
==== Front
Mol Metab
Mol Metab
Molecular Metabolism
2212-8778
Elsevier

S2212-8778(24)00133-9
10.1016/j.molmet.2024.102002
102002
Original Article
Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity
Wang Qian 1
Farhadipour Mona 1
Thijs Theo 1
Ruilova Sosoranga Emily 2
Van der Schueren Bart 34
Ceulemans Laurens J. 5
Deleus Ellen 6
Lannoo Matthias 6
Tack Jan 27
Depoortere Inge inge.depoortere@kuleuven.be
1⁎
1 Gut Peptide Research Lab, Translational Research for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
2 Translational Research for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
3 Department of Endocrinology, University Hospitals Leuven, Leuven, Belgium
4 Laboratory of Clinical and Experimental Endocrinology, University of Leuven, Leuven, Belgium
5 Leuven Intestinal Failure and Transplantation (LIFT) Center, University Hospitals Leuven, Leuven, Belgium
6 Department of Abdominal Surgery, University Hospitals Leuven, Leuven, Belgium
7 Department of Gastroenterology and Hepatology, University Hospitals Leuven, Leuven, Belgium
⁎ Corresponding author. inge.depoortere@kuleuven.be
05 8 2024
10 2024
05 8 2024
88 10200226 6 2024
1 8 2024
1 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Objective

Growth differentiation factor 15 (GDF15), a stress related cytokine, was recently identified as a novel satiety signal acting via the GFRAL receptor located in the hindbrain. Bitter compounds are known to induce satiety via the release of glucagon-like peptide 1 (GLP-1) through activation of bitter taste receptors (TAS2Rs, 25 subtypes) on enteroendocrine cells in the gut. This study aimed to investigate whether and how bitter compounds induce a stress response in intestinal epithelial cells to affect GDF15 expression in patients with obesity, thereby facilitating satiety signaling from the gut.

Methods

The acute effect of oral intake of the bitter-containing medication Plaquenil (hydroxychloroquine sulfate) on plasma GDF15 levels was evaluated in a placebo-controlled, double-blind, randomized, two-visit crossover study in healthy volunteers. Primary crypts isolated from the jejunal mucosa from patients with obesity were stimulated with vehicle or bitter compounds, and the effect on GDF15 expression was evaluated using RT-qPCR or ELISA. Immunofluorescence colocalization studies were performed between GDF15, epithelial cell type markers and TAS2Rs. The role of TAS2Rs was tested by 1) pretreatment with a TAS2R antagonist, GIV3727; 2) determining TAS2R4/43 polymorphisms that affect taste sensitivity to TAS2R4/43 agonists.

Results

Acute intake of hydroxychloroquine sulfate increased GDF15 plasma levels, which correlated with reduced hunger scores and plasma ghrelin levels in healthy volunteers. This effect was mimicked in primary jejunal cultures from patients with obesity. GDF15 was expressed in enteroendocrine and goblet cells with higher expression levels in patients with obesity. Various bitter-tasting compounds (medicinal, plant extracts, bacterial) either increased or decreased GDF15 expression, with some also affecting GLP-1. The effect was mediated by specific intestinal TAS2R subtypes and the unfolded protein response pathway. The bitter-induced effect on GDF15/GLP-1 expression was influenced by the existence of TAS2R4 amino acid polymorphisms and TAS2R43 deletion polymorphisms that may predict patient's therapeutic responsiveness. However, the effect of the bitter-tasting antibiotic azithromycin on GDF15 release was mediated via the motilin receptor, possibly explaining some of its aversive side effects.

Conclusions

Bitter chemosensory and pharmacological receptors regulate the release of GDF15 from human gut epithelial cells and represent potential targets for modulating metabolic disorders or cachexia.

Highlights

• A bitter pill increases GDF15 plasma levels and decreases hunger scores in healthy volunteers.

• GDF15 is expressed in enteroendocrine and goblet cells with higher expression levels in patients with obesity.

• Various bitter compounds affect GDF15/GLP-1 expression in primary intestinal cultures from patients with obesity.

• TAS2R4 and TAS2R43 in the gut represent novel targets to increase or decrease GDF15 and/or GLP-1 expression.

• The motilin receptor, not TAS2Rs, mediates azithromycin-induced GDF15 expression, possibly explaining some side effects.

Keywords

GDF15
GLP-1
Obesity
Bitter
Motilin receptor
Integrated stress response pathway
TAS2R polymorphisms
==== Body
pmc1 Introduction

Growth differentiation factor 15 (GDF15), an atypical member of the TGF-β superfamily, is a cellular stress-induced cytokine involved in mitochondrial stress and the unfolded protein response (UPR) [1]. Plasma GDF15 levels are increased in a wide range of diseases, such as cancer [2], inflammation [2], cardiovascular disease [3], and during dysregulation of energy homeostasis [4]. GDF15 is therefore currently considered as a biomarker predicting adverse disease outcomes [3] but also as a target to treat metabolic disorders [5] or cancer-induced cachexia [6]. Indeed, body weight and food intake were reduced in obese mice and monkeys overexpressing GDF15 or administered long-acting recombinant GDF15 [7]. Neutralization of GDF15 or its receptor with a monoclonal antibody improved cancer or chemotherapy-induced anorexia and weight loss in mice and/or nonhuman primates [8,9]. A recent 12-week multiple ascending dose study in overweight or obese individuals showed that administration of a long-lasting analog of GDF15, LY3463251, decreased food intake and appetite scores, resulting in a modest reduction in body weight [10]. Additionally, GDF15 delayed gastric emptying through a vagal-mediated mechanism [7] which may contribute to the decrease in hunger signaling and initiated lipolysis via the sympathetic nervous system [7,8].

The anorexic effects of GDF15 are mediated by binding to glial cell-derived neurotrophic factor family receptor α-like (GFRAL) and the recruitment of the receptor tyrosine kinase RET [11]. GFRAL is located exclusively in the area postrema and the nucleus tractus solitarius of the hindbrain and plays a distinct role in processing peripheral feeding signals to regulate food intake compared to the hypothalamus [12].

The area postrema is also referred to as the chemoreceptor trigger zone and has a specialized role in monitoring toxic substances and regulating vomiting. Several studies have suggested that GDF15 acts as a defense signal to promote a behavioral program of toxin avoidance such as aversion if threshold levels are exceeded [13]. Many toxic substances and medical drugs taste bitter, such as the anti-diabetic medicine metformin or the flavonoid resveratrol [14]. Both drugs increase GDF15 expression [15,16] and induce body weight loss [15,17]. Studies in both mice and humans showed that body weight loss induced by metformin is mediated via GDF15-GFRAL signaling pathway [15,18].

Ingestion of bitter compounds activates bitter taste receptors on taste buds of the tongue that will initiate an aversive response to prevent the further ingestion of harmful substances. Bitter taste receptors are G protein-coupled receptors (GPCRs) that belong to the taste two receptor family (TAS2Rs) [19], of which roughly 25 subtypes exist in humans [20]. However, TAS2Rs are also present outside the oral cavity, such as the gastrointestinal tract and adipose tissue, and have been suggested as possible therapeutic targets to treat obesity [21,22]. In the gut activation of TAS2Rs on enteroendocrine cells by bitter compounds regulates the release of appetite regulating hormones, such as ghrelin [23] and glucagon-like peptide 1 (GLP-1), [24] in humans. In diet-induced obese mice, chronic intra-gastric administration of bitter compounds decreased body weight and improved multiple metabolic parameters [21,24]. In healthy individuals, intra-gastric administration of denatonium benzoate or quinine reduced hunger scores by suppressing the release of gut hormones and gastric motility and decreased hedonic eating by altering brain activity in homeostatic and hedonic brain regions [[25], [26], [27], [28], [29]]. A recent phase 1 clinical trial confirmed that oral ingestion of encapsulated denatonium acetate (ARD-101) elevated circulating levels of GLP-1 in healthy volunteers, with most exposure of the drug being restricted to the gut [30].

This study aimed to investigate whether the effect of bitter compounds on satiety signaling is not only mediated via the release of gut hormones but may also be facilitated via a separate anorexic neuronal system involving the release of GDF15. We showed that oral ingestion of Plaquenil, containing the bitter compound hydroxychloroquine, increased GDF15 levels correlating with a decrease in hunger scores in healthy volunteers. Using primary jejunal cell cultures from patients with obesity, we confirm that various bitter compounds in the gut induce a stress response resulting in an increase or decrease in GDF15 release, often accompanied by an effect on GLP-1 secretion. The effect of certain bitter compounds was mediated through the activation of TAS2Rs, while others, like the bitter antibiotic azithromycin, act via activation of the motilin receptor, possibly explaining some of its side effects. Amino acid/deletion polymorphisms in TAS2Rs were used to show a role for TAS2R4 and TAS2R43 in the effect of quorum sensing molecules and aloin on GDF15 and/or GLP-1 expression. This insight holds promise for predicting therapeutic responsiveness in patients with obesity who may benefit from the combined release of endogenous GDF15 and GLP-1 acting on different neuronal pathways to control satiety.

2 Results

2.1 Oral intake of Plaquenil increases GDF15 plasma levels in normal-weight volunteers which inversely correlate with hunger scores and ghrelin plasma levels

The acute effects of oral intake of Plaquenil, containing the bitter drug hydroxychloroquine sulphate (HCQS), on plasma GDF15 levels were measured in healthy volunteers (n = 10) in a placebo-controlled, double-blind, randomized, two-visit crossover study. At baseline (t = −10 min), no difference was observed in plasma GDF15 levels between placebo conditions (369 ± 31 pg/mL) and Plaquenil conditions (387 ± 33 pg/mL). The trendline showing the time-dependent changes in ln-fold change in plasma GDF15 levels was significantly (P < 0.01) different between the placebo and Plaquenil condition (Figure 1A). Post-hoc analysis showed that Plaquenil administration significantly (P < 0.05) increased GDF15 plasma levels in healthy individuals at 90 min. The increase in GDF15 levels was not due to the consumption of the milkshake (hedonic food intake) at 60 min, as extrapolation of GDF15 plasma levels from the fasted period (0–60 min) did not differ from the GDF15 plasma levels in the fed state at 90 min.Figure 1 Effect of oral intake of Plaquenil on GDF15 and ghrelin plasma levels, and hunger scores in healthy volunteers.

