Cell Line STC-1 Research Articles

Imaging flow cytometry reveals LPS-induced changes to intracellular intensity and distribution of α-synuclein in a TLR4-dependent manner in STC-1 cells

BackgroundParkinson’s disease is a chronic neurodegenerative disorder, where pathological protein aggregates largely composed of phosphorylated α-synuclein are implicated in disease pathogenesis and progression. Emerging evidence suggests that the interaction between pro-inflammatory microbial factors and the gut epithelium contributes to α-synuclein aggregation in the enteric nervous system. However, the cellular sources and mechanisms for α-synuclein pathology in the gut are still unclear. MethodsThe STC-1 cell line, which models an enteroendocrine population capable of communicating with the microbiota, immune and nervous systems, was treated with a TLR4 inhibitor (TAK-242) prior to microbial lipopolysaccharide (LPS) exposure to investigate the role of TLR4 signalling in α-synuclein alterations. Antibodies targeting the full-length protein (α-synuclein) and the Serine-129 phosphorylated form (pS129) were used. Complex, multi-parametric image analysis was conducted through confocal microscopy (with Zen 3.8 analysis) and imaging flow cytometry (with IDEAS® analysis). ResultsConfocal microscopy revealed heterogenous distribution of α-synuclein and pS129 in STC-1 cells, with prominent pS129 staining along cytoplasmic processes. Imaging flow cytometry further quantified the relationship between various α-synuclein morphometric features. Thereafter, imaging flow cytometry demonstrated a dose-specific effect of LPS, where the low (8 μg/mL), but not high dose (32 μg/mL), significantly altered measures related to α-synuclein intensity, distribution, and localisation. Pre-treatment with a TLR4 inhibitor TAK-242 alleviated some of these significant alterations. ConclusionThis study demonstrates that LPS-TLR4 signalling alters the intracellular localisation of α-synuclein in enteroendocrine cells in vitro and showcases the utility of combining imaging flow cytometry to investigate subtle protein changes that may not be apparent through confocal microscopy alone. Further investigation is required to understand the apparent dose-dependent effects of LPS on α-synuclein in the gut epithelium in healthy states as well as conditions such as Parkinson’s disease.

Effect of hypoxia on GLP-1 secretion – an in vitro study using enteroendocrine STC-1 -cells as a model

Glucagon-like peptide (GLP)-1 is a hormone released by enteroendocrine L-cells after food ingestion. L-cells express various receptors for nutrient sensing including G protein-coupled receptors (GPRs). Intestinal epithelial cells near the lumen have a lower O2 tension than at the base of the crypts, which leads to hypoxia in L-cells. We hypothesized that hypoxia affects nutrient-stimulated GLP-1 secretion from the enteroendocrine cell line STC-1, the most commonly used model. In this study, we investigated the effect of hypoxia (1% O2) on alpha-linolenic acid (αLA) stimulated GLP-1 secretion and their receptor expressions. STC-1 cells were incubated for 12 h under hypoxia (1% O2) and treated with αLA to stimulate GLP-1 secretion. 12 h of hypoxia did not change basal GLP-1 secretion, but significantly reduced nutrient (αLA) stimulated GLP-1 secretion. In normoxia, αLA (12.5 μM) significantly stimulated (~ 5 times) GLP-1 secretion compared to control, but under hypoxia, GLP-1 secretion was reduced by 45% compared to normoxia. αLA upregulated GPR120, also termed free fatty acid receptor 4 (FFAR4), expressions under normoxia as well as hypoxia. Hypoxia downregulated GPR120 and GPR40 expression by 50% and 60%, respectively, compared to normoxia. These findings demonstrate that hypoxia does not affect the basal GLP-1 secretion but decreases nutrient-stimulated GLP-1 secretion. The decrease in nutrient-stimulated GLP-1 secretion was due to decreased GPR120 and GPR40 receptors expression. Changes in the gut environment and inflammation might contribute to the hypoxia of the epithelial and L-cells.Graphical

Dubosiella newyorkensis Modulates Tryptophan and Serotonin Production and Related Signaling Pathways in the Enteroendocrine Cell Line STC1

