high gastric ghrelin expression that decreased throughout the small and large intestine [5,31] which is in line with decreasing gastrointestinal ghrelin tissue concentration through the gastrointestinal tract [5,6].Here, we provide a more detailed description of mucosal ghrelin mRNA expression along the entire length of the human intestinal tract showing robust expression in the duodenum with gradually decreasing expression levels along the small intestine and uniformly low expression levels along the large intestine and in the rectum.Interestingly, in small intestinal segments 7 and 8 (Fig. 2C), we observed higher ghrelin mRNA expression levels in individuals with T2D compared to healthy controls.As circulating levels of ghrelin have been reported to be diminished in T2D [22,32,33] and overweight/obesity [34], respectively, this may be a chance finding.Unpublished data from another study of ours using similar methodology show mean ghrelin mRNA expression levels of 375 RPKM in the gastric cardia of seven healthy individuals (mean age: 63.0 years, mean BMI: 23.8 kg/m2; mean HbA1c: 33.6 mmol/mol), i.e., ~40-fold higher than the duodenal expression levels observed in the present study in which gastric biopsies were not retrieved.
In our liver samples, we observed non-detectable or negligible levels of ghrelin mRNA (Fig. 2D), which is in contrast to previous findings showing some expression in healthy individuals and individuals with hepatic steatosis [3,4,23,35].Most biopsies analyzed in these studies were collected during autopsies performed 12-24 h postmortem [3] or from patients undergoing laparoscopic surgery [35].Ultrasound-guided transcutaneous liver biopsies retrieved in the current study were obtained from participants recruited for research purposes only, performed by experienced radiologists and immediately distributed into RNAlater and frozen at − 20⁰C until analysis [25].Hence, differences in biopsy retrieval and handling could account for some of the discrepancies found in hepatic ghrelin mRNA expression.
As several gut and liver-derived hormones exert important paracrine effects, we evaluated the intestinal expression profile and the hepatic expression of GHSR, the common receptor of ghrelin and LEAP2.GHSR was expressed in the liver and small and large intestine in rats [5,31].In mice, GHSR expression in the small and large intestine, but no hepatic expression was observed [18].In line with previous studies in humans, we did not find significant gene expression of GHSR in the liver [3,4,36] or along the intestinal tract [3,4].Despite a recent study demonstrating the presence of GHSR in human fibrotic liver tissue [23], the present and previous findings do not support GHSR-mediated paracrine effects of LEAP2 and ghrelin in the human intestinal tract or in the human liver.
Strengths of the present study include: 1) the frequent sampling of mucosal biopsies throughout the intestinal tract using double-balloon enteroscopy in dedicated study participants with T2D and matched healthy controls providing unprecedented granularity to the intestinal distribution pattern of LEAP2 and ghrelin mRNA; 2) investigation of transcutaneous liver biopsies sampled from men with obesity (otherwise healthy) and lean healthy men recruited for the purpose; and 3) the protocolized and, thus, uniform, handling and processing of biopsy material.Limitations include 1) the low number of individuals included, 2) lack of more specific confirmatory gene expression analysis (i.e.RT-PCR), and 3) the limited amount of tissue in the individual samples preventing us from undertaking quantitative or immunohistochemical analysis of peptides/proteins of interest.

Conclusion
In conclusion, we provide unique and detailed mRNA expression profiling of LEAP2, ghrelin and GHSR mRNA throughout the intestines of individuals with T2D and healthy controls, as well as comparisons of hepatic LEAP2, ghrelin and GHSR mRNA expression, respectively, in men with and without obesity.

Financial support
This work was supported by a research grant from the Danish Diabetes and Endocrine Academy, which is funded by the Novo Nordisk Foundation, grant number NNF22SA0079901.

Declaration of Generative AI and AI-assisted technologies in the writing process
During the preparation of this work, ChatGPT4, developed by OpenAI, was used to enhance English language and to assist in coding figures in RStudio.After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of Competing Interest
A.E has nothing to declare.H.G.V has received research support from Zealand Pharma.M.P.S and, L.S.G have nothing to declare.T.V. has served on scientific advisory panels or speakers' bureaus or has served as a consultant to or received research support from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Eli Lilly, Gilead, Merck Sharp & Dohme, Mundipharma, Novo Nordisk, Sanofi, and SunPharma.F.K.K. is employed by Novo Nordisk A/S as of December 1st, 2023.Before joining Novo Nordisk A/S, F.K.K. has served on scientific advisory panels and/or been part of speaker's bureaus for, served as a consultant to, and/or received research support from 89bio, Amgen, AstraZeneca, Boehringer Ingelheim, Carmot Therapeutics, Eli Lilly, Gubra, MedImmune, MSD/ Merck, Mundipharma, Norgine, Novo Nordisk, Sanofi, Zealand Pharma and Zucara.