A) Time-dependent changes in plasma GDF15 levels after oral ingestion of Plaquenil or placebo in healthy volunteers (n = 10; ∗P < 0.05: versus baseline [−10 min], ##P < 0.01: versus the placebo condition; ANCOVA mixed model). Dashed line: trendline in the fasted state; dotted line: extrapolation from trendline in the fasted state.

B and C) Oral intake of Plaquenil, but not placebo, resulted in a negative correlation between GDF15 plasma levels and hungers scores measured between 0 and 90 min after administration. (n = 10; Pearson correlation coefficient within individuals was transformed into Fisher z to calculate the average coefficient [r] and P values).

D and E) Oral intake of Plaquenil, but not placebo, resulted in a negative correlation between GDF15 plasma levels and ghrelin plasma levels measured between 0 and 90 min after administration. (n = 10; Pearson correlation coefficient within individuals was transformed into Fisher z to calculate the average coefficient [r] and P values).All data were presented as mean ± SEM.

Figure 1

Pearson's correlation coefficients between ln-fold change in GDF15 plasma levels and changes in hunger scores or plasma levels of the hunger hormone ghrelin were calculated for each participant (0–90 min) and the average was assessed after Fisher z-transformation. A negative correlation between ln-fold change in GDF15 plasma levels and delta hunger scores was observed in the Plaquenil condition (Fisher z-transformed r = −0.61, P < 0.01), but not in the placebo condition (Fisher z-transformed r = −0.07, P = 0.78) (Figure 1B, C). In addition, changes in GDF15 plasma levels showed a significant negative correlation with changes in ghrelin plasma levels in the Plaquenil condition (Fisher z-transformed r = −0.53, P < 0.05) but not in the placebo condition (Fisher z-transformed r = 0.03, P = 0.89) (Figure 1D, E).

Taken together, these findings suggest that the bitter drug Plaquenil prompts the release of GDF15, and that this effect cannot be reversed by consumption of a rewarding chocolate milkshake ad libitum. Moreover, the inverse relationship observed between GDF15 plasma levels and delta hunger scores or ghrelin plasma levels, suggests that the release of GDF15 along with a reduction in ghrelin plasma levels play a role in reducing hunger scores after Plaquenil administration in healthy volunteers.

2.2 GDF15 is expressed in goblet and enteroendocrine cells from the jejunum, with higher expression levels in patients with obesity

We hypothesized that the gut may be a potential source of circulating plasma GDF15 levels after intragastric administration of Plaquenil. In addition, because bitter agonists and GDF15 are considered possible targets for the treatment of obesity, we compared the GDF15 mRNA expression in the proximal gut of normal-weight individuals and patients with obesity [23,31]. The mRNA expression of GDF15 differed between both populations in a region-dependent manner (population × region: P < 0.001) (Figure 2A). In normal-weight individuals, the mRNA expression levels of GDF15 were evenly distributed, with only the antrum showing significantly higher expression levels (P < 0.05) compared with the jejunum. In patients with obesity, GDF15 expression levels were markedly increased (P < 0.001) in the jejunum relative to the other three parts of the stomach. Moreover, the expression levels of GDF15 in the jejunum were 82-fold higher (P < 0.001) in patients with obesity compared with normal-weight individuals. Even though analysis of GDF15 at the protein level showed no difference in the number of GDF15+ cells, a significant increase (P < 0.05) in the intensity of the staining of individual GDF15+ cells was observed in patients with obesity compared with normal-weight individuals (Figure 2B, C).Figure 2 Distribution and characterization of GDF15 expressing epithelial cells in the proximal gut of normal-weight individuals and patients with obesity.

A) Relative mRNA expression (efficiency−ΔΔCt method) of GDF15 in resection specimens from the fundus (nNW = 7, nOB = 10), corpus (nNW = 5, nOB = 8), antrum (nNW = 5, nOB = 8), and jejunum (nNW = 5, nOB = 7) of normal-weight individuals and patients with obesity. (Proc Mixed Model with Šidák correction for multiple comparisons).

B) Representative single-immunofluorescence staining for GDF15 in jejunal sections (10 μm) of a normal-weight individual and a patient with obesity. GDF15+ cells were stained with Cy3 (green) and nuclei were stained with DAPI (blue). Scale bars: 25 μm.

C) Average number and intensity of GDF15+ cells in jejunal tissue sections from both normal-weight individuals and patients with obesity (nNW = 5, nOB = 5; two-tailed unpaired Student's t-test).

D–H) Representative double-immunofluorescence staining for GDF15 (green [Cy3 or Alexa594]) with markers for (D) goblet (MUC2: red [Cy5]), (E) Paneth (α-defensin 6: red [Cy5]) or (F–G) enteroendocrine (CHGA: red [Alexa488]) and ghrelin (red [Alexa488]) cells in jejunal sections (10 μm) from normal-weight individuals.

Arrows indicate co-localization. Normal rabbit serum was used as negative control. Nuclei were labeled with DAPI (blue). Scale bars: 25 μm. NW: normal-weight, OB: obese. Data of figure A, C represent mean ± SEM and single values are plotted. ∗P < 0.05: versus jejunum in normal-weight individuals, $$$P < 0.001: versus jejunum in patients with obesity, #P < 0.05, ###P < 0.001: versus normal-weight individuals.

Figure 2

To investigate which epithelial cell types produce GDF15 in the small intestine, double immunofluorescence studies were performed between GDF15 and several markers for gut epithelial cells in jejunal tissue sections from normal-weight individuals. 80 ± 4% of MUC2-positive cells (goblet cell marker) co-localized with GDF15, while no co-staining was observed between α-defensin 6-containing cells (Paneth cell marker) and GDF15 (Figure 2D, E). Moreover, 66 ± 7% of chromogranin A (CHGA) positive cells (enteroendocrine cell marker) were GDF15-positive, whereas co-staining between ghrelin jejunal cells and GDF15 was rare (13 ± 9%) (Figure 2F, G). In summary, GDF15 is produced in secretory goblet and enteroendocrine cells of the proximal intestine in normal-weight individuals. Obesity specifically increases GDF15 expression in the jejunum but not in the stomach.

2.3 Bitter compounds affect GDF15 and GLP-1 levels in primary jejunal crypts from patients with obesity

The jejunum is the primary site for the expression of GDF15 in patients with obesity, we therefore aimed to investigate whether Plaquenil, but also other bitter compounds, could influence GDF15 expression in vitro in primary jejunal crypts. Bitter compounds that trigger the endogenous release of two satiety hormones hold therapeutic promise for patients with obesity. Consequently, their impact on the expression of GLP-1, another satiety hormone, was studied in parallel (for an overview see Table 1, Figure 4F).Table 1 Overview of the effect of different bitter agonists on GDF15 or GLP-1 mRNA expression in primary crypts from patients with obesity.

Table 1Bitter compounds	Origin	TAS2Rs subtype	Conc. (mM)	Ref.	GDF15 mRNA expression
% of vehicle (n)	GLP-1 mRNA expression
% of vehicle (n)	
Generalists	
Quinine (QUI)	Medical drug	4, 7, 10, 14, 31, 39, 40, 43, 46	0.5	[32]	118 ± 21 (6)	15 ± 4∗∗∗ (11)	
Denatonium benzoate (DB)	Synthetic	4, 8, 10, 13, 30, 39, 43, 46	0.5	[32]	143 ± 14∗∗ (9)	116 ± 11 (9)	
Intermediates	
N-octanoyl-L-homoserine lactone (C8-AHL)	Bacterial (quorum sensing)	4, 14	0.1	[33]	101 ± 5 (6)	91 ± 17 (5)	
N-(3-oxododecanoyl)-L-homoserine lactone (C12-O-AHL)	Bacterial (quorum sensing)	4, 14, 20	0.15	[33]	128 ± 11 (7)	99 ± 7 (14)	
Azithromycin (AZI)	Medical drug	4, 14, 20	0.075	[34]	163 ± 8∗∗ (5)	105 ± 9 (7)	
1	[34]	437 ± 44∗∗∗ (4)	66 ± 16∗∗∗ (6)	
Tobramycin (TOB)	Medical drug	14, 20	1	[34]	91 ± 7 (5)	144 ± 41 (4)	
Resveratrol	Plant-derived (polyphenol)	14, 39	0.3	[35]	102 ± 19 (5)	N/A	
Emetine	Plant-derived	14, 46	0.3	[36]	91 ± 18 (4)	28 ± 6∗∗ (6)	
Aloin	Plant-derived (aloe vera)	43,44	0.1	[32]	89 ± 6 (6)	N/A	
Specialists	
Gallic acid (GA)	Plant-derived (phenolic acid)	4	0.01	[37]	112 ± 10 (5)	138 ± 45 (4)	
1	[37]	314 ± 31∗∗∗ (5)	75 ± 9∗∗∗ (16)	
1,10-Phenanthroline (PHE)	Synthetic	5	0.1	[32]	175 ± 13∗∗∗ (5)	74 ± 15 (5)	
Erythromycin A (EM)	Medical drug	10	0.3	[32]	120 ± 11 (4)	N/A	
1	[32]	408 ± 46∗∗∗ (5)	30 ± 15∗∗∗ (3)	
Cucurbitacin E (CuE)	Plant-derived	10	0.01	[32]	124 ± 14 (6)	N/A	
Diclofenac (DIC)	Medical drug	14	0.15	[38]	119 ± 15 (5)	88 ± 14 (5)	
Flufenamic acid (FFA)	Medical drug	14	0.01	[32]	158 ± 37 (4)	110 ± 25 (4)	
Salicylic acid (SA)	Medical drug	14	1	[39]	120 ± 2 (4)	113 ± 41 (3)	
Sodium benzoate (SB)	Synthetic	14	1	[32]	106 ± 10 (6)	N/A	
Phenylthiocarbamide (PTC)	Synthetic	38	1	[32]	110 ± 6 (6)	99 ± 11 (6)	
Acetaminophen (ACE)	Medical drug	39	3	[32]	66 ± 4∗ (5)	84 ± 9 (4)	
Berberine (BERB)	Plant-derived (alkaloid)	46	0.1	[40]	163 ± 6∗∗∗ (4)	100 ± 12 (4)	
Data shown as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: versus vehicle group. Proc Mixed Model was used for the statistical analysis with Šidák correction for multiple comparisons.