Dubosiella newyorkensis Modulates Tryptophan and Serotonin Production and Related Signaling Pathways in the Enteroendocrine Cell Line STC1

Abstract 2236 Dubosiella newyorkensis modulates tryptophan and serotonin production and related signaling pathways in the enteroendocrine cell line STC1

Abstract 2236 Dubosiella newyorkensis modulates tryptophan and serotonin production and related signaling pathways in the enteroendocrine cell line STC1

In Vitro Hypoglycemic Activities of Lactobacilli and Bifidobacterium Strains from Healthy Children's Sources and Their Effect on Stimulating GLP-1 Secretion in STC-1 Cells.

A long-term use of chemical drugs cannot cure type II diabetes mellitus (T2DM) and their numerous toxic side effects can be harmful to human health. In recent years, probiotics have emerged as a natural resource to replace chemical drugs in alleviating many human ailments. Healthy children's intestines have a lot of colonized Lactobacilli and Bifidobacterium, and these beneficial bacteria can help promote overall health. The objective of this study was to isolate potential antidiabetic probiotic strains from healthy children and evaluate their application prospects. Firstly, Lactobacillus and Bifidobacterium strains were isolated from healthy children's feces and identified by the pheS or clpC genes with their respective 16S rRNA genes. Then, hydrophobicity, artificial gastrointestinal fluid tolerance, α-Glucosidase and Dipeptidyl peptidase IV (DPP-IV) inhibitory activities of isolated strains were determined, and antioxidant activities and promoting secretion of GLP-1 in STC-1 cells of candidate strains were tested. Results showed that 6 strains of Lactobacillus and Bifidobacterium were obtained from the feces of healthy children aged 3 years, respectively, including Lacticaseibacillus paracasei L-21 and L-25, Levilactobacillus brevis L-16, Lentilactobacillus buchneri L-9, Lactiplantibacillus plantarum L-8 and L-3, Bifidobacterium bifidum 11-1 and B-84, Bifidobacterium longum subsp. longum 6-1, 6-2, B42 and B53. The hydrophobicity and auto-aggregation levels of all these strains were higher than 30% and 50%, respectively, and the decrease in the number of colonies of all strains in the artificial gastrointestinal fluid was less than 2 log CFU/mL. Strains L-3, L-8, L-9, L-21, 6-1, 11-1, B53 and B84 were selected based on their high α-glucosidase inhibitory activity and DPP-IV inhibitory activity, and results of the antioxidant capacity assay showed that the remaining strains all had intense comprehensive antioxidant activity. Additionally, Lacticaseibacillus paracasei L-21 and Bifidobacterium longum subsp. longum B-53 had the most substantial prompting effect on GLP-1 secretion in the STC-1 cell line. These results indicated that Lacticaseibacillus paracasei L-21 and Bifidobacterium longum subsp. longum B-53 could be used as a potential antidiabetic strain; thus, its application as a food supplement and drug ingredient could be recommended after in vivo mitigation of type II diabetes test.

Free fatty acid receptor 1 stimulates cAMP production and gut hormone secretion through Gq-mediated activation of adenylate cyclase 2

ObjectiveFree fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2+ and cAMP pathways. Whereas IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear, although originally proposed to be Gs mediated. Methods and resultsWhen stimulated with AM5262, we observe that FFAR1 can activate the majority of the Gα proteins, except - surprisingly - members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in pro-glucagon expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293 cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion were both blocked by YM253890. ConclusionsOur results show that Gq signaling is responsible not only for the IP3/Ca2+ but also the cAMP response, which together are required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the Gq-driven cAMP response.

