Combination of Citrus aurantifolia Fruit Rind and Theobroma cacao Seed Extracts Stimulates Glucagon-Like Peptide-1 Secretion by Activating hTAS2Rs and the Phospholipase C-Mediated Signaling Pathway in NCI-H716 Cells

Abstract

LN19183 is a proprietary blend containing Citrus aurantifolia fruit rind and Theobroma cacao seed extracts in a 2:1 ratio. Recently, LN19183 was found to improve serum glucagon-like peptide-1 (GLP-1) levels after 16 weeks of intake in overweight adults. However, its specific mechanisms remain unknown. Therefore, the present study evaluated whether LN19183 acts through the bitter taste receptor-phospholipase C (PLC)-calcium signaling pathway in the human enteroendocrine L-cell line NCI-H716. The results showed that LN19183 dose-dependently increased relative GLP-1 secretion and reached statistical significance at 50 µg/mL (P<0.05). At the same dose, LN19183 selectively amplified the mRNA expression of bitter taste receptors (hTAS2R3, hTAS2R45, and most strongly, hTAS2R46) and concurrently increased the mRNA expression of α-gustducin. In addition, LN19183 upregulated the mRNA expression of proglucagon and prohormone convertase 1/3. To determine its mechanisms, cells were treated with U73122 (a PLC inhibitor) and 2-aminoethoxydiphenyl borate (an inositol 1,4,5-trisphosphate receptor inhibitor). These treatments inhibited the mRNA expression of proglucagon and secretion of GLP-1. Collectively, these findings indicate that LN19183 activates hTAS2R46-α-gustducin-PLC signaling to stimulate the biosynthesis and secretion of GLP-1. These data provide a molecular rationale for the increase in GLP-1 observed in previous animal and human studies of LN19183, thereby positioning this botanical as a promising adjunct for the prevention or comanagement of obesity and related metabolic diseases.

INTRODUCTION

Obesity is one of the most pressing health issues worldwide. According to the World Health Organization, over two billion adults (nearly 43% of the global population) are overweight (Boutari and Mantzoros, 2022). Obesity’s high prevalence is related to chronic diseases, including diabetes, cancers, cardiovascular diseases, and liver disorders (Hakkak and Bell, 2016). Obesity results from complex interactions among genetic, environmental, and endocrine factors, including the dysregulation of appetite-regulating hormones (Barness et al., 2007; Chearskul et al., 2012). Among these factors, hormonal imbalance plays an important role in obesity development. Therefore, hormone-based therapies may be a promising treatment strategy for this disease.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is released from intestinal L-cells after nutrient intake. It helps decrease blood glucose by enhancing insulin secretion in a glucose-dependent manner, reducing appetite, and promoting weight loss (Shah and Vella, 2014). Because of these effects, GLP-1 is an important target for the treatment of type 2 diabetes and obesity (Heppner and Perez-Tilve, 2015).

According to recent studies, TAS2Rs, which are known for detecting bitterness on the tongue, are also expressed in intestinal L-cells and may trigger the secretion of GLP-1 in response to specific bitter stimuli (Jensterle et al., 2021). Upon activation, hTAS2Rs initiate a signaling cascade involving G protein subunits, especially G α-gustducin. This process triggers the activation of phospholipase C-β (PLCβ), leading to the production of inositol trisphosphate (IP₃) and release of calcium ions (Ca²⁺) from intracellular stores. The resulting increase in intracellular Ca²⁺ stimulates the exocytosis and secretion of GLP-1 from L-cells. This pathway can be disrupted by inhibiting key signaling molecules (e.g., PLCβ or IP₃), thereby reducing GLP-1 secretion. This mechanism highlights TAS2Rs as potential therapeutic targets in the treatment of obesity and metabolic disorders (Jang et al., 2007; Kim et al., 2014).