In agreement with our in vivo study, HCQS (100 μM) increased GDF15 mRNA expression (P < 0.05) in primary crypts from patients with obesity after 4 h of stimulation (Figure 3A). In contrast, HCQS decreased (P = 0.05) GLP-1 mRNA expression (Figure 3B). Previous studies showed that weight loss induced by the anti-diabetic drugs metformin and phenformin was linked to elevated GDF15 levels [16]. Since these drugs taste bitter, we explored their potency to increase GDF15 mRNA expression in the gut. Our results confirmed a 2.8-fold increase (P < 0.001) in GDF15 mRNA expression after stimulation of primary jejunal crypts from patients with obesity with the more soluble form phenformin (2.5 mM), but not with metformin (2.5–5 mM) (Supplemental Fig. 1).Figure 3 Effects of bitter generalists and intermediates on GDF15 or GLP-1 expression in jejunal crypts from patients with obesity.

A) Effect of 4-hour stimulation with 100 μM HCQS on relative GDF15 mRNA expression (efficiency−ΔΔCt method) (n = 5; two-tailed paired Student's t-test).

B) Effect of 4-hour stimulation with 100 μM HCQS on relative GLP-1 mRNA expression (efficiency−ΔΔCt method) (n = 5; two-tailed paired Student's t-test).

C) Effect of bitter generalists on fold change in relative GDF15 mRNA expression after 4 h of stimulation (n = 6–9; Proc Mixed Model with Šidák correction for multiple comparisons).

D) Effect of bitter intermediates on fold change in relative GDF15 mRNA expression after 4 h of stimulation (n = 4–6; Proc Mixed Model with Šidák correction for multiple comparisons).

E) Relative GDF15 secretion in the supernatant of primary crypts from patients with obesity stimulated with 1 mM azithromycin. Azithromycin was removed after 4 h for the 24-hour timepoint (n = 3–6; one-tailed paired Student's t-test).

F) Relative GLP-1 secretion in the supernatant of primary crypts from patients with obesity stimulated with 1 mM azithromycin for 6 h (n = 3; two-tailed paired Student's t-test).

All data are present as mean ± SEM and single values are plotted. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: versus the vehicle group.

Figure 3

The 25 human TAS2R subtypes have a wide range of activation thresholds and are sensitive to bitter compounds that are structurally very different [32]. Additional bitter compounds were tested and grouped according to the number of TAS2Rs they activate: generalists (activating >5 TAS2Rs); intermediates (activating 2–4 TAS2Rs), and specialists (specific for 1 TAS2R). Furthermore, compounds belonged to different categories (synthetic, bacterial quorum sensing, plant-derived) including medical drugs that induce satiety and nausea as side effects (Table 1).

The generalist denatonium benzoate, widely recognized as one of the most bitter tasting compounds(activating 8 TAS2R subtypes), induced a 1.4-fold increase (P < 0.01) in GDF15 mRNA expression (Figure 3C). Another generalist, quinine (0.5 mM), activating 9 TAS2Rs (5 in common with DB), did not affect GDF15 expression compared with vehicle-treated crypts (Figure 3C). However, quinine but not DB induced a 6.7-fold (P < 0.001) decrease in GLP-1 mRNA expression (Table 1).

Among the intermediates, only the antibiotic azithromycin, a TAS2R4, -14, -20 agonist, induced a substantial increase (4.4-fold, P < 0.001) in GDF15 mRNA expression and a decrease (1.5-fold, P < 0.001) in GLP-1 mRNA levels in a concentration-dependent manner (Figure 3D and Table 1). GDF15 secretion in the primary crypt supernatant increased in a time-dependent manner after stimulation with 1 mM azithromycin (Figure 3E). Despite the decrease in GLP-1 mRNA expression, GLP-1 secretion in response to stimulation with azithromycin was increased (P < 0.01) in the cell culture supernatant (Figure 3F).

The bitter-intermediate emetine, a TAS2R14 and TAS2R46 agonist, caused a notable reduction in GLP-1 mRNA expression (P < 0.01), but did not affect GDF15 mRNA expression levels (Table 1). Other bitter intermediates, such as the quorum sensing homoserine lactones C8-AHL (TAS2R4, -14) and C12-O-AHL (TAS2R4, -14, -20), the antibiotic tobramycin (TAS2R14, -20), the polyphenol resveratrol (TAS2R14, -39), and the plant extract aloin (TAS2R43, -44), had no significant effect on either GDF15 or GLP-1 mRNA expression (Figure 3D and Table 1).

The bitter specialist, gallic acid (1 mM), a phenolic acid that activates TAS2R4, strongly increased (3.1-fold, P < 0.001) GDF15 mRNA expression and decreased (1.3-fold, P < 0.001) GLP-1 mRNA expression (Figure 4A and Table 1). The effect of gallic acid on the secretion of GDF15 was evaluated via immunofluorescence staining since the brown color of gallic acid is interfering with the GDF15 ELISA (Figure 4B). Gallic acid decreased the average fluorescence intensity of GDF15+ cells by 38 ± 8% (P < 0.001; Figure 4C) at 24 h after a 4 h stimulation with gallic acid, pointing towards increased release of GDF15. Moreover, the antibiotic erythromycin A, structurally related to azithromycin but a selective TAS2R10 agonist, increased (4.1-fold, P < 0.001) GDF15 mRNA expression but decreased (3.3-fold, P < 0.001) GLP-1 mRNA expression (Figure 4A). In addition, bitter specialists, such as the synthetic TAS2R5 agonist, 1,10-phenanthroline (0.1 mM) and the plant alkaloid, berberine (0.1 mM), acting as a TAS2R46 agonist, significantly (P < 0.001) upregulated GDF15 mRNA expression 1.8-fold and 1.6-fold, respectively (Figure 4A). Of the bitter specialists targeting TAS2R14, only the nonsteroidal anti-inflammatory drug (NSAID), flufenamic acid increased (1.6-fold, P = 0.05) GDF15 expression (Figure 4A). None of the TAS2R5, TAS2R14 or TAS2R46 agonists affected GLP-1 mRNA expression.Figure 4 Effects of bitter specialists on GDF15 mRNA or protein expression in primary jejunal crypts from patients with obesity.

A) Effect of specialists on relative GDF15 mRNA expression after 4 h of stimulation (n = 4–7; Proc Mixed Model with Šidák correction for multiple comparisons).

B) Representative single-immunofluorescence staining for GDF15 (green [Cy3]) in primary crypts treated with 1 mM gallic acid or vehicle after 24 h with 4 h stimulation. Nuclei were labeled with DAPI (blue). Scale bars: 20 μm.

C) Average intensity of GDF15+ cells in primary crypts treated with 1 mM gallic acid or vehicle. Gallic acid was removed after 4 h of stimulation and staining was performed 20 h later (n = 4; two-tailed paired Student's t-test).

D) Relative GDF15 secretion in the supernatant of primary crypts stimulated with 3 mM acetaminophen for 6 h (n = 3; one-tailed paired Student's t-test).

E) Relative GDF15 secretion in the supernatant of primary crypts stimulated with 3 mM acetaminophen. Acetaminophen was removed after 4 h for the 24-hour timepoint (n = 3; one-tailed paired Student's t-test).

F) Venn diagram of bitter compounds that affected GDF15 and/or GLP-1 mRNA expression after stimulation or primary crypts for 4 h.

Asterisk (∗) indicates an inhibitory effect on mRNA expression; the use of hashtags (#) signifies potential variations in the effects on GDF15 or GLP-1 mRNA, depending on the genotype of the patients. Data of figure A, C, D, E are present as mean ± SEM and single values are plotted. ∗∗P < 0.01, ∗∗∗P < 0.001: versus the vehicle group.

Figure 4

Interestingly, acetaminophen, targeting TAS2R39, significantly decreased (−1.5-fold, P < 0.01) GDF15 mRNA expression (Figure 4A) and tended (P = 0.07) to decrease the relative GDF15 secretion in the culture supernatant (Figure 4D, E). These results indicate that bitter taste receptor agonists may not only activate but also inhibit GDF15 expression.

In summary, bitter compounds stimulate or inhibit GDF15 expression in a TAS2R subtype-dependent manner. In addition, bitter compounds activating TAS2R4 and TAS2R10 influence the expression of both GDF15 and GLP1 while others (emetine and quinine) are selective for GLP-1 alone (Figure 4F).

2.4 TAS2Rs mediate the effects of bitter agonists on GDF15 and GLP-1 mRNA expression

To confirm the involvement of TAS2Rs in the effect of bitter compounds on the mRNA expression of GDF15 and GLP-1, a bitter taste receptor antagonist GIV-3727 (110 μM), targeting TAS2R4, -7, -20, -31, -40, and -43, was used [41]. GIV-3727 blocked the increase in GDF15 mRNA expression but not the inhibition of GLP-1 mRNA expression induced by gallic acid (1 mM) (Figure 5A, B). Similarly, the effect of azithromycin (75 μM) on GDF15 mRNA expression was not blocked by GIV-3727 (Figure 5C).Figure 5 Role of TAS2Rs and the motilin receptor in the effect of bitter agonists on GDF15 or GLP-1expression in primary crypts from patients with obesity.

A) The TAS2R antagonist, GIV3727 (110 μM) blocked the effect of gallic acid (1 mM) on GDF15 mRNA expression (efficiency−ΔΔCt method) (n = 4; Proc Mixed Model with Šidák correction for multiple comparisons).

B) GIV3727 (110 μM) did not block the effect of gallic acid (1 mM) on GLP-1 mRNA expression (efficiency−ΔΔCt method) (n = 4; Proc Mixed Model with Šidák correction for multiple comparisons).

C) GIV3727 (110 μM) did not block the effect of azithromycin (75 μM) on GDF15 mRNA expression (efficiency−ΔΔCt method) (n = 3; Proc Mixed Model with Šidák correction for multiple comparisons).

D) The motilin receptor antagonist MA-2029 blocked the effect of azithromycin (75 μM) on GDF15 mRNA expression (efficiency−ΔΔCt method) (n = 4; Proc Mixed Model with Šidák correction for multiple comparisons).

E) C12-O-AHL (0.15 mM) increased relative GDF15 mRNA expression in primary crypts from obese patients with TAS2R4(GG/CG) (n = 9) but not TAS2R4(CC) (n = 4) genotype (Proc Mixed Model with Šidák correction for multiple comparisons).

F) C12-O-AHL (0.15 mM) decreased GLP-1 mRNA expression in primary crypts from obese patients with the TAS2R4(GG/CG) (n = 9) but not the TAS2R4(CC) genotype (n = 4). (Proc Mixed Model with Šidák correction for multiple comparisons).

