Proglucagon mRNA Research Articles

Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for β-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of β-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Isosinensetin Stimulates Glucagon-like Peptide-1 Secretion via Activation of hTAS2R50 and the Gβγ-Mediated Signaling Pathway.

Bitter taste receptors (TAS2Rs) are G protein-coupled receptors localized in the taste buds of the tongue. They may also be present in non-lingual organs, including the brain, lung, kidney, and gastrointestinal (GI) tract. Recent studies on bitter taste receptor functions have suggested TAS2Rs as potential therapeutic targets. The human bitter taste receptor subtype hTAS2R50 responds to its agonist isosinensetin (ISS). Here, we demonstrated that, unlike other TAS2R agonists, isosinensetin activated hTAS2R50 as well as increased Glucagon-like peptide 1 (GLP-1) secretion through the Gβγ-mediated pathway in NCI-H716 cells. To confirm this mechanism, we showed that ISS increased intracellular Ca2+ and was suppressed by the IP3R inhibitor 2-APB as well as the PLC inhibitor U73122, suggesting that TAS2Rs alters the physiological state of enteroendocrine L cells in a PLC-dependent manner. Furthermore, we demonstrated that ISS upregulated proglucagon mRNA and stimulated GLP-1 secretion. ISS-mediated GLP-1 secretion was suppressed in response to small interfering RNA-mediated silencing of Gα-gust and hTAS2R50 as well as 2-APB and U73122. Our findings improved the understanding of how ISS modulates GLP-1 secretion and indicates the possibility of using ISS as a therapeutic agent in the treatment of diabetes mellitus.

Dietary carbohydrate modifies the density of L cells in the chicken ileum.

Glucagon-like peptides (GLPs) are secreted from intestinal L cells and stimulate various physiological functions in the gastrointestinal tract. The secretion of GLPs is influenced by macronutrient ingestion. This study aims to clarify the effects of dietary carbohydrate (CHO) on L cells in the chicken ileum. Six-week-old, male White Leghorn chickens were divided into three groups: control, low-CHO and CHO-free, with five chickens in each group. Paraffin sections were made from the proximal and distal ileum of each animal and subjected to immunohistochemistry for GLP-1 and GLP-2 peptides and in situ hybridization for proglucagon (PG) mRNA. A significant reduction of GLP-1- and GLP-2-immunoreactive cells was observed in the two experimental groups compared with that in the control. A reduction of cells expressing PG mRNA was observed in the proximal and distal ileum of the CHO-free group compared with that in the control. The ratio of GLP-1-immunoreactive cells showing Ki-67 immunoreactivity was significantly lower in the distal ileum of the CHO-free group than that in the control group. These data suggest that dietary CHO is an effective stimulator for modifying L cell density in the chicken ileum.

Dietary carbohydrate influences the colocalization pattern of Glucagon-like Peptide-1 with neurotensin in the chicken ileum

Glucagon-like peptide (GLP)-1 colocalizes with neurotensin (NT) in the same enteroendocrine cells (EECs) of the chicken ileum. The present study was designed to clarify the influence of dietary carbohydrate (CHO) on the colocalization pattern of GLP-1 with NT in the chicken distal ileum. Male White Leghorn chickens at 6 weeks of age (n=15) were divided into three groups, a control and two experimental (low-CHO and CHO-free), with five chickens in each, and fed control or experimental diets for 7 d. Distal ileum was collected from each bird as a tissue sample and subjected to double immunofluorescence staining to detect GLP-1 and NT. Three types of EEC, GLP-1+/NT+, GLP-1+/NT- and GLP-1-/NT+, were demonstrated in the chicken ileum. GLP-1+/NT+ cells in the control group had a spindle-like shape with a long cytoplasmic process, but those in the experimental groups were round and lacked a cytoplasmic process. The ratio of GLP-1+/NT+ cells was significantly decreased in the two experimental groups compared with that in the control group. The ratio of GLP-1+/NT+ cells was significantly lower than those of GLP-1+/NT- and GLP-1-/NT+ cells in the two experimental groups. Most cells that were immunoreactive for GLP-1 and NT antisera lacked signals of proglucagon (PG) and NT precursor (NTP) mRNA in the experimental groups. The number of EECs expressing PG and NTP mRNA signals showed tendencies for decreases with a reduction of dietary CHO level. These findings suggest that dietary CHO could be a significant regulator of the pattern of colocalization pattern of GLP-1 with NT in the chicken ileum.