Citrus aurantifolia fruit rind and Theobroma cacao seed contain polyphenols and alkaloids that are responsible for bitterness and exhibit various pharmacological effects (Ammatalli et al., 2024). C. aurantifolia is abundant in hesperidin and naringin, which promote fatty acid oxidation and inhibit fat accumulation (Sui et al., 2018; Liao et al., 2023). Meanwhile, T. cacao contains theobromine and epicatechin, which are known to enhance lipase activity and stimulate thermogenesis (Loizzo et al., 2012; Castellanos et al., 2018). According to recent studies, the combination of C. aurantifolia and T. cacao extracts (LN19183) decreased body weight and improved body composition in overweight adults after 16 weeks of intake, including an increase in serum GLP-1 levels (Chadalavada et al., 2025). However, the mechanisms specifically related to GLP-1 secretion induced by LN19183 remain unclear and warrant further investigation.

To the best of our knowledge, this study is the first to demonstrate that LN19183 enhances GLP-1 secretion by activating specific human bitter taste receptors in enteroendocrine L-cells. Moreover, we investigated whether LN19183 stimulates GLP-1 secretion via the hTAS2R-G α-gustducin-PLC-IP₃/Ca²⁺ signaling pathway. The present study provides insights into the mechanisms underlying LN19183-mediated GLP-1 secretion and suggests that LN19183 is a promising nutraceutical for the management of obesity and metabolic disorders.

MATERIALS AND METHODS

Test material

LN19183 (also referred to as CL19183 or Theolim^TM) was prepared as previously described (Chadalavada et al., 2025). Dried C. aurantifolia was pulverized and extracted with 50% aqueous ethanol at 50°C－60°C for approximately 14－16 h. Dried T. cacao was extracted with water at 50°C－55°C for 9 h. The extracts were concentrated to either thick paste or dry powder under vacuum and blended at a 2:1 ratio. The final product contained 80% herbal blend and 20% excipients (18% GLUCIDEX and 2% SYLOID) (Dodda et al., 2024).

Reagents

Roswell Park Memorial Institute (RPMI) 1640 Medium and fetal bovine serum (FBS) were obtained from Gibco, and antibiotic-antimycotic (AA) was purchased from GenDEPOT (Barker). U73122, 2-aminoethoxydiphenyl borate (2-APB), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-[2H]-tetrazolium bromide (MTT) was obtained from Invitrogen. The RNeasy Mini Kit and GLP-1 ELISA Kit were purchased from Qiagen and Abcam, respectively.

Cell culture

The human enteroendocrine L-cell line NCI-H716 was obtained from the Korean Cell Line Bank. NCI-H716 cells were cultured in suspension at 37°C in a humidified 5% CO₂ incubator in RPMI 1640 Medium supplemented with 10% FBS and 1% AA (Gagnon et al., 2015). To perform the NCI-H716 cell experiments, the culture plates (Corning) were first coated with Matrigel and seeded with the appropriate cell density. After two days, the medium was removed and replaced with RPMI 1640 Medium containing 0.5% FBS and 1% AA solution to induce serum starvation. Then, the cells were treated with the test samples.

MTT assay

To assess cell viability, NCI-H716 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well. After 48 h of incubation at 37°C in 5% CO₂, the cells were treated with LN19183 (1, 2.5, 5, 10, and 50 µg/mL) and cultured for an additional 16 h. Subsequently, each well was added with MTT reagent (0.5 mg/mL) and incubated for 4 h. After removing the medium, the resulting formazan crystals were fully dissolved in DMSO, and the absorbance was measured at 540 nm using an Epoch^TM microplate reader (BioTek Instruments).

GLP-1 kit assay

NCI-H716 cells were seeded at a density of 1×10⁶ cells/well into Matrigel-coated six-well plates to determine whether LN19183 induces GLP-1 secretion through the PLC pathway. After a starvation period, the cells were treated with either LN19183 alone (5, 10, or 50 µg/mL), LN19183 (50 µg/mL) plus the PLC inhibitor U73122 (10 µM), or LN19183 (50 µg/mL) plus the IP₃-receptor blocker 2-APB (10 µM) for 1 h (Jin et al., 1994; Bootman et al., 2002). The supernatants were collected, and GLP-1 levels were quantified using a GLP-1 ELISA Kit.