G) Aloin at 30 μM (white dots) and 100 μM (black dots) decreased relative GDF15 mRNA expression in primary crypts from obese patients with the TAS2R43(+)GG/CG (n = 9) but not the TAS2R43(+)CC (n = 6) or TAS2R43(−) (n = 6) genotype (Proc Mixed Model with Šidák correction for multiple comparisons).

H) Aloin at 30 μM (white dots) and 100 μM (black dots) did not affect the relative GLP-1 mRNA expression in primary crypts from obese patients with the TAS2R43(+) GG/GC (n = 9), TAS2R43(+) CC (n = 6), or TAS2R43(−) (n = 4) genotype (Proc Mixed Model with Šidák correction for multiple comparisons).

I–K) Representative double-immunofluorescence images for GDF15 (green [Cy3]) and TAS2R4 (red [Cy5]), TAS2R43 (red [Cy5]) or TAS2R10 (red [Cy5]) in jejunal sections (10 μm thickness) from normal-weight populations. Arrows indicate co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 20 μm.

M–O) Representative double-immunofluorescence images for GDF15 (green [Cy3]) and TAS2R4 (red [Cy5]), TAS2R43 (red [Cy5]) or TAS2R10 (red [Cy5]) in primary jejunal crypt from patients with obesity.

Arrows indicate co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 20 μm. (L and P) Normal rabbit serum as a negative control. Nuclei were labeled with DAPI (blue). Scale bars: 20 μm. Data of figure A–H are presented as mean ± SEM and single values are plotted. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: versus vehicle group, ##P < 0.01, ###P < 0.001: bitter treatment × antagonist, $P < 0.05: versus TAS2R4(CC) population, &P < 0.05: versus TAS2R43(−) population.

Figure 5

Due to the lack of more specific TAS2R antagonists, the presence of several TAS2R single nucleotide polymorphisms (SNPs) were used to assess the role of TAS2Rs in the regulation of GDF15 expression [42]. The TAS2R4 gene polymorphism rs2234001 (G286-C), which results in an amino acid change at position 96 from valine to leucine, is associated with a phenotype that allows to perceive either the bitter (GG/CG genotype) or sweet taste (CC genotype) of stevioside, respectively [43]. Significant differences between TAS2R4 genotypes were observed in the effects of C12-O-AHL (TAS2R4, -14, -20), which initially did not affect GDF15 or GLP-1 mRNA expression due to high variability (Figure 3D). In patients with the bitter sensitive TAS2R4 (GG/CG) genotype, C12-O-AHL increased (P < 0.05) GDF15 mRNA expression but not in patients with the TAS2R4 (CC) genotype (Figure 5E, F). Similarly, C12-O-AHL decreased (P < 0.05) GLP-1 mRNA expression in TAS2R4 (GG/CG) patient, while tending to even increase (P = 0.06) GLP-1 mRNA expression in TAS2R4 (CC) patients (Figure 5F).

In addition, we evaluated the effect of the SNP (rs68157013 (C104-G)) within the TAS2R43 gene, leading to a tryptophan–serine alteration at position 35. This alteration determines oral sensitivity to the TAS2R43 agonist aloin [44], with (TAS2R43(+) GG/CG) individuals exhibiting heigh sensitivity to the bitterness of aloin, while those with the (TAS2R43(+) CC) genotype being less sensitive. In addition, a deletion polymorphism exists for TAS2R43 (TAS2R43(−)). In our population of patients with obesity, 29% was TAS2R43(−). To obtain sufficient data for this sub-analysis, we combined the results from the experiments conducted with two concentrations (30 and 100 μM) of aloin on GDF15 mRNA expression. Aloin which initially did not affect GDF15 mRNA expression, significantly inhibited (P < 0.05) the relative GDF15 mRNA expression in the highly sensitive TAS2R43(+) GG/CG patients but not in TAS2R43(+) CC or TAS2R43(−) patients with obesity (Figure 5G). In contrast, the TAS2R43 genotype did not affect GLP-1 mRNA expression after the stimulation of aloin (Figure 5H).

Double immunofluorescence co-localization studies confirmed colocalization between TAS2Rs and GDF15 in jejunal tissue sections from normal-weight individuals and in primary jejunal crypt cultures from patients with obesity. GDF15+ cells colocalized for 48 ± 10% with TAS2R4+, for 51 ± 14% with TAS2R10+, and for 38 ± 13% with TAS2R43+ cells in tissue sections from normal-weight individuals (Figure 5I–K). This co-staining between GDF15+ and TAS2R4+ (67 ± 9%), TAS2R10+ (84 ± 9%) or TAS2R43+ (64 ± 8%) was also confirmed in primary jejunal crypt cultures from patients with obesity (Figure 5M–O).

In conclusion, TAS2Rs are involved in the regulation of GDF15 mRNA expression by gallic acid, but not by azithromycin. C12-O-AHL affects both GDF15 and GLP-1 mRNA expression via TAS2R4, but the effect is influenced by genetic variants. Aloin inhibits GDF15 mRNA expression but does not affect GLP-1 mRNA expression via activation of TAS2R43 in a genotype-dependent manner.

2.5 Motilin receptor mediates the effects of azithromycin on GDF15 mRNA expression

The antibiotics azithromycin and erythromycin are known to accelerate gastric emptying via activation of the motilin receptor (MTLR) [45]. Since the effect of azithromycin was not blocked by the TAS2R antagonist, GIV3727, we hypothesized that the azithromycin-induced increase in GDF15 mRNA expression is mediated via the MTLR. Indeed, the MTLR antagonist MA-2029 [46], blocked the azithromycin-induced increase in GDF15 mRNA expression (Figure 5D). MA-2029 alone did not affect GDF15 mRNA expression. Taken together, the MTLR, but not TAS2Rs, mediate the increase in GDF15 mRNA expression induced by azithromycin.

2.6 The unfolded protein response signaling pathway regulates gallic acid-induced GDF15 mRNA expression

Our previous transcriptomic study unveiled that the bitter agonist, denatonium benzoate, induced an unfolded protein response and upregulated ATF4 and DDIT3 (gene of CHOP) mRNA expression in primary crypts from patients with obesity [47]. The relationship between ATF4 and DDIT3 mRNA expression and the induction of GDF15 mRNA expression by various bitter agonists (denatonium benzoate, azithromycin, gallic acid, 1,10-phenanthroline, erythromycin, acetaminophen, and berberine) was further investigated in crypt preparations from patients with obesity. The effect of the different bitter agonists tested on GDF15 mRNA expression showed a significant positive correlation with DDIT3 (r = 0.90, P < 0.05) and ATF4 (r = 0.70, P = 0.055) mRNA expression (Figure 6A, B). Furthermore, a significant correlation (r = 0.94; P < 0.001) was observed between the expression of transcription factor ATF4 and DDIT3 expression induced by bitter stimulation (Figure 6C). ISRIB, an inhibitor of eIF2α kinase that suppresses the translation of ATF4 and consequently CHOP, blocked (P < 0.01) the effect of gallic acid on GDF15 mRNA expression and confirmed a role for the unfolded protein response pathway in the effect of bitter agonists on GDF15 mRNA expression (Figure 6D). However, ISRIB failed to block the inhibitory effect of gallic acid on GLP-1 mRNA expression (Figure 6E). ISRIB itself did not show any effects on either GDF15 or GLP-1 mRNA expression. In conclusion, gallic acid activates TAS2Rs and the unfolded protein response pathway to increase the expression of GDF15 but not of GLP-1.Figure 6 Role of the unfolded protein response pathway in the effect of bitter agonists on GDF15 and GLP1 mRNA expression in primary crypts from patients with obesity.

A) Positive correlation between Log2 fold change of GDF15 and DDIT3 mRNA expression induced by bitter agonists (bitter: DB, AZI, GA, PHE, EM, ACE, BERB; n = 4–5/bitter treatment; Pearson correlation coefficient [r]; P value on graph).

B) Positive correlation between Log2 fold change of GDF15 and ATF4 mRNA expression induced by bitter agonists from figure A (n = 4–5/bitter treatment; Pearson correlation coefficient [r]; P value on graph).

C) Positive correlation between Log2 fold change of DDIT3 and ATF4 mRNA expression induced by bitter agonist from figure A (n = 4–5/bitter treatment; Pearson correlation coefficient [r]; P value on graph).

D) ISRIB (5 μM) blocked the effect of gallic acid (1 mM) on GDF15 mRNA expression (n = 3; Proc Mixed Model with Šidák correction for multiple comparisons).

E) ISRIB (5 μM) did not affect the effects of gallic acid (1 mM) on GLP-1 mRNA expression (n = 2; Proc Mixed Model with Šidák correction for multiple comparisons).

Data are presented as mean ± SEM and single values are plotted. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: versus vehicle group, ##P < 0.01: bitter treatment × antagonist.

Figure 6

3 Discussion

This study is the first to show that intragastric administration of the bitter compound, Plaquenil increased GDF15 levels in vivo in healthy volunteers, correlating with a reduction in hunger scores. The effect on GDF15 expression in response to a variety of bitter compounds was mimicked in vitro in gut epithelial cells from patients with obesity. For the polyphenol gallic acid, the effect was mediated through TAS2Rs and the integrated stress response pathway. Bacterial quorum sensing molecules increased the expression of both GDF15 and GLP-1 in a TAS2R4 genotype-dependent manner, suggesting a dual role of TAS2R4 in the induction of satiety. In contrast, we identified TAS2R39 and TAS2R43 as receptors that selectively inhibit the expression of GDF15 indicating their putative role in the treatment of cachexia. Finally, we show for the first time that the motilin receptor, and not TAS2Rs, in primary crypts regulates the release of GDF15 in response to the bitter-tasting antibiotic azithromycin. It is tempting to speculate that this may be the mechanism for the side effects, such as nausea, induced by macrolide antibiotics apart from their gastrointestinal side effects which are mediated by the motilin receptor in the smooth muscle layer. In summary, the expression of GDF15 in the gut is controlled by a wide range of compounds via pleiotropic mechanisms involving chemosensory and pharmacological receptors.

Our study showed that oral intake of Plaquenil, containing the bitter compound hydroxychloroquine, reduced hunger scores in normal-weight volunteers, which correlated with both decreased ghrelin levels and increased GDF15 levels. Previous studies already showed that intragastric administration of the bitter compounds, quinine or denatonium benzoate, in the fasted state reduced hunger contractions and hunger scores which were accompanied by a decrease in plasma levels of the orexigenic hormones motilin and/or ghrelin [25,26]. An effect on the release of GLP-1 was also observed after intragastric administration of quinine before a mixed-nutrient drink [27]. Thus, both orexigenic and anorexigenic peptides, including the stress-related cytokine GDF15, are affected after administration of bitter compounds in the gut to decrease appetite signaling.