Genistein and Momordica charantia L. prevent oxidative stress and upregulate proglucagon and insulin receptor mRNA in diabetic rats.

Type 2 diabetes occurs as a result of insulin resistance and dysfunction in insulin signaling. Controlling hyperglycemia and activation of insulin signaling are important in the management of type 2 diabetes. This study aimed to evaluate the effect of genistein and Momordica charantia L. fruit (MCF) on oxidative stress, markers of inflammation, and their role in proglucagon and insulin receptor messenger RNA (mRNA) expression by real-time PCR in diabetic rats. Thirty-five albino rats were divided into 7 groups (n= 5). Group I (non-diabetic) and group II (diabetic control) were treated with distilled water, and groups III and IV received 250mg/kg and 500mg/kg lyophilized MCF, respectively. Groups V and VI received 10mg/kg and 20mg/kg genistein, respectively, while group VII received 500mg/kg metformin. The administration lasted for 28days. MCF and genistein significantly reduced interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) levels, which were elevated in the serum of diabetic rats. Treatment with MCF and genistein significantly increased the expression of proglucagon mRNA in the small intestine and insulin receptor mRNA in the liver of diabetic rats. In conclusion, MCF and genistein ameliorate type 2 diabetes complications by preventing the loss of insulin-positive cells, inhibiting IL-1β and TNF-α, and upregulating proglucagon and insulin receptor mRNA expression. Novelty: MCF and genistein have an inhibitory effect on diabetic induced IL-1β and TNF-α production. MCF and genistein upregulate proglucagon and insulin receptor mRNA expression.

Chronodisruption by chronic jetlag impacts metabolic and gastrointestinal homeostasis in male mice.

Chronodisruption desynchronizes peripheral clocks and leads to metabolic diseases. Feeding cues are important synchronizers of peripheral clocks and influence rhythmic oscillations in intestinal microbiota and their metabolites. We investigated whether chronic jetlag, mimicking frequent time zone travelling, affected the diurnal fluctuations in faecal short-chain fatty acid (SCFA) levels, that feed back to the gut clock to regulate rhythmicity in gut function. Rhythms in faecal SCFAs levels and in the expression of clock genes and epithelial markers were measured in the colonic mucosa of control and jetlagged mice. The entraining effect of SCFAs on the rhythm in clock gene mRNA expression was studied in primary colonic crypts. The role of the circadian clock in epithelial marker expression was studied in Arntl-/- mice. Chronic jetlag increased body weight gain and abolished the day/night food intake pattern which resulted in a phase-delay in the rhythm of faecal SCFAs that paralleled the shift in the expression of mucosal clock genes. This effect was mimicked by stimulation of primary colonic crypts from control mice with SCFAs. Jetlag abolished the rhythm in Tnfα, proglucagon and ghrelin expression but not in the expression of tight junction markers. Only a dampening in plasma glucagon-like peptide-1 but not in ghrelin levels was observed. Rhythms in ghrelin but not proglucagon mRNA expression were abolished in Arntl-/- mice. The altered food intake pattern during chronodisruption corresponds with the changes in rhythmicity of SCFA levels that entrain clock genes to affect rhythms in mRNA expression of gut epithelial markers.