Real-time polymerase chain reaction (PCR)

NCI-H716 cells were seeded at a density of 1×10⁶ cells/well in Matrigel-coated six-well plates. After a starvation period, the cells were treated with LN19183 (5, 10, or 50 µg/mL) for 1 h. After treatment, the supernatants were removed, and total RNA was isolated using the RNeasy kit. The RNA concentrations were measured using a Nanodrop spectrophotometer (BioTek Instruments), and cDNA was synthesized using a PCR master mix (Toyobo) at 45°C for 60 min, followed by 95°C for 5 min. Gene expression was quantified on a real-time PCR system (Bio-Rad) using SYBR Green Master Mix (Toyobo). Table 1 shows the primer sequences.

Table 1.

Primers for 21 hTAS2Rs, α-gustducin, GCG, and PC1/3

Gene	Forward/reverse	Sequence	Expected amplicon (bp)
hTAS2R1	Forward	GCCCCATGCCTTTATTGTTA	130
	Reverse	GAGGTGAGACTCTAGCATTT
hTAS2R3	Forward	AGTGAGGAGATTCTATGTAT	140
	Reverse	TGACCAGAATTCCCAGTGTG
hTAS2R5	Forward	TCGCTTGAAGACAGATTACG	161
	Reverse	GAGAAATTCAACCACTGCCA
hTAS2R8	Forward	TTAACCTGTTTGCAATTGTC	140
	Reverse	TGGCTCTCACATGAACTTCT
hTAS2R9	Forward	CTGTCATTTTCCCATCAAGC	120
	Reverse	GGTCTCTATGGAACAAAAGGC
hTAS2R10	Forward	CATTTCCCTTTGGAGACACA	76
	Reverse	ATGAGCTTCTGTGTTGGAGT
hTAS2R14	Forward	CCTCACTGCTTTGGCAATCT	66
	Reverse	ACACACACCAGCTTCCGAAT
hTAS2R19	Forward	CGAACCATTTCAGCATGTGG	108
	Reverse	CCCCAACAGTATCACCAGAA
hTAS2R20	Forward	AGATGCGACCAAAAGAAATT	120
	Reverse	CACCTGCCACAAAACTGAAA
hTAS2R30	Forward	TTCAGCTATCCTTCAACCCA	124
	Reverse	GCCCCTCTTGTGAATCTATG
hTAS2R31	Forward	CAGCACCAAGGTCCACATAA	66
	Reverse	GTAAACGGCACATAACAAGA
hTAS2R38	Forward	TGGCAACCAGGTCTTTAGAT	120
	Reverse	ACTCCAGGACTGAAATGAAC
hTAS2R39	Forward	ATTACTGGATTGATACCCTGGC	147
	Reverse	TGTTTTCTTAGTGGAGTTGGAGG
hTAS2R40	Forward	TGCCGGCCACTCAGTACAA	60
	Reverse	ACCGCTTCCAGGCTCTTCTC
hTAS2R41	Forward	CCATGCAGAACGACTTTTAC	92
	Reverse	TTGAGGTTGCTGAAGATGAG
hTAS2R42	Forward	ACTGGTAAACTGCTCTGAAGG	102
	Reverse	ATGTGAAGCAAGTCCCACTAG
hTAS2R43	Forward	GACCACGAACCCACCTG	123
	Reverse	ACAAATGTAACCACTACCAG
hTAS2R45	Forward	CCTTTGCTGACCAAATTGTCACT	72
	Reverse	TAATAATAACACCCAGAGCAAACCAA
hTAS2R46	Forward	CAAAGGATCTCAAGATCCCAGCATG	122
	Reverse	CCAGACTCTCAAAACTCCAAACTGAC
hTAS2R50	Forward	GTTGTCATGGTTAGCAAGGC	124
	Reverse	GAGTTGAGAGTTTCAGGTCT
hTAS2R60	Forward	ACGGAGCTACTGTGAGAAAT	123
	Reverse	CGGAATCCTGAGGTTGTAAG
GAPDH	Forward	GGAGCGAGATCCCTCCAAAAT	197
	Reverse	GGCTGTTGTCATACTTCTCATGG
GNAT3	Forward	AGAGCAAGGAGTCAGCCAAAAG	66
	Reverse	CGCTCAGCATCCTCCTGAA
GCG	Forward	GAGGAAGGCGAGATTTCCCAG	66
	Reverse	GAACCATCAGCATGTCTGCG
PC1/3	Forward	CAGAAGGCTTTTGAATATGGTGT	123
	Reverse	GGAGGCACTGCTGATGGAGAT

GCG, proglucagon; PC1/3, prohormone convertase-1/3; GNAT, G protein α-gustducin.