Investigating the effect of B. thetaiotaomicron and its derivatives on the expression of tlr2 and tlr4 genes in STC-1 cell line

Investigating the effect of B. thetaiotaomicron and its derivatives on the expression of tlr2 and tlr4 genes in STC-1 cell line

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

An Investigation of Insulinotropic Potential of Herbal Plants for Management of Diabetes

The Insulinotropic hormone glucagon-like peptide-1, which has been proposed as a modern treatment for management of diabetes, is metabolized extremely with the aid of dipeptidyl peptidase-IV. Inhibitors on dipeptidyl peptidase-IV enhance the level of glucagon-like peptide-1, as have elevated glucose tolerance and improved insulin secretion. Recently, incretin-based treatments have end up a beneficial tool to treat diabetic patients, and distinctive research have focused on the identification of glucagon-like peptide-1 receptor agonists, which includes these of herbal origin.
 Aims: This study was aimed to explorations the effect of methanolic extract of plants to inhibit dipeptidyl peptidase-IV and comparing its inhibitory activities with Diprotin, as a reference standard additionally, this study focuses to the influences of glucagon-like peptide-1secretions which accelerated glucose tolerance and provocation of insulin biosynthesis and secretion on STC-1 cell line.
 Study Design: In-vitro model.
 Place and Duration of Study: Department of pharmacology, Karnataka College of pharmacy, Bangalore, India, between Jan 2022 to April 2022.
 Methodology: The test drug undertaking of different medicinal plants extract of management over diabetes right here we were the methanolic extraction of three plants; “Aegle marmelos, Moringa oleifera, then Syzygium cumini” have been tested in-vitro for dipeptidyl peptidase-IV inhibitory activity and have an impact on of incretin system like glucagon-like peptide-1. An in-vitro assay to measure glucagon-like peptide-1release from cultured murine EEC’s under fatty acid stimulation. dipeptidyl peptidase-IV is involved in the inactivation over glucagon-like peptide-1, a potent Insulinotropic peptide.
 Results: The current study underlines up to expectation the extracts inhibits the dipeptidyl peptidase-IV and enhances the glucagon-like peptide-1for diabetes. Results established so the extracts on Aegle marmelos, Moringa oleifera, and Syzygium cumini had dipeptidyl peptidase-IV inhibitory activity on 42.57μg/mL, 42.38/mL, and then 41.48μg/mL respectively. Diprotin A confirmed an IC50 virtue on 29.83μg/mL, as is used as positive controls. Similarly, the study additionally demonstrates that on a cellular level of Aegle marmelos, Syzygium cumini potentially stimulate glucagon-like peptide-1secretion, however Moringa oleifera indicate decent rises into glucagon-like peptide-1secretion.
 Conclusion: The outcomes assure the inhibitory impact about plants regarding dipeptidyl peptidase-IV, glucagon-like peptide-1secretion, and the main in conformity with stand a novel, efficient and fair strategy for the management on diabetes.

844-P: Jatupalathika Extract Acts via G-Protein-Coupled Receptors to Stimulate GLP-1 Secretion in STC-1 Cell Line

One of the most important molecular targets for the prevention and treatment of diabetes is glucagon-like peptide-1 (GLP-1) , a molecule secreted by enteroendocrine L cells in the intestine. It has been reported that nutrients, as well as non-nutritional dietary factors, can activate GLP-1 secretion. Jatupalathika (JPT) is a traditional herbal formulation of four dried fruits: Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula Retz., Terminalia arjuna (Roxb.) Wight & Arn., and Phyllanthus emblica Linn. JPT extracts have traditionally been used for their anti-oxidant, anti-aging, body fat depletion, and weight loss properties. However, its effects on glucose metabolism and GLP-1 are not known. Thus, we evaluated the antidiabetes potential of JPT aqueous extracts by assessing its ability to induce GLP-1 secretion in a mouse intestinal cell line (STC-1) . The MTT assay was used to assess the cytotoxicity, and the LUMINEX multiplex assays were used to assess GLP-1 hormone secretion. When compared to control, JPT extracts (125 µg/ml) increased GLP-1 secretion after 45-120 minutes of incubation without any effects on cell survival. The mechanism of action of GLP-1 secretion activity in cells was studied using real-time PCR. We discovered an increase in expression of six genes related to GLP-1 secretion: SLC5A1 (encoding SGLT1) , FFAR2 (encoding GPR43) , FFAR3 (encoding GPR41) , GPR119, GPBAR1, and CABP1. This finding demonstrated that JTP extract significantly increased GLP-1 hormone production via G-protein-coupled receptor activation, which also promotes Ca2+ release from the endoplasmic reticulum. Phytochemical profiling and molecular docking study revealed that Ellagic acid and Orientin are abundant metabolites with a favorable ability to bind the FFAR2/3 protein, acting as potential agents that aid in GLP-1 production. The findings showed that JPT might be useful in the prevention and treatment of diabetes. However, more research on in vivo effects is required. Disclosure C.Chaichana: None. N.Khumkhana: None. P.Wongshaya: None. W.Deekum: None. P.Pramyothin: Research Support; Novo Nordisk, Speaker's Bureau; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk.