First evidence for the presence of amino acid sensing mechanisms in the fish gastrointestinal tract

This study aimed to characterize amino acid sensing systems in the gastrointestinal tract (GIT) of the carnivorous fish model species rainbow trout. We observed that the trout GIT expresses mRNAs encoding some amino acid receptors described in mammals [calcium-sensing receptor (CaSR), G protein-coupled receptor family C group 6 member A (GPRC6A), and taste receptors type 1 members 1 and 2 (T1r1, T1r2)], while others [taste receptor type 1 member 3 (T1r3) and metabotropic glutamate receptors 1 and 4 (mGlur1, mGlur4)] could not be found. Then, we characterized the response of such receptors, as well as that of intracellular signaling mechanisms, to the intragastric administration of l-leucine, l-valine, l-proline or l-glutamate. Results demonstrated that casr, gprc6a, tas1r1 and tas1r2 mRNAs are modulated by amino acids in the stomach and proximal intestine, with important differences with respect to mammals. Likewise, gut amino acid receptors triggered signaling pathways likely mediated, at least partly, by phospholipase C β3 and β4. Finally, the luminal presence of amino acids led to important changes in ghrelin, cholecystokinin, peptide YY and proglucagon mRNAs and/or protein levels. Present results offer the first set of evidence in favor of the existence of amino acid sensing mechanisms within the fish GIT.

Glucolipotoxicity and GLP-1 secretion

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide...

In vitro modulation of glucagon-like peptide release by DPP-IV inhibitory polyphenol-polysaccharide conjugates of sprouted quinoa yoghurt

Glucagon-like peptide-1 (GLP-1) is an important signal in the peripheral and neural systems, which contributes to the maintenance of glucose and energy homeostasis. In this study, 1H NMR validated polyphenols and polysaccharides extracted from sprouted quinoa yoghurt were used as isolates and conjugates to upregulate the stimulation of GLP-1 release in NCI-H716 cells. In addition, we explored their effect on proglucagon and prohormone convertase 3 mRNA expressions, HNF-3γ and CCK-2R gene protein expression, as well as cytosolic calcium release. Variations in concentration showed a dose-dependent GLP-1 stimulation, and were significantly optimized by germination. Proglucagon mRNA expression in NCI-H716 cells was upregulated, and was relatively highest with QYPSP1 treatments in a 2.68 fold. The results suggested that the conjugates had greater potential to stimulate GLP-1 release than their isolates. Sprouted quinoa yoghurt could therefore be a potential functional food useful to regulate glucose and energy homeostasis.

NFAT5 is involved in GRP‐enhanced secretion of GLP‐1 by high sodium

Organ to organ communication is important in the maintenance of normal fluid and electrolyte balance and blood pressure (BP). Neural mechanisms and gastrointestinal (GIT) hormones mediate the natriuresis of an oral sodium load. The GIT is the first organ exposed to ingested nutrients and most likely to react initially to a sodium load. Sensing the amount of ingested sodium by the GIT may be an important mechanism by which sodium balance is regulated. We have reported that gastrin secreted by G‐cells in the stomach and duodenum is involved in the regulation of sodium balance and BP. Besides gastrin, glucagon‐like peptide‐1 (GLP‐1) is another gut hormone, which may also participate in regulation of sodium balance and BP. It is well known that glucose‐induced GLP‐1 secretion is sodium‐dependent. The increase in intracellular calcium needed for GLP‐1 secretion is also due to sodium‐dependent cell excitability. However, the specific effect of varying sodium concentration on GLP‐1 secretion and its potential mechanism remain to be elucidated. This study aimed to determine the effect of varying sodium concentrations on GLP‐1 secretion and its potential mechanism. Here, we show that sodium can stimulate GLP‐1 secretion and that gastrin‐releasing peptide (GRP) enhances the high sodium‐induced increase in GLP‐1 secretion in a concentration and time‐dependent manner in L‐cells. The exposure of L‐cells to high (170 mM) Na+ increases GLP‐1 protein (produced by degradation of glucagon) or proglucagon mRNA expression, while Low (90 mM) and high (170 mM) Na+ increase NFAT5 and GRP receptor (GRPR) mRNA and protein expression. Silencing NFAT5 abrogates the GRP‐mediated enhancement of high sodium‐induced increase in GLP‐1 secretion and blocks the sodium‐induced increase in GRPR mRNA and protein expressions in L‐cells. Furthermore, NFAT5 silencing reduces the GLP‐1 protein or proglucagon expression in L‐cells exposed to high sodium concentration. NFAT5 silencing also reduces GRPR expression, using the promoter reporter assay; n=3/group for all studies. These results suggest that the high sodium‐induced NFAT5 expression increases expression of GRPR; GRP acting on the increased GRPR expression increases GLP‐1 secretion from L‐cells. These data give a new perspective on the mechanisms of GLP‐1 secretion, and the direct relationship between sodium and GLP‐1. These may contribute to better utilization of existing GLP‐1 based drugs and further GLP‐1‐based drug development.Effect of NaCl or GRP on GLP‐1 secretion in CCL‐251 L cells (human colorectal adenocarcinoma cells=GLP‐1 secreting cells).The same amount of cells were exposed to 90 mM, 145 mM, or 170 mM NaCl medium, and A, treated with varying concentrations of GRP (10, 100, 500, or 1000 nmol) for 4 h, or B, treated with 1000 nmol GRP at different times (1 h, 2 h, 3 h, or 4 h). GLP‐1 in the cell culture supernatant was measured by ELISA. The results are expressed as ng/ml. *P<0.05 vs. 90 mM with or without GRP. #P<0.05 vs. others, one‐way ANOVA, Holm‐Sidak test, n=3/treatment.Figure 1