There is some controversy in the literature to whether either intragastric or intraduodenal administration of quinine is most effective to reduce hunger scores and/or the release of gut hormones [48,49]. Our study indicated that GDF15 was expressed in both the stomach and jejunum of normal-weight individuals, but that only in patients with obesity expression levels were markedly higher in the jejunum. In addition, we previously showed that bitter induced the release of ghrelin in duodenal explants from normal-weight individuals but not in patients with obesity [23]. Therefore, the optimal site of delivery of bitter agonists in the gut to induce the release of GDF15 and also other gut hormones might differ according to the patient group and should be carefully considered. In fact, GDF15 plasma levels are elevated in several pathological conditions, including obesity, driven by cellular stress and low-grade inflammation, and imply a state of resistance that could reduce the efficacy of GDF15 [50]. However, unlike other obesity-induced cytokines such as leptin, GDF15 promotes weight loss and improves glycemic control in obese mice and monkeys [7,51]. Furthermore, GDF15 also provides a protective function by regulating the host's immune response from a type 1 to a type 2 inflammatory response [52].

Combination therapy that relies on the synergistic potential of several gut hormones, such as GLP-1, GIP and glucagon, are currently considered as the next generation of obesity treatments [53]. A study in rats showed that combined GDF15 and semaglutide (GLP-1 agonist) treatment, produced greater food intake and body weight suppression than either treatment alone, without enhancing malaise, likely because these agonists act on distinct pathways [54]. Thus, adding agonists that target the GFRAL receptor to the emerging list of gut hormone-based combination therapies may be a promising approach. In this regard, our findings show that bitter compounds represent a natural form of combination therapy as they release different hormones that induce satiety. For instance, the effect of the acyl homoserine-lactone quorum-sensing molecule (C12-O-AHL) released by bacteria, affected GDF15 and GLP-1 mRNA in patients with the bitter sensitive TAS2R4 (GG/GC) genotype but not in patients with the TAS2R4 (CC) genotype. Thus, TAS2R4 was identified as a potential natural target for combination therapy in which the TAS2Rs SNPs may be used to determine the patient responsiveness.

Several medications have a bitter taste. For instance, semaglutide has a special coating that allows for oral administration but patients taking oral semaglutide complain of its bitter taste [55], which is also true of the antidiabetic medication metformin [56]. The take home message from our study is that the bitter taste of these medications should not be avoided since they may contribute to the weight-lowering effect of these drug by inducing the release of both GDF15 and GLP-1. However, masking the initial bitter perception in the oral cavity, but retaining the ability to taste bitterness in the gut through complexation of metformin with a pH-dependent masking agent, has proven to be a viable approach to improve the palatability of drug intake for patients [56].

Intriguingly, our findings suggest the involvement of distinct downstream signaling pathways in the modulation of GDF15 and GLP-1 levels by bitter compounds. Specifically, the bitter antagonist GIV-27327 inhibited the gallic acid-induced upregulation of GDF15 expression. This regulation of GDF15 seemed to involve the unfolded protein response (UPR) pathway, as evidenced by ISRIB, an inhibitor of eIF2α phosphorylation upstream of ATF4 and DDIT3. The association of the UPR pathway with GDF15 aligns with previous research [57]. However, neither GIV-27327 nor ISRIB affected the gallic acid-induced modulation of GLP-1, suggesting that gallic acid's effect on GLP-1 was not mediated through TAS2R activation and the UPR pathway.

It is important to note that activation of TAS2Rs does not only stimulate but can also inhibit GDF15 release. Acetaminophen-induced TAS2R39 activation and aloin-induced TAS2R43 activation inhibited GDF15 expression without affecting GLP-1. The effect of aloin was genotype-dependent and only observed in patients that are TAS2R43 positive but not in those that have a deletion polymorphism of TAS2R43. GDF15 is a stress cytokine implicated in several diseases, including cancer types in which elevated serum levels are associated with weight loss and reduced survival rates [[58], [59], [60]]. Targeting TAS2R39 or TAS2R43 to lower GDF15 levels, therefore, holds therapeutic promise for managing cancer cachexia. Notably, a phase two clinical trial is currently ongoing with Ponsegromab, a humanized monoclonal antibody that directly targets GDF15, with the aim of improving cachexia-related symptoms [61]. Recently, Fejzo et al. showed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and hyperemesis gravidarum [62]. Targeting TAS2R39 and TAS2R43 receptors may also be of interest in this patient group. To enhance therapeutic effectiveness, the genotype of the patients should be taken into account. However, since most bitter compounds increase GDF15 expression, which can lead to appetite suppression, it is desirable to advise pregnant women not to consume food with bitter flavors.

We previously confirmed a role for aloin on innate immune responses in primary crypts from patients with obesity [47]. The effect of aloin on Escherichia coli growth and the release of the mucus glycoprotein CLCA1 was affected by the TAS2R43 deletion polymorphism as well. Immunofluorescence studies confirmed the expression of TAS2R43 in Paneth cells and goblet cells. Interestingly, we found that GDF15 is not only localized in chromogranin A containing enteroendocrine cells but also in goblet cells. Previous studies showed that GDF15 is involved in counteracting microbiota-associated mucosal barrier dysfunction by regulating mucin production from goblet cells [63]. Indeed, studies in GDF15 knockout mice showed that Gdf15 deficiency depleted colonic mucin secretion [63]. We found that quorum-sensing molecules (C12-O-AHL) secreted by Gram-negative bacteria could regulate the expression of GDF15 in a TAS2R4 genotype-dependent manner. In view of the colocalization between TAS2R4 and GDF15 and the presence of GDF15 in goblet cells, it is therefore tempting to speculate that C12-O-AHL may affect mucus production in human primary crypts as well. Taken together, activation of TAS2R4 could result in increased GDF15 release from goblet cells, thereby enhancing the innate immune system, while also releasing both GDF15 and GLP1 from enteroendocrine cells for appetite regulation.

We were intrigued by the finding that the marked effect of the antibiotic azithromycin on GDF15 mRNA expression was not blocked by the bitter antagonist GIV3727. Macrolide antibiotics, such as azithromycin and erythromycin, are known to be motilin receptor agonist [64]. The motilin receptor is expressed in the smooth muscle cells and the myenteric plexus of the human gastrointestinal tract [65]. Motilin is considered as the physiological initiator of the migrating motor complex, a pattern of strong contractions starting in the stomach and migrating distally to remove food remnants during the fasting state [66]. It was shown that macrolides mimic the effect of motilin on gastrointestinal motility in the fasted state and accelerate gastric emptying [67,68]. The effects on gastrointestinal motility by these antibiotics may be responsible for the side effects such as upper abdominal pain and bloating [69,70]. We now provide the first evidence that azithromycin also activates a MTLR in the mucosa to induce the release of GDF15. Studies have also linked GDF15 to nausea and vomiting by engaging emetic neurocircuitry, known side effects of the antibiotics [13,65,71]. Moreover, erythromycin serves as a prokinetic agent in clinical settings, aimed at enhancing gastric emptying among ICU patients receiving enteral nutrition to prevent gastric stasis and intolerance [72,73]. However, prolonged use of erythromycin may result in the release of GDF15, potentially contradicting erythromycin's primary goal of accelerating gastric emptying and instead inducing feeding intolerance characterized by nausea, vomiting and abdominal discomfort [74].

The lack of specific TAS2R subtype receptor antagonists prevented us from identifying the TAS2R subtype involved for all bitter compounds that affected GDF15. Furthermore, we demonstrated the role of the TAS2R4 and TAS2R43 genotype in determining the sensitivity to bitter compounds in the intestine. In fact, there are 169 identified TAS2R SNPs particularly affecting receptor function [42]. It remains to be investigated whether they also affect the sensitivity to bitter in the gut. Additionally, future research should include GFRAL knockout mice to further define the role of GDF15 in the effect of bitter compounds on food intake and body weight.

In conclusion, our findings suggest that TAS2Rs are interesting therapeutic targets for combination therapy since they alter endogenous release of GDF15 and GLP-1 in patients with obesity. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responsiveness. Furthermore, we provide a novel role for the MTLR in the gut mucosa that regulates the release of GDF15 in response to antibiotics.

4 Materials and methods

4.1 Sex as a biological variable

The effects of oral ingestion of Plaquenil on plasma levels of GDF15 were investigated exclusively in female healthy volunteers, given their higher degree of gastrointestinal sensitivity to bitter compounds [26,75]. Stomach and jejunal tissues used in the in vitro study were retrieved from both male and female individuals.

4.2 Study approval

Written informed consent was obtained from all participants with obesity recruited at the University Hospital UZ Leuven (Leuven, Belgium) for this study. According to Belgian laws, every citizen is a potential donor, and no specific consent is needed for organ or tissue donation. In cases of donation, tissue can be used for scientific research if the local ethics committee agrees. Ethics approval for the study was provided by the Medical Ethics Committee UZ/KU Leuven (S61870 [healthy volunteer], S56978 [organ donors] and S57826 [patients with obesity]) and performed in accordance with Declaration of Helsinki principles.

4.3 Clinical trial

GDF15 measurements were performed in samples from ten female healthy volunteers (age: 24 ± 1 years; BMI: 22.8 ± 0.3 kg/m2) from a clinical trial (NCT04005768) testing the effect of oral ingestion of Plaquenil (hydroxychloroquine sulfate) on hunger scores and gut hormones in a placebo-controlled, double-blind, randomized, two-visit crossover design [76]. In brief, after an overnight fast of at least 12 h, volunteers arrived at the hospital and a baseline blood sample (t = −10 min) was taken. Ten minutes later, participants received two tablets of Plaquenil® (total concentration 310 mg) or placebo with 240 mL water. Blood samples were taken every 10 min for one hour (t = 10 to t = 60 min). Thereafter, volunteers were instructed to drink a rewarding chocolate milkshake ad libitum until they felt fully satisfied, the last blood samples were taken 30 min later (t = 90 min). During the entire study, volunteers were requested to score their hunger and symptoms every 10 min on a 100 mm VAS. Blood samples were collected in LiHep tube supplemented with 500 kIU/mL aprotinin, centrifuged, separated, and stored at −80 °C until analysis. Human plasma levels of GDF15 were measured using the Human GDF-15 Quantikine ELISA Kit (DGD150; R&D systems) following the manufacturer's instructions.