ZD-2, a novel DPP4 inhibitor, protects islet β-cell and improves glycemic control in high-fat-diet-induced obese mice

AimsDipeptidyl peptidase-4 (DPP-4) inhibitors have been extensively used for the treatment of type 2 diabetes mellitus. Nevertheless, side effects like sore throat and diarrhea also occur in DPP-4 inhibitors treatment. The study aims to identify and develop novel DPP-4 inhibitors with better therapeutic profiles. Materials and methodsHere we synthesized a series of vildagliptin analogs, and among which, ZD-2 showed the moderate inhibition of DPP-4 activity compared with vildagliptin. High-fat-diet (HFD) mice were treated with ZD-2 (4.5 and 7.5 mg/kg) or vildagliptin (6 mg/kg) for 7 weeks following the examinations of metabolic index and pancreatic β-cell function. Mouse pancreatic cell line MIN6 was used to evaluate β-cell function, and intestinal enteroendocrine cell line STC-1 was used to evaluate the expression of gut hormones. Key findingsThe IC50 of ZD-2 was over 30-fold higher than vildagliptin. However, both ZD-2 and vildagliptin treatment showed comparable effects on improving glucose tolerance and reducing the steatosis of liver and fat mass in HFD mice. Moreover, ZD-2 exerted β-cell-protective actions by preserving islet β-cell mass and increasing the expression of functional β-cell-related genes. Additionally, ZD-2 also stimulated the expression of gut hormones in STC-1 cells. SignificanceZD-2 showed comparable anti-diabetic activities in HFD-fed mice although its lower potency on inhibition of DPP-4 compared with vildagliptin. Protection of β-cell function might contribute to its anti-diabetic effects.

Dietary Medium-Chain Triglyceride Decanoate Affects Glucose Homeostasis Through GPR84-Mediated GLP-1 Secretion in Mice.

BackgroundDietary triglycerides are an important energy source; however, their excess intake causes metabolic diseases such as obesity and type 2 diabetes. Medium-chain triglycerides (MCTs) as triglyceride forms of medium-chain fatty acids (MCFAs) are applied to meet the energy demands of athletes, the elderly, and people with stunted growth, because MCFAs are efficiently converted into energy for immediate utilization by the organs and do not accumulate as fat. Although the intake of each MCT type (octanoate; C8:0, decanoate; C10:0, and dodecanoate; C12:0) exhibits beneficial metabolic effects, individual functional differences remain unclear.MethodsMCTs or MCFAs were administrated to male GPR84-deficient mice with a C57BL/6J background and mouse enteroendocrine cell line STC-1, and the effects on glucose homeostasis and gut hormone GLP-1 secretion were evaluated.ResultsC10:0 intake improves glucose metabolism through the MCFA receptor GPR84-mediated GLP-1 secretion. Each MCT intake showed resistance to obesity and improved metabolic parameters compared with lard intake. Moreover, oral administration of MCTs enhanced glucose tolerance, especially C10:0 administration, which sufficiently increased plasma GLP-1 levels. Additionally, C10:0 stimulation promoted GLP-1 secretion via GPR84 in STC-1, enhanced glucose tolerance through GPR84-mediated GLP-1 secretion, and showed resistance to high-fat diet (HFD)-induced obesity in mice.ConclusionsDietary MCT (C10:0) intake efficiently may protect against obesity and improve insulin resistance via GLP-1 secretion.