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA—mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

Potentiation of Glucagon-Like Peptide-2 Dynamics by Methotrexate Administration in Rat Small Intestine.

The aim of this study was to clarify the relationship between chemotherapy-induced mucositis and endogenous glucagon-like peptide-2 (GLP-2) dynamics in the small intestine following treatment with either methotrexate or 5-fluorouracil. Rats were injected intraperitoneally with a single dose of either 50 mg/kg methotrexate or 100 mg/kg 5-fluorouracil. At 24 and 72 h after drug administration, ileal tissues and plasma were used to investigate GLP-2 dynamics. Administration of methotrexate caused moderate but not significant intestinal injury within 72 h, while administration of 5-fluorouracil caused severe injury in a time-dependent manner. Methotrexate significantly increased proglucagon mRNA expression and the number of anti-GLP-2 antibody-positive cells in the ileal tissue at 24 h after administration. Methotrexate also significantly induced GLP-2 receptor, insulin-like growth factor-1 (IGF-1), and transforming growth factor-β2 (TGF-β2) mRNA expression in ileal tissue. In contrast, 5-fluorouracil significantly inhibited proglucagon, GLP-2 receptor, IGF-1, and TGF-β2 mRNA expression as well as the number of anti-GLP-2 antibody-positive cells. Methotrexate slightly increased dipeptidyl peptidase IV (DPP-4) mRNA expression, whereas 5-fluorouracil significantly increased DPP-4 mRNA expression. These results suggest that potentiation of endogenous GLP-2 dynamics by methotrexate is associated with a mechanism that preserves gastrointestinal mucosal integrity at a moderate level.

A New Player in Intra‐islet Communication: The Hyperglycemic Effects of Neuronostatin through Increased Glucagon Production and Reduced Insulin Release

The tight regulation of blood glucose with insulin and antidiabetic agents greatly increases the risk and dangers of hypoglycemia in patients with Type 1 and Type 2 Diabetes. In addition, repeated bouts of hypoglycemia diminish the sympathetic arms of the counterregulatory response and blunt awareness of symptoms, creating a vicious cycle of unawareness and increasingly severe hypoglycemia. The only option in preventing or reversing hypoglycemia unawareness is careful avoidance of hypoglycemia through close monitoring– an often unobtainable goal. There is great need for other therapies and better understanding of diabetic hypoglycemia. We recently identified a novel peptide, neuronostatin (NST), encoded in the somatostatin preprohormone and found in all somatostatin‐positive tissues and cells. However, the activity of NST is functionally distinct from other preprosomatostatin products. Furthermore, it appears the physiologic stimuli of the peptides are different and that the peptides are sequestered in separate secretory vesicles. Our group identified the orphan GPCR, GPR107, as a cognate receptor of NST. Through an interaction with GPR107, NST enhanced proglucagon mRNA expression and glucagon release from isolated islets and immortalized alpha cells. NST also reduced beta cell release of insulin in response to high glucose conditions in isolated islets. Based off our findings, we hypothesized that NST is a pancreatic component of the counterregulatory response to hypoglycemia. In support of our hypothesis, an infusion of NST substantially increased plasma glucose levels over a 30‐minute time period in male rats. Additionally, NST delayed glucose clearance and diminished the insulin response to hyperglycemia in a glucose tolerance test. Fasting blood glucose raised plasma NST levels in rats, and exposure to low glucose stimulated NST release from human islets. Further experiments will confirm the role of NST in preventing or reversing hypoglycemia and determine how NST plays a role in the complex relationships of islet regulation and signaling. We propose that NST could represent a novel therapeutic target for the treatment and prevention of hypoglycemia in diabetes.Support or Funding InformationThis work was completed with funding support from the NIH and NIDDK, and the Helmsley Charitable Trust.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Orosensory Detection of Dietary Fatty Acids Is Altered in CB₁R-/- Mice.