4.4 Human tissues

The stomach or jejunum was obtained either from normal-weight (BMI < 25) multiorgan donors [donation after circulatory death (DCD): 35% or donation after brain death (DBD): 65%], or non-diabetic patients with obesity undergoing the Roux-en-Y gastric bypass surgery (jejunum) or sleeve gastrectomy (stomach). Detailed protocols for human tissue procurement have been described previously [47]. Demographics of organ donors and patients with obesity are summarized in Supplemental Table 1.

4.5 Primary human cell culture

Primary crypts were isolated from the proximal small intestine of obese patients with obesity by collagenase digestion. Fresh jejunal tissue was dissected, cleared of fat, muscle layer, and submucosa, then minced and repeatedly digested with 0.35 mg/mL collagenase from Clostridium histolyticum. The resulting jejunal cell suspensions were centrifuged, re-suspended in complete DMEM (10% FBS, 1% penicillin and streptomycin, 1% Glutamax, v/v), and filtered to obtain crypts. Subsequently, primary crypts were seeded onto 1.4% (v/v) matrigel-coated 12-well plates or glass coverslips and incubated for 24 h at 37 °C.

4.6 Bitter stimulation in primary crypts

Primary crypts were incubated with DMEM or 0.1% (v/v) DMSO (vehicle group) or bitter agonists in parallel for 4 h at 37 °C. The concentration of bitter agonists was determined according to their potency to induce a Ca2+ response in HEK-293T cells transfected with TAS2Rs (Table 1). The potential cytotoxic effects of the compounds at the determined concentrations on jejunal crypts were excluded by a neutral red uptake assay [77].

To investigate potential signaling pathways, cells were pre-incubated for 30 min with antagonists [4-(2,2,3-trimethylcyclopentyl) butanoic acid (GIV3727, 110 μM); N,N′-trans- 1,4-cyclohexanediylbis[2-(4-chlorophenoxy)-acetamide (ISRIB, 5 μM)] or vehicle, and then replaced by a mixture of the test compound in combination with the antagonist and incubated for 4 h at 37 °C.

4.7 Immunofluorescence staining of human intestinal tissue sections or primary crypts

The jejunal mucosa from normal-weight organ donors was fixed with 4% (w/v) paraformaldehyde (2 h), cryoprotected overnight in 30% (w/v) sucrose at 4 °C, frozen and sliced (10 μm sections). Primary jejunal crypts from patients with obesity were fixed with 4% (w/v) paraformaldehyde for 30 min. Both preparations were subjected to an antigen retrieval step (sodium citrate buffer (10 mM, pH = 6) for 30 min at 80 °C) followed by 2 h pre-incubation at room temperature (RT) with 10% (v/v) normal donkey serum and 0.3% Triton X-100. Slides were incubated overnight at 4 °C with either one or two (co-localization studies) of the following primary antibodies: mouse anti-human GDF15 (1:100; MA5-31346; Thermo Fisher Scientific); rabbit anti-human mucin2 (MUC2; 1:150; 27675-1-AP; Proteintech); rabbit anti-human α-defensin 6 (1:100; 17923-1-AP; Proteintech); goat anti-human chromogranin A (CHGA; 1:250; sc-1488; Santa Cruz); goat anti-human total ghrelin (1:250; ab 104307; Abcam); rabbit anti-human TAS2R4 (1:100; OSR00153W; Osenses); rabbit anti-human TAS2R10 (1:250; PA5-39708; Thermo Fisher Scientific); rabbit anti-human TAS2R43 (1:100; PA5-103257; Thermo Fisher Scientific). After washing, samples were incubated for 2 h at RT with donkey anti-rabbit Cy5 (1:800; 711-175-152; Jackson), donkey anti-mouse Cy3 (1:800; 715-165-150; Jackson), donkey anti-goat Alexa488 (1:800; 705-545-003; Jackson) or donkey anti-mouse Alexa594 (1:800; 715-586-151; Jackson) depending upon the primary antibodies used. After washing, samples were incubated for 2 min with 0.5 mg/mL DAPI (1:2000; D1306; Thermofisher), washed, mounted with Mowiol, and imaged using a Zeiss AXIO Scan Z1 fluorescence microscope (VIB Bio-Imaging Core Leuven).

In the quantification studies, images were consistently captured at the same exposure duration. The median fluorescence intensity of stained cells was measured across a minimum of 10 distinct views from either two tissue sections or one coverslip (for crypts preparation) per patient (n = 5), employing ImageJ for analysis (NIH).

4.8 RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA from the tissue sections of stomach/small intestine mucosa was isolated by the RNeasy Mini Kit (Qiagen) following a treatment of Turbo DNA-free™ kit (Thermo Fisher Scientific) to remove genomic DNA. Total RNA from the primary jejunal crypts was extracted by the ReliaPrep miRNA Cell and Tissue miniprep system (Promega). All the RNA samples were reverse transcribed with qScript® cDNA SuperMix (Quantabio). Real-time PCR was performed using the Lightcycler 480 (Roche), with LightCycler480 SYBR Green 1 Master mix (Roche Diagnostics). Primers are listed in Supplemental Table 2. Raw data were analyzed using the software LinRegPCR to determine primer efficiency. Ct values were normalized to a calibrator to correct inter-run variations and to the geometric mean of the endogenous control genes S18, β-actin, and GAPDH.

4.9 TAS2R4 and TAS2R43 polymorphisms analysis

Single nucleotide polymorphisms (SNP) for TAS2R4 and TAS2R43 were determined from cDNA preparation from primary crypts in each patient. PCR was performed using the GoTaq® G2 Green Master Mix kit (Promega) to amplify the gene of TAS2R4 and TAS2R43 by designed primers (Table S2). PCR products were run on a 1% (w/v) agarose gel with 0.01% (v/v) SYBR safe (ThermoFisher Scientific). Selected amplicons were extracted using the QiaQuick Gel Extraction Kit (QIAGEN) and sequenced (LGC Genomics). The SNP was identified by Chromas DNA sequencing software 2.6.6. For TAS2R4 SNPs, patients with a G allele at position 286 (TAS2R4 (GG/CG)) were identified as sensitive to bitterness, whereas those with a C allele (TAS2R4 (CC)) were considered to be less sensitive to bitterness [43]. For TAS2R43 SNP, the nucleotide G at position 104 (TAS2R43(+) GG/GC) resulted in a genotype, which is highly sensitive to aloin [44]. Conversely, a C at this position (TAS2R43(+)) results in a CC genotype, which is less sensitive to aloin. Some patients are not sensitive to TAS2R43 because of a deletion polymorphism.

4.10 GDF15 and GLP-1 secretion in primary crypts

The cell culture supernatant of primary crypts stimulated with bitter agonists was collected, cells were lysed with a lysis buffer (1 M Tris–HCl, 5 M NaCl, 1% v/v Igepal CA-630, 0.5% w/v sodium deoxycholate monohydrate, and 1 tablet of EDTA-free protease inhibitor cocktail) and stored at −80 °C until future use. GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer's protocol. GLP-1 secretion was quantified using a V-PLEX GLP-1 (total) Kit (Meso Scale Discovery), following the manufacturer's instructions. GDF15 and GLP-1 secretion was expressed as a fraction of the total hormone content per well or normalized (%) to basal hormone secretion, which was measured in parallel wells stimulated with the appropriate vehicle.

4.11 Statistical analysis

Results are shown as mean ± SEM. Data were assessed for normality of distribution. Non-normally or non-homogeneously distributed data underwent log transformation prior to statistical analysis. Differences in age and BMI between normal-weight individuals and patients with obesity were analyzed using a two tailed unpaired student t-test. Sex difference between the two populations was analyzed using the chi-squared test.

To analyze plasma levels in the in-vivo study, a paired t-test was first performed to compare the difference in the initial baseline value (absolute value at −10 min) between the conditions (treatment: Plaquenil or Placebo). GDF15 plasma concentration was analyzed as a ln-fold change from baseline, as there was no significant difference in baseline values among conditions. The statistical analysis was done using the ANCOVA mixed model (SAS/Stat 14.1, SAS Institute Inc., Cary, NC) with patient as random variable. In this model, time and conditions were random factors, factor time was considered having an autoregressive order 1 (AR1) covariance structure. Due to the various feeding states of patients, we split the whole period as three groups: control (baseline, −10 min), fasted state (10–60 min), and fed state (90 min). For the quadratic covariance analysis, we used the interaction effects (factors: treatment∗group∗time) as fixed effects, setting “time” and “time2” as a continuous variable. Post hoc, LRT tests were performed to analyze the differences of trendline in conditions, setting 5% level as significance, and Satterthwaite method was used to calculate the freedom degrees. We also compared the differences in GDF15 plasma level at different individual timepoint versus control in different conditions. By extrapolating the trendline based on the fasted state, we could compare the difference of GDF15 plasma level between the fasted and fed state. Fisher z-transformation was performed to obtain average coefficient (r) between GDF15 plasma level and Δ hunger sensations (vs. baseline at −10 min) within volunteers by Pearson correlation analysis, followed a one-tailed paired t-test to compare the r in conditions.

Differences in GDF15 mRNA tissue distribution between patients with obesity and normal-weight individuals (factors: body weight∗tissue region) or differences between the effect of various bitter agonists on GDF15 or GLP-1 mRNA expression in primary crypts compared to vehicle-treated groups (factors: treatment) were analyzed using a proc Mixed Model (SAS/Stat 14.1, SAS Institute Inc., Cary, NC) followed by planned comparisons with Šidák correction for multiple testing. The effect on GDF15 and GLP-1 secretion and the quantification of the fluorescence immunostaining was analyzed using a one or two tailed paired student's t-test. The correlation analysis between GDF15 or GLP-1 mRNA expression and cellular stress related gene expression induced by bitter agonists was evaluated by two-tailed Pearson test. P values lower than 0.05 are considered significant.

Funding sources

This work was supported by a FWO research project (G081523N ), a fellowship of the Chinese Scholarship Council (CSC) (grant no. 202008370227 ), a Methusalem grant from the KU Leuven and the FWO Research Network (W001620N ).