Transcellular propagation of fibrillar \u03b1-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent

Parkinson’s disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

Isothiocyanates in Brassica Vegetables Induce Glucagon Like Peptide-1 Secretion in Enteroendocrine Cells Through Intestinal Bitter Taste Receptors

Abstract Objectives Bitter taste receptors (T2Rs) have been found in the gastrointestinal tract and their activation was linked with improving glucose homeostasis, mainly through inducing the secretion of gut hormones. Brassicaceae vegetables are rich in glucosinolates (GSLs), which upon hydrolysis, produce bioactive isothiocyanates (ITCs) with a bitter and pungent flavor. We hypothesized that GSLs and/or ITCs may interact with the intestinal T2Rs and induce the secretion of glucagon-like peptide-1 (GLP-1). Methods The murine enteroendocrine cell line STC-1 cells were treated with allyl isothiocyanate (AITC), phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), sulforaphane (SF), or sinigrin (SN) at 6.25, 12.5, and 25.0 μM, or vehicle (0.1% DMSO) for 1 hour for active GLP-1 measurement. To test the mechanism of ITCs interacting with T2Rs to induce GLP-1 secretion, 2-APB (IP3 receptor antagonist, 50 μM), A967079 (TRPA1 channel inhibitor, 30 μM), TPPO (TRPM5 channel inhibitor, 10 μM) and EGTA (extracellular Ca2+ chelator,1 mM) were added with AITC (12.5 μM) or vehicle (0.1% DMSO) to cells for 1 hour and active GLP-1 levels were measured. Student's t-test was used to compare two treatments. A P-value < 0.05 was considered statistically significant. Results AITC (25.0 μM), BITC (12.5, 25.0 μM), and SF (6.25, 12.5, 25.0 μM) increased GLP-1 secretion compared to the vehicle (P < 0.05), whereas PEITC and SN showed no impact (P > 0.05). When comparing different ITCs, SF (6.25 μM) tended to induce higher GLP-1 secretion than AITC (6.25 μM) (P = 0.05). 2-APB, A967079 and EGTA significantly reduced GLP-1 secretion in AITC treated cells compared to AITC only (P < 0.05), whereas no changes were observed in GLP-1 levels in the vehicle group with/without the addition of the inhibitors 2-APB and EGTA. Conclusions AITC at 25.0 μM is more potent at inducing GLP-1 secretion compared to its parent GSL, SN. Other ITCs, including BITC and SF, also increased GLP-1 secretion. AITC induces GLP-1 secretion, potentially through T2R pathways mediated by IP3, TRPA1, and the extracellular Ca2+. This study may provide evidence for a novel therapeutic mechanism explaining brassica vegetables improving glucose homeostasis. Funding Sources This study was supported in part by the MOE program, DNS, UIUC. Anqi Zhao was supported by the USDA NIFA Grant (2019–38,420-28,973) to the DNS, UIUC.

Gut microbial short-chain fatty acids-mediated olfactory receptor 78 stimulation promotes anorexigenic gut hormone peptide YY secretion in mice

Olfactory receptor 78 (Olfr78), which is also known as a receptor for short-chain fatty acids (SCFAs) produced via gut microbial fermentation from indigestible polysaccharides such as dietary fibers, is expressed in the enteroendocrine cells of the colon. However, the role of Olfr78 in gut hormone secretion remains unknown. Here, we aimed to investigate the function and mechanism of action of Olfr78 in vivo and in vitro. Toward this, we assessed the expression of Olfr78 in several tissues, affinity of Olfr78 to various monocarboxylates, and the secretion of anorexigenic gut hormone peptide YY (PYY) via Olfr78 using various molecular and biochemical techniques. Olfr78 was abundantly expressed in the colon and mouse enteroendocrine cell line STC-1 and showed specific affinity to SCFAs such as acetate and propionate, but not butyrate, in a monocarboxylate ligand screening assay using a heterologous expression system. Acetate promoted PYY secretion in STC-1 cells via Olfr78-protein kinase A signaling, whereas the effects were abolished by Olfr78 RNA interference. Colonic SCFAs production via oral administration of fructo-oligosaccharide significantly increased plasma PYY levels, whereas this effect was abolished in Olfr78-deficient and germ-free mice. These results suggested that the SCFA receptor Olfr78 is important for anti-obesity and anorexigenic effects of the gut microbiota and dietary fibers.