Obesity is one of the major public health issues, and its prevalence is steadily increasing all the world over. The endocannabinoid system (ECS) has been shown to be involved in the intake of palatable food via activation of cannabinoid 1 receptor (CB1R). However, the involvement of lingual CB1R in the orosensory perception of dietary fatty acids has never been investigated. In the present study, behavioral tests on CB1R−/− and wild type (WT) mice showed that the invalidation of Cb1r gene was associated with low preference for solutions containing rapeseed oil or a long-chain fatty acid (LCFA), such as linoleic acid (LA). Administration of rimonabant, a CB1R inverse agonist, in mice also brought about a low preference for dietary fat. No difference in CD36 and GPR120 protein expressions were observed in taste bud cells (TBC) from WT and CB1R−/− mice. However, LCFA induced a higher increase in [Ca2+]i in TBC from WT mice than that in TBC from CB1R−/− mice. TBC from CB1R−/− mice also exhibited decreased Proglucagon and Glp-1r mRNA and a low GLP-1 basal level. We report that CB1R is involved in fat taste perception via calcium signaling and GLP-1 secretion.

Molecular identification of single hormone-encoding proglucagon cDNA isoforms from squamates and their abundant expression

Among ectothermic reptiles, the order Squamata has adapted most successfully to the terrestrial environment. However, the physiological background of this success remains unknown. Since the regulation of energy metabolism provides an important insight into terrestrial adaption by ectothermic animals, we focused on proglucagon-derived peptides (PGDPs). In the process of cloning proglucagon mRNA in geckos, we identified several novel proglucagon (PG) cDNA isoforms. They were tissue-specifically and strongly expressed in the pancreas and small intestine of the geckos, suggesting their biological relevance. Therefore, in order to clarify whether these novel cDNA isoforms are phylogenetically conserved, we performed the additional molecular characterization of proglucagon cDNAs from several representative species of the Squamata and Testudine clade and examined the expression of proglucagon mRNAs in the small intestine and pancreas.In the present study, a total of 7 proglucagon cDNA isoforms were identified and divided into two groups (Classes A and B) based on the 3′-UTR sequence of each isoform. The longest isoform of each group (named PG-A1 and PG-B1, respectively) had the same molecular characteristics as those previously reported from chickens and reptiles, namely, PG-A and PG-B. Other 5 isoforms were novel-type cDNAs, and were the products of exon skipping (named PG-A2, PG-A2s, PG-B2, PG-B2s, and PG-B3). Some of these isoforms coded for only one peptide hormone (GLP-1 or GLP-2). This is the first identification of single hormone-encoding proglucagon cDNAs in vertebrates. Moreover, an expression analysis of these isoforms revealed that single hormone-encoding proglucagon mRNAs were predominantly expressed with tissue and lineage specificities in the reptile clade.Collectively, the present results suggest an independent regulatory system for GLP-1 and GLP-2 secretion and indicate the plasticity of proglucagon genes in expressing different isoforms in different tissues in Squamata. These results also provide insights into the plastic energy metabolic system of Squamata in accordance with various habitats in the terrestrial environment, supporting their successful prosperity.

Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3

One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut.

Size Effect on Lipid Nanocapsule-Mediated GLP-1 Secretion from Enteroendocrine L Cells.