CRediT authorship contribution statement

Qian Wang: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Mona Farhadipour: Writing – review & editing. Theo Thijs: Formal analysis, Data curation. Emily Ruilova Sosoranga: Resources, Methodology. Bart Van der Schueren: Resources. Laurens J. Ceulemans: Resources. Ellen Deleus: Resources. Matthias Lannoo: Resources. Jan Tack: Resources, Methodology. Inge Depoortere: Writing – review & editing, Supervision, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare no conflicts of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgments

The authors thank L. Nys and S. Boel (Gut Peptide Research Lab, TARGID, KU Leuven) for their skillful technical assistance. We like to thank the doctors from the bariatric surgery team and LIFT Center (University Hospitals Leuven) for collecting jejunal tissue from patients. The authors also gratefully acknowledge A. Kerstens and N. Peredo from the VIB Bio Imaging Core (hosted by the center for Brain & Disease research) for their training, support, and access to the machinery park.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2024.102002.
==== Refs
References

1 Bootcov M. Bauskin A.R. Valenzuela S.M. Moore A.T. Bansal M. He X. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily Proc Natl Acad Sci USA 94 21 1997 11514 11519 10.1073/pnas.94.21.11514 9326641
2 Silviya Lodi Rathna Yu B. Xia L. Liu F. Roles and regulation of growth differentiation factor-15 in the immune and tumor microenvironment Hum Immunol 82 12 2021 937 944 10.1016/j.humimm.2021.06.007 34412918
3 Wollert K.C. Kempf T. Wallentin L. Growth differentiation factor 15 as a biomarker in cardiovascular disease Clin Chem 63 1 2017 140 151 10.1373/clinchem.2016.255174 28062617
4 Asrih M. Wei S. Nguyen T.N. Yi H.-S. Ryu D. Gariani Karim Overview of growth differentiation factor 15 in metabolic syndrome J Cell Mol Med 27 9 2023 1157 1167 10.1111/jcmm.17725 36992609
5 Sarkar S. Melchior J.T. Henry H.R. Syed F. Mirmira R.G. Nakayasu E.S. GDF15: a potential therapeutic target for type 1 diabetes Expert Opin Ther Targets 26 1 2022 57 67 10.1080/14728222.2022.2029410 35138971
6 Ling T. Zhang J. Ding F. Ma L. Role of growth differentiation factor 15 in cancer cachexia (Review) Oncol Lett 26 5 2023 462 10.3892/ol.2023.14049 37780545
7 Xiong Y. Walker K. Min X. Hale C. Tran T. Komorowski R. Long-acting MIC-1/GDF15 molecules to treat obesity: evidence from mice to monkeys Sci Transl Med 9 412 2017 eaan8732 10.1126/scitranslmed.aan8732 29046435
8 Suriben R. Chen M. Higbee J. Oeffinger J. Ventura R. Li B. Antibody-mediated inhibition of GDF15–GFRAL activity reverses cancer cachexia in mice Nat Med 26 8 2020 1264 1270 10.1038/s41591-020-0945-x 32661391
9 Breen D.M. Kim H. Bennett D. Calle R.A. Collins S. Esquejo R.M. GDF-15 neutralization alleviates platinum-based chemotherapy-induced emesis, anorexia, and weight loss in mice and nonhuman primates Cell Metab 32 6 2020 938 950.e6 10.1016/j.cmet.2020.10.023 33207247
10 Benichou O. Coskun T. Gonciarz M.D. Garhyan P. Adams A.C. Du Y. Discovery, development, and clinical proof of mechanism of LY3463251, a long-acting GDF15 receptor agonist Cell Metab 35 2 2023 274 286.e10 10.1016/j.cmet.2022.12.011 36630958
11 Yang L. Chang C.-C. Sun Z. Madsen D. Zhu H. Padkjær S.B. GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand Nat Med 23 10 2017 1158 1166 10.1038/nm.4394 28846099
12 Hsu J.-Y. Crawley S. Chen M. Ayupova D.A. Lindhout D.A. Higbee J. Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15 Nature 550 7675 2017 255 259 10.1038/nature24042 28953886
13 Borner T. Shaulson E.D. Ghidewon M.Y. Barnett A.B. Horn C.C. Doyle R.P. GDF15 induces anorexia through nausea and emesis Cell Metab 31 2 2020 351 362.e5 10.1016/j.cmet.2019.12.004 31928886
14 Dagan-Wiener A. Di Pizio A. Nissim I. Bahia M.S. Dubovski N. Margulis E. BitterDB: taste ligands and receptors database in 2019 Nucleic Acids Res 47 D1 2018 D1179 D1185 10.1093/nar/gky974
15 Coll A.P. Chen M. Taskar P. Rimmington D. Patel S. Tadross J.A. GDF15 mediates the effects of metformin on body weight and energy balance Nature 578 7795 2019 444 448 10.1038/s41586-019-1911-y 31875646
16 Baek S.J. Wilson L.C. Eling T.E. Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53 Carcinogenesis 23 3 2002 425 432 10.1093/carcin/23.3.425 11895857
17 Arzola-Paniagua M.A. García-Salgado López E.R. Calvo-Vargas C.G. Guevara-Cruz M. Efficacy of an orlistat-resveratrol combination for weight loss in subjects with obesity: a randomized controlled trial Obesity 24 7 2016 1454 1463 10.1002/oby.21523 27221771
18 Day E.A. Ford R.J. Smith B.K. Mohammadi-Shemirani P. Morrow M.R. Gutgesell R.M. Metformin-induced increases in GDF15 are important for suppressing appetite and promoting weight loss Nat Metab 1 12 2019 1202 1208 10.1038/s42255-019-0146-4 32694673
19 Adler E. Hoon M.A. Mueller K.L. Chandrashekar J. Ryba N.J.P. Zuker C.S. A novel family of mammalian taste receptors Cell 100 6 2000 693 702 10.1016/s0092-8674(00)80705-9 10761934
20 Wooding S.P. Ramirez V.A. Behrens M. Bitter taste receptors: genes, evolution, and health Evol Med Public Health 9 1 2021 431 447 10.1093/emph/eoab031 35154779
21 Avau B. Bauters D. Steensels S. Vancleef L. Laermans J. Lesuisse J. The gustatory signaling pathway and bitter taste receptors affect the development of obesity and adipocyte metabolism in mice PLoS One 10 12 2015 e0145538 10.1371/journal.pone.0145538
22 Wang Q. Liszt I.K. Depoortere I. Extra-oral bitter taste receptors: new targets against obesity? Peptides 127 2020 170284 10.1016/j.peptides.2020.170284
23 Wang Q. Liszt K.I. Deloose E. Canovai E. Thijs T. Farré R. Obesity alters adrenergic and chemosensory signaling pathways that regulate ghrelin secretion in the human gut FASEB J 33 4 2019 4907 4920 10.1096/fj.201801661rr 30629462
24 Kok B.P. Galmozzi A. Littlejohn N.K. Albert V. Godio C. Kim W. Intestinal bitter taste receptor activation alters hormone secretion and imparts metabolic benefits Mol Metab 16 2018 76 87 10.1016/j.molmet.2018.07.013 30120064
25 Deloose E. Corsetti M. Van Oudenhove L. Depoortere I. Tack J. Intragastric infusion of the bitter tastant quinine suppresses hormone release and antral motility during the fasting state in healthy female volunteers Neurogastroenterol Motil 30 1 2017 e13171 10.1111/nmo.13171
26 Deloose E. Janssen P. Corsetti M. Biesiekierski J. Masuy I. Rotondo A. Intragastric infusion of denatonium benzoate attenuates interdigestive gastric motility and hunger scores in healthy female volunteers Am J Clin Nutr 105 3 2017 580 588 10.3945/ajcn.116.138297 28148502
27 Bitarafan V. Fitzgerald P.C.E. Little T.J. Meyerhof W. Jones K.L. Wu T. Intragastric administration of the bitter tastant quinine lowers the glycemic response to a nutrient drink without slowing gastric emptying in healthy men Am J Physiol Regul Integr Comp Physiol 318 2 2020 R263 R273 10.1152/ajpregu.00294.2019 31774306
28 Iven J. Biesiekierski J.R. Zhao D. Deloose E. O'Daly O.G. Depoortere I. Intragastric quinine administration decreases hedonic eating in healthy women through peptide-mediated gut-brain signaling mechanisms Nutr Neurosci 22 12 2018 850 862 10.1080/1028415x.2018.1457841 29607741
29 Avau B. Rotondo A. Thijs T. Andrews C.N. Janssen P. Tack J. Targeting extra-oral bitter taste receptors modulates gastrointestinal motility with effects on satiation Sci Rep 5 2015 15985 10.1038/srep15985
30 Niethammer A.G. Zheng Z. Timmer A. Lee T. First-in-human evaluation of oral denatonium acetate (ARD-101), a potential bitter taste receptor agonist: a randomized, double-blind, placebo-controlled phase 1 trial in healthy adults Clin Pharmacol Drug Dev 11 8 2022 997 1006 10.1002/cpdd.1100 35509219
31 Hale C. Véniant M.M. Growth differentiation factor 15 as a potential therapeutic for treating obesity Mol Metab 46 2021 101117 10.1016/j.molmet.2020.101117
32 Meyerhof W. Batram C. Kuhn C. Brockhoff A. Chudoba E. Bufe B. The molecular receptive ranges of human TAS2R bitter taste receptors Chem Senses 35 2 2009 157 170 10.1093/chemse/bjp092 20022913
33 Jaggupilli A. Singh N. Jesus V.C.D. Duan K. Chelikani P. Characterization of the binding sites for bacterial acyl homoserine lactones (AHLs) on human bitter taste receptors (T2Rs) ACS Infect Dis 4 7 2018 1146 1156 10.1021/acsinfecdis.8b00094 29799189
34 Jaggupilli A. Singh N. De Jesus V.C. Gounni M.S. Dhanaraj P. Chelikani P. Chemosensory bitter taste receptors (T2Rs) are activated by multiple antibiotics FASEB J 33 1 2018 501 517 10.1096/fj.201800521rr 30011231
35 Roland W.S.U. van Buren L. Gruppen H. Driesse M. Gouka R.J. Smit G. Bitter taste receptor activation by flavonoids and isoflavonoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39 J Agric Food Chem 61 44 2013 10454 10466 10.1021/jf403387p 24117141