Short Chain Fatty Acids Enhance Expression and Activity of the Umami Taste Receptor in Enteroendocrine Cells via a Gαi/o Pathway.

The short chain fatty acids (SCFAs) acetate, butyrate and propionate, are produced by fermentation of non-digestible carbohydrates by the gut microbiota and regulate appetite, adiposity, metabolism, glycemic control, and immunity. SCFAs act at two distinct G protein coupled receptors (GPCRs), FFAR2 and FFAR3 and are expressed in intestinal enteroendocrine cells (EECs), where they mediate anorectic gut hormone release. EECs also express other GPCRs that act as nutrient sensors, thus SCFAs may elicit some of their health-promoting effects by altering GPCR expression in EECs and enhance gut sensitivity to dietary molecules. Here, we identify that exposure of the murine EEC STC-1 cell line or intestinal organoids to physiological concentrations of SCFAs enhances mRNA levels of the umami taste receptors TASR1 and TASR3, without altering levels of the SCFA GPCRs, FFAR2 and FFAR3. Treatment of EECs with propionate or butyrate, but not acetate, increased levels of umami receptor transcripts, while propionate also reduced CCK expression. This was reversed by inhibiting Gαi/o signaling with pertussis toxin, suggesting that SCFAs act through FFAR2/3 to alter gene expression. Surprisingly, neither a FFAR3 nor a FFAR2 selective ligand could increase TASR1/TASR3 mRNA levels. We assessed the functional impact of increased TASR1/TASR3 expression using unique pharmacological properties of the umami taste receptor; namely, the potentiation of signaling by inosine monophosphate. Activation of umami taste receptor induced inositol-1-phosphate and calcium signaling, and butyrate pretreatment significantly enhanced such signaling. Our study reveals that SCFAs may contribute to EEC adaptation and alter EEC sensitivity to bioactive nutrients.

Dipeptidyl peptidase-4 and GLP-1 interplay in STC-1 and GLUTag cell lines

Glucagon like peptide-1 (GLP-1) is an incretin hormone, secreted from L-cells of distal ileum and colon in response to nutrient ingestion in human. GLP-1 plays a major role in gut motility, appetite regulation, and insulin secretion. Dipeptidyl peptidase-4 (DPP4), a serine peptidase, cleaves N-terminal dipeptides of GLP-1, rendering it inactive and responsible for its short half-life. DPP4 is widely expressed in numerous tissues in a membrane bound or soluble form. The enteroendocrine cell lines STC-1 and GLUTag are extensively used as models for in vitro studies, however, the basic parallel characterization between these cell lines is still missing. Previously, we demonstrated that these cell lines exhibit different responses to α-linolenic acid (αLA)-induced GLP-1 secretion. Therefore, we examined the basal and stimulated GLP-1 and DPP4 secretion between the two cell lines. GPR120 and GPR40 are known to bind long chain fatty acids. We found that STC-1 cells secreted significantly more basal and αLA-induced GLP-1 than GLUTag cells. In addition, STC-1 secreted DPP4 and expressed higher amounts of DPP4 and GPR120 than GLUTag cells, while GLUTag cells expressed higher GPR40 protein levels than STC-1 cells. Interestingly, the secreted soluble DPP4 did not change the active GLP-1 concentrations in the buffer group, and only 5.5 % of GLP-1 was degraded in the αLA stimulated group. These results suggested that STC-1 cells have a higher potential to secrete GLP-1 and DPP4 than GLUTag cells, and the membrane bound DPP4 may play a more significant role in the inactivation of GLP-1 secretion.

Release of Cholecystokinin from Rat Intestinal Mucosal Cells and the Enteroendocrine Cell Line STC-1 in Response to Maleic and Succinic Acid, Fermentation Products of Alcoholic Beverages.