Statistical analysis

All in vitro experiments were performed in duplicate and independently repeated thrice. Data are presented as the mean±standard error of the mean, and the standard deviation was also reported. Statistical analyses were performed using ordinary one-way analysis of variance, followed by Dunnett’s or Tukey’s multiple comparisons tests. Statistical significance was considered at P<0.05, and P<0.01 and <0.001 were considered highly significant.

RESULTS AND DISCUSSION

Effects of LN19183 on GLP-1 secretion

The cytotoxicity of LN19183 was measured using MTT assay in NCI-H716 cells. LN19183 exhibited no cytotoxicity up to 50 µg/mL, maintaining cell viability >93% of the control (NT) (Fig. 1). Based on these data, the relative GLP-1 release after LN19183 treatment at 5, 10, and 50 µg/mL was measured using the GLP-1 ELISA Kit. GLP-1 secretion increased with a significant elevation at 50 µg/mL compared with NT (P<0.05) (Fig. 2). PLC and IP₃ are important components of the bitter-taste-receptor-mediated signaling cascade in enteroendocrine L-cells (Bootman et al., 2002; Wang et al., 2025). The PLC inhibitor U73122 prevents the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and IP₃, thereby blocking downstream calcium mobilization. Likewise, 2-APB antagonizes IP₃Rs, inhibiting IP₃-induced release of calcium from the endoplasmic reticulum. In the present study, cotreatment of NCI-H716 cells with LN19183 and either U73122 or 2-APB abolished the LN19183-induced enhancement of GLP-1 release, decreasing the secretion to NT levels (Fig. 2). This complete reversal indicates that the stimulatory action of LN19183 on GLP-1 secretion strictly depends on PLCβ activation and subsequent IP₃-mediated intracellular calcium signaling.

Fig. 1.

Effects of LN19183 on the viability of NCI-H716 cells. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-[2H]-tetrazolium bromide assay was assessed after 16 h exposure to 1－50 µg/mL of LN19183. All experiments were performed in triplicate, and the data are presented as the mean±standard error of the mean. NT, control.

Fig. 2.

Increase in the secretion of glucagon-like peptide-1 (GLP-1) following LN19183 treatment. Secreted GLP-1 levels after treatment with 5, 10, and 50 µg/mL of LN19183 were quantified by colorimetric enzyme-linked immunosorbent assay (ab184857, Abcam). Phospholipase C-β (PLCβ) blockade abolishes LN19183-induced upregulation of GLP-1 by treatment with 10 µM U73122 or 10 µM 2-aminoethoxydiphenyl borate (2-APB). Values with different superscript letters (a-c) in a column are significantly different (P<0.05), as determined by one-way analysis of variance followed by Tukey’s post hoc test. NT, control.

Profiling of hTAS2R gene expression

We examined the mRNA expression levels of 21 human bitter taste receptor genes using real-time quantitative PCR to investigate the expression of hTAS2R genes in differentiated NCI-H716 cells treated with LN19183 (Fig. 3). The results revealed a significant expression of hTAS2R3, hTAS2R45, and hTAS2R46, with hTAS2R46 being the most abundantly expressed. Thus, hTAS2R46 emerges as the most likely target receptor for LN19183. LN19183 contains various polyphenols and alkaloids, including hesperidin and theobromine (Zheng et al., 2004; Chadalavada et al., 2025), whose bitter taste properties may activate hTAS2R46. Considering that bitter taste receptor signaling involves the G protein α-gustducin (GNAT3), we next quantified GNAT3 expression (Jang et al., 2007). LN19183 markedly increased the mRNA expression of GNAT3 (P<0.001), suggesting the activation of the taste receptor pathway (Fig. 4).

Fig. 3.

mRNA expression profiling of hTAS2Rs in differentiated NCI-H716 cells treated with 50 µg/mL of LN19183 for 1 h. The expression levels of all 21 hTAS2Rs were normalized to that of GAPDH. All experiments were performed in triplicate. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s post hoc test. Values are expressed as the mean±standard error of the mean. ***P<0.001 and *P<0.05 vs. NT. NT, control.