36 Margulis E. Slavutsky Yuli Lang T. Behrens M. Benjamini Yuval Niv M.Y. BitterMatch: recommendation systems for matching molecules with bitter taste receptors J Cheminf 14 1 2022 45 10.1186/s13321-022-00612-9
37 Sterneder S. Stoeger V. Dugulin C.A. Liszt K.I. Di Pizio A. Korntheuer K. Astringent gallic acid in red wine regulates mechanisms of gastric acid secretion via activation of bitter taste sensing receptor TAS2R4 J Agric Food Chem 69 36 2021 10550 10561 10.1021/acs.jafc.1c03061 34460245
38 Levit A. Nowak S. Peters M. Wiener A. Meyerhof W. Behrens M. The bitter pill: clinical drugs that activate the human bitter taste receptor TAS2R14 FASEB J 28 3 2013 1181 1197 10.1096/fj.13-242594 24285091
39 Lossow K. Hübner S. Roudnitzky N. Slack J.P. Pollastro F. Behrens M. Comprehensive analysis of mouse bitter taste receptors reveals different molecular receptive ranges for orthologous receptors in mice and humans J Biol Chem 291 29 2016 15358 15377 10.1074/jbc.m116.718544 27226572
40 Wiener A. Shudler M. Levit A. Niv M.Y. BitterDB: a database of bitter compounds Nucleic Acids Res 40 D1 2011 D413 D419 10.1093/nar/gkr755 21940398
41 Slack J.P. Brockhoff A. Batram C. Menzel S. Sonnabend C. Born S. Modulation of bitter taste perception by a small molecule hTAS2R antagonist Curr Biol 20 12 2010 1104 1109 10.1016/j.cub.2010.04.043 20537538
42 Wooding S.P. Ramirez V.A. Global population genetics and diversity in the TAS2R bitter taste receptor family Front Genet 13 2022 952299 10.3389/fgene.2022.952299
43 Risso D. Morini G. Pagani L. Quagliariello A. Giuliani C. De Fanti S. Genetic signature of differential sensitivity to stevioside in the Italian population Genes Nutr 9 3 2014 401 10.1007/s12263-014-0401-y 24705770
44 Pronin A.N. Xu H. Tang H. Zhang L. Li Q. Li X. Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin Curr Biol 17 16 2007 1403 1408 10.1016/j.cub.2007.07.046 17702579
45 Broad J. Sanger G.J. The antibiotic azithromycin is a motilin receptor agonist in human stomach: comparison with erythromycin Br J Pharmacol 168 8 2013 1859 1867 10.1111/bph.12077 23190027
46 Ozaki K. Onoma Mitsu Muramatsu H. Sudo H. Yoshida S. Shiokawa Rie An orally active motilin receptor antagonist, MA-2029, inhibits motilin-induced gastrointestinal motility, increase in fundic tone, and diarrhea in conscious dogs without affecting gastric emptying Eur J Pharmacol 615 1–3 2009 185 192 10.1016/j.ejphar.2009.04.059 19445919
47 Liszt K.I. Wang Q. Farhadipour M. Segers A. Thijs T. Nys L. Human intestinal bitter taste receptors regulate innate immune responses and metabolic regulators in obesity J Clin Investig 132 3 2022 e144828 10.1172/JCI144828
48 Rezaie P. Bitarafan V. Rose B.D. Lange K. Rehfeld J.F. Horowitz M. Quinine effects on gut and pancreatic hormones and antropyloroduodenal pressures in humans-role of delivery site and sex J Clin Endocrinol Metab 107 7 2022 e2870 e2881 10.1210/clinem/dgac182 35325161
49 Verbeure W. Deloose E. Tóth J. Rehfeld J.F. Van Oudenhove L. Depoortere I. The endocrine effects of bitter tastant administration in the gastrointestinal system: intragastric versus intraduodenal administration Am J Physiol Endocrinol Metab 321 1 2021 E1 E10 10.1152/ajpendo.00636.2020 34029163
50 Keipert S. Ost M. Stress-induced FGF21 and GDF15 in obesity and obesity resistance Trends Endocrinol Metab 32 11 2021 904 915 10.1016/j.tem.2021.08.008 34526227
51 Mullican S.E. Lin-Schmidt X. Chin C.-N. Chavez J.A. Furman J.L. Armstrong A.A. GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates Nat Med 23 10 2017 1150 1157 10.1038/nm.4392 28846097
52 Reyes J. Yap G.S. Emerging roles of growth differentiation factor 15 in immunoregulation and pathogenesis J Immunol 210 1 2023 5 11 10.4049/jimmunol.2200641 36542831
53 Camilleri M. Acosta A. Newer pharmacological interventions directed at gut hormones for obesity Br J Pharmacol 181 8 2023 1153 1164 10.1111/bph.16278 37917871
54 Ghidewon M. Wald H.S. McKnight A.D. De Jonghe B.C. Breen D.M. Alhadeff A.L. Growth differentiation factor 15 (GDF15) and semaglutide inhibit food intake and body weight through largely distinct, additive mechanisms Diabetes Obes Metabol 24 6 2022 1010 1020 10.1111/dom.14663
55 Okada S. Okada J. Okada K. Yamada E. Saito T. Kikkawa Koji Hot water eliminates the bitter taste of oral semaglutide: a report of four cases Intern Med 2024 10.2169/internalmedicine.2608-23
56 Bhoyar P.K. Amgaonkar Y.M. Taste masking and molecular properties of metformin hydrochloride-indion 234 complexes J Young Pharm 3 2 2011 112 118 10.4103/0975-1483.80294 21731355
57 Patel S. Alvarez-Guaita A. Melvin A. Coll A. Savage D. O'Rahilly S. GDF15 provides an endocrine signal of nutritional stress in mice and humans Cell Metab 29 3 2019 707 718 10.1016/j.cmet.2018.12.016 30639358
58 Lerner L. Hayes T.G. Tao N. Krieger B. Feng B. Wu Z. Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients J Cachexia Sarcopenia Muscle 6 4 2015 317 324 10.1002/jcsm.12033 26672741
59 Lerner L. Tao J. Liu Q. Nicoletti R. Feng B. Krieger B. MAP3K11/GDF15 axis is a critical driver of cancer cachexia J Cachexia Sarcopenia Muscle 7 4 2015 467 482 10.1002/jcsm.12077 27239403
60 Alessio Molfino. Ida Amabile Maria Imbimbo G. Rizzo V. Pediconi Federica Catalano C. Association between growth differentiation factor-15 (GDF-15) serum levels, anorexia and low muscle mass among cancer patients Cancers 13 1 2020 99 10.3390/cancers13010099 33396237
61 Groarke J.D. Crawford J. Collins S.M. Lubaczewski S.L. Breen D.M. Harrington M.A. Phase 2 study of the efficacy and safety of ponsegromab in patients with cancer cachexia: PROACC-1 study design J Cachexia Sarcopenia Muscle 15 3 2024 1054 1061 10.1002/jcsm.13435 38500292
62 Fejzo M. Rocha N. Cimino I. Lockhart S.M. Petry C.J. Kay R.G. GDF15 linked to maternal risk of nausea and vomiting during pregnancy Nature 625 7996 2023 760 767 10.1038/s41586-023-06921-9 38092039
63 Ray N. Jun Park Seung Jung H. Kim J. Korcsmaros Tamas Moon Y. Stress-responsive Gdf15 counteracts renointestinal toxicity via autophagic and microbiota reprogramming Commun Biol 6 1 2023 602 10.1038/s42003-023-04965-1 37270567
64 Peeters T. Matthijs G. Depoortere I. Cachet T. Hoogmartens J. Vantrappen G. Erythromycin is a motilin receptor agonist Am J Physiol Gastrointest Liver Physiol 257 3 1989 G470 G474 10.1152/ajpgi.1989.257.3.g470
65 Feighner S.D. Receptor for motilin identified in the human gastrointestinal system Science 284 5423 1999 2184 2188 10.1126/science.284.5423.2184 10381885
66 Deloose E. Verbeure W. Depoortere I. Tack J. Motilin: from gastric motility stimulation to hunger signalling Nat Rev Endocrinol 15 4 2019 238 250 10.1038/s41574-019-0155-0 30675023
67 Janssens J. Peeters T.L. Vantrappen G. Tack J. Urbain J.L. De Roo M. Improvement of gastric emptying in diabetic gastroparesis by erythromycin N Engl J Med 322 15 1990 1028 1031 10.1056/nejm199004123221502 2320062
68 Tomomasa T. Kuroume T. Arai H. Wakabayashi K. Itoh Z. Erythromycin induces migrating motor complex in human gastrointestinal tract Dig Dis Sci 31 2 1986 157 161 10.1007/bf01300701 3943442
69 Sarna S.K. Soergel K.H. Koch T.R. Stone J.E. Wood C.M. Ryan R.P. Gastrointestinal motor effects of erythromycin in humans Gastroenterology 101 6 1991 1488 1496 10.1016/0016-5085(91)90383-v 1955115
70 Seifert Cf. Swaney Rj. Ra B.-M. Intravenous erythromycin lactobionate-induced severe nausea and vomiting DICP 23 1 1989 40 44 10.1177/106002808902300108 2655295
71 Principi N. Esposito S. Comparative tolerability of erythromycin and newer macrolide antibacterials in paediatric patients Drug Saf 20 1 1999 25 41 10.2165/00002018-199920010-00004 9935275
72 Landzinski J. Kiser T.H. Fish D.N. Wischmeyer P.E. MacLaren R. Gastric motility function in critically ill patients tolerant vs intolerant to gastric nutrition J Parenter Enteral Nutr 32 1 2008 45 50 10.1177/014860710803200145
73 Lewis K. Alqahtani Z. Mcintyre L. Almenawer S. Alshamsi F. Rhodes A. The efficacy and safety of prokinetic agents in critically ill patients receiving enteral nutrition: a systematic review and meta-analysis of randomized trials Crit Care 20 1 2016 259 10.1186/s13054-016-1441-z 27527069
74 Casaer M.P. Van den Berghe G. Nutrition in the acute phase of critical illness N Engl J Med 370 13 2014 1227 1236 10.1056/nejmra1304623 24670169
75 Bartoshuk L.M. Duffy V.B. Miller I.J. PTC/PROP tasting: anatomy, psychophysics, and sex effects Physiol Behav 56 6 1994 1165 1171 10.1016/0031-9384(94)90361-1 7878086
76 Ruilova Sosoranga E. Verbeure W. Geysen H. Thijs T. Matthys C. Depoortere I. The acute effect of hydroxychloroquine sulfate on hunger, the plasma concentration of orexigenic peptides and hedonic food intake: a pilot study Nutrients 15 19 2023 4264 10.3390/nu15194264 37836548
77 Repetto G. del Peso A. Zurita J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity Nat Protoc 3 7 2008 1125 1131 10.1038/nprot.2008.75 18600217