Alcoholic beverages stimulate pancreatic enzyme secretions by inducing cholecystokinin (CCK) release. CCK is the major stimulatory hormone of pancreatic exocrine secretions, secreted from enteroendocrine I-cells of the intestine. Fermentation products of alcoholic beverages, such as maleic and succinic acids, influence gastric acid secretions. We hypothesize that maleic and succinic acids stimulate pancreatic exocrine secretions during beer and wine ingestion by increasing CCK secretions. Therefore, the effects of maleic and succinic acids on CCK release were studied in duodenal mucosal cells and the enteroendocrine cell line STC-1. Mucosal cells were perfused for 30 min with 5 min sampling intervals, STC-1 cells were studied under static incubation for 15 min, and supernatants were collected for CCK measurements. Succinate and maleate-induced CCK release were investigated. Succinate and maleate doses dependently stimulated CCK secretions from mucosal cells and STC-1 cells. Diltiazem, a calcium channel blocker, significantly inhibited succinate and maleate-induced CCK secretions from mucosal cells and STC-1 cells. Maleate and succinate did not show cytotoxicity in STC-1 cells. Our results indicate that succinate and maleate are novel CCK-releasing factors in fermented alcoholic beverages and could contribute to pancreatic exocrine secretions and their pathophysiology.

The effects of different concentrations of glucose on glucose sensors and GLP-1 secretion in the enteroendocrine cell line STC-1

Glucose triggers glucagon-like peptide (GLP)-1 secretion from L cells involving several glucose sensors including sodium-glucose transporter (SGLT)1, glucose transporter (GLUT)2, and sweet taste receptors (STRs). This study investigated the effects of different glucose concentrations on GLP-1 secretion, intracellular concentrations of Ca2+ and cAMP, glucose uptake, and protein levels of SGLT1, GLUT2, and STRs in STC-1 cells. Low glucose (5.6 mM) increased GLP-1 secretion, intracellular Ca2+ concentration, and SGLT1 protein level compared with glucose-free group. GLP-1 secretion and intracellular Ca2+ concentration triggered by low glucose were inhibited by the SGLT1 inhibitor. GLP-1 secretion or intracellular Ca2+ concentration in high-glucose (25, 100, 200 mM) groups was significantly higher than that of low-glucose group. Elevation of cAMP level was observed in concentration-dependent manner, and decreased glucose uptake was observed in 100 or 200 mM glucose group. High glucose increased protein levels of STRs and GLUT2 in comparison to low-glucose group. GLP-1 secretion and intracellular levels of Ca2+ and cAMP triggered by high glucose were inhibited in the presence of the GLUT2 or STR inhibitor. These results suggest that SGLT1 is dominantly responsible for GLP-1 secretion triggered by low glucose, and that STRs and GLUT2 are involved in GLP-1 secretion induced by high glucose.

Inorganic mesoporous particles for controlled α-linolenic acid delivery to stimulate GLP-1 secretion in vitro

Novel treatment methods for obesity are urgently needed due to the increasing global severity of the problem. Gastrointestinal hormones, such as GLP-1 and PYY, are secreted by the enteroendocrine cells, playing a critical role in regulating food intake. Digested nutrients trigger the secretion of these hormones, which have a very short half-life. α-Linolenic acid (αLA) has been shown to stimulate GLP-1 secretion, however, chemical instability and fast uptake in the small intestine hinder its use in body weight management. We developed a novel delivery system based on inorganic mesoporous particles for αLA to increase secretion of gastrointestinal peptides. αLA was loaded to thermally hydrocarbonized porous silicon particles (THCPSi). 47.9 ± 3.84% and 30.7 ± 2.86% of αLA was released during 6 h from 3.0% and 9.2% loading degree (w/w) samples in vitro, respectively. Native αLA (50 µM) significantly increased GLP-1 secretion from enteroendocrine STC-1 and GLUTag cell lines. αLA loaded THCPSi significantly and dose dependently stimulated GLP-1 secretion from STC-1 cells, whereas empty particles did not. We demonstrated in vitro that THCPSi particles have the potential to be used as a controlled delivery system for nutrients such as αLA, increasing GLP-1 secretion. Our results justify further in vivo investigations.