Fig. 4.

Expression of G protein α-gustducin gene (GNAT3) in differentiated NCI-H716 cells treated with 5, 10, and 50 µg/mL of LN19183 for 1 h. The expression levels of GNAT3 were normalized to that of GAPDH. All experiments were performed in triplicate. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s post hoc test. Values are expressed as the mean±standard error of the mean. ***P<0.001 vs. NT. NT, control.

TAS2R46 responds to a broad spectrum of bitter substances, including sesquiterpene lactones, diterpenes, strychnine, and denatonium benzoate (Brockhoff et al., 2007). Tangeretin and nobiletin, which are present in C. aurantifolia peel (Permadi et al., 2024; Wani et al., 2024), have also been associated with TAS2R46 activation (Kuroda et al., 2016). According to a recent study, the activation of intestinal TAS2R46 may attenuate metabolic disorders by regulating the production of liver lipoproteins, suggesting a possible therapeutic use against obesity and metabolic syndrome (Ding et al., 2025). Although an animal study supported the antiobesity effects of TAS2R46 activation, the findings in the present study highlight TAS2R46 as a potential target for LN19183-mediated weight management.

LN19183 stimulates the secretion of proglucagon (GCG)-encoding GLP-1

We analyzed the mRNA levels of GCG and prohormone convertase-1/3 (PC1/3) by real-time PCR to clarify the molecular basis of GLP-1 release. LN19183 treatment at 50 µg/mL significantly upregulated GCG expression (P<0.05), whereas PC1/3 showed an upward trend (Fig. 5A and 5B). When PLC signaling was blocked with U73122 or 2-APB, the LN19183-induced increase in GCG mRNA expression was completely abolished (Fig. 5C). These results confirmed that LN19183-induced GLP-1 secretion occurs through the PLC-IP₃R pathway, which is essential for GCG gene transcription and hormone secretion (Huang et al., 2022; Lee et al., 2023). This mechanistic insight aligns with previous findings that quercetin and other phytochemicals promote the release of GLP-1 through TAS2R-mediated activation of PLC (Wang et al., 2025).

Fig. 5.

Dose-dependent expression of (A) proglucagon gene (GCG) and (B) prohormone convertase-1/3 gene (PC1/3) in differentiated NCI-H716 cells treated with 5, 10, and 50 µg/mL of LN19183 for 1 h. (C) Phospholipase C-β blockade abolishes the LN19183-induced upregulation of proglucagon mRNA by treatment with 10 µM U73122 or 10 µM 2-aminoethoxydiphenyl borate (2-APB). The expression levels of GCG and PC1/3 were normalized to that of GAPDH. All experiments were performed in triplicate. Values are expressed as the mean±standard error of the mean. Statistical analyses for (A) and (B) were performed using one-way analysis of variance followed by Dunnett’s post hoc test (***P<0.001 vs. NT). For (C), one-way analysis of variance followed by Tukey’s post hoc test was performed. Values with different superscript letters (a-c) within a column are significantly different (P<0.05). NT, control.

Collectively, our findings demonstrated that LN19183 stimulated GLP-1 secretion via the hTAS2R-α-gustducin-PLC-IP₃/Ca²⁺ signaling cascade in intestinal L-cells (Fig. 6). The current data provide a molecular explanation for the GLP-1 secretion observed in 16-week human studies with LN19183. In addition, according to a previous animal study, LN19183-induced GLP-1 secretion enhanced peripheral GLP-1 signaling and upregulated GLP-1 receptors in target tissues, including the liver (Chadalavada et al., 2025). The present study corroborated those observations and substantiated LN19183 as a safe (Dodda et al., 2024) and potent adjunct for preventing or comanaging obesity and its associated metabolic disorders.

Fig. 6.

Proposed mechanism behind the effects of LN19183 in stimulating glucagon-like peptide-1 (GLP-1) secretion via the hTAS2R-α-gustducin-phospholipase C (PLC)-IP₃/Ca²⁺ signaling cascade in human enteroendocrine L-cells. 2-APB, 2-aminoethoxydiphenyl borate; PC1/3, prohormone convertase-1/3; ER, endoplasmic reticulum; GCG, proglucagon.