L cells are enteroendocrine cells located throughout the gastrointestinal tract that secrete physiologically important peptides. The most characterized peptides secreted by L cells are the peptide YY (PYY) and the glucagon-like peptides 1 (GLP-1) and 2 (GLP-2). These peptides are released rapidly into the circulation after oral nutrient ingestion. Recently, lipid-based nanoparticles (NP) have been described as triggers for GLP-1 secretion by L cells. NP physicochemical properties play a key role in the NP-cell interaction, and drive NP cell internalization. We herein hypothesize that lipid-based NP with appropriate size would not only be able to deliver drugs into blood circulation but also act like endogenous ligands to stimulate GLP-1 secretion. We tested five different size (25, 50, 100, 150, and 200 nm) lipid nanocapsules (LNC) on murine L cells in vitro to confirm this hypothesis. Our study showed that GLP-1 secretion was induced only by the 200 nm size LNC, highlighting the importance of LNC particle size on the secretion of GLP-1 by L cells. The different formulations did not affect proglucagon mRNA expression, suggesting that there was not an increased GLP-1 synthesis. As a proof of concept, we further demonstrated in normoglycemic mice that 200 nm LNC administration increases GLP-1 levels by 4- and 3-fold compared to untreated control mice 60 and 180 min after the administration, respectively. Our study suggests that 200 nm LNC as a nanocarrier to encapsulate drug candidates and as a ligand to induce endogenous GLP-1 secretion might represent a promising strategy for type 2 diabetes mellitus treatment.

P 158 - GLP-2 in the capacity of (-)-epicatechin and anthocyanidins to improve insulin sensitivity

Glucagon-like peptide-2 (GLP-2) is an incretin involved in energy balance through the regulation of postprandial glycaemia. GLP-2 is generated from the post-translational cleavage of proglucagon, being rapidly degraded in the bloodstream by dipeptidyl peptidase IV (DPP-IV). We currently investigated the effects of (-)-epicatechin (EC) and anthocyanidins (AC) to modulate GLP-2 using in vivo and in vitro approaches. To assess in vivo effects on proglucagon mRNA and plasma GLP-2 concentrations, male C57BL/6 J mice were fed for 14 w control (C) or high fat (HF) diets without or with 20 mg EC/kg or 40 mg AC/kg BW. Supplementation with EC and AC mitigated HF-induced insulin resistance (improved GTT, ITT, plasma insulin and glucose levels). Plasma GLP-2 levels were 148 and 200% higher in C mice supplemented with EC and AC compared to non-supplemented C mice, and 83 and 46% higher in HF mice supplemented with EC and AC compared to non-supplemented HF mice. Colon proglucagon mRNA levels were higher (84%) in AC-supplemented vs unsupplemented C mice, while EC had no effect. In vitro, cyanidin, but not EC, inhibited DPP-IV activity. The improvement of insulin sensitivity by EC and AC in a model of high fat diet-induced type 2 diabetes could be in part due to GLP-2 upregulation. The underlying mechanisms for AC involve an increase in proglucagon expression and decreased GLP-2 degradation.

Ghrelin Is a Regulator of Glucagon-Like Peptide 1 Secretion and Transcription in Mice.

The gut hormones ghrelin, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) have been intensively studied for their role in metabolism. It is, however, not well known whether the hormones interplay and regulate the secretion of each other. In this study, we studied the effect of ghrelin on GLP-1, GIP, and insulin secretion during an oral glucose tolerance test (OGTT) in mice. Intravenous administration of ghrelin caused increased GLP-1 secretion during the OGTT. On the other hand, ghrelin had no effect on circulating levels of glucose, insulin, and GIP. Furthermore, ghrelin treatment reduced proglucagon mRNA expression in GLUTag cells. The effect of ghrelin on GLP-1 secretion and proglucagon transcription was reinforced by the presence of GHS-R1a in human and mouse ileal L-cells, as well as in GLUTag cells. In summary, ghrelin is a regulator of GLP-1 secretion and transcription, and interfering with GHS-R1a signaling may be a way forward to enhance endogenous GLP-1 secretion in subjects with type 2 diabetes.