Functionality and antidiabetic utility of ß- and L-cell containing pseudoislets.

Unavailability of tissue and poor engraftment remain significant obstacles to clinical islet transplantation. Here, the therapeutic potential of pseudoislets generated from the insulin and GLP-1 releasing cell-lines MIN6 and GLUTag was investigated. Glucose and other secretagogues evoked 1.3-5.7 fold increases in insulin secretion from both pseudoislet types. Secretion expressed in relation to basal values did not greatly differ between configurations. Exposure of both types of pseudoislets to ninhydrin, H2O2, streptozotocin or cytokine cocktails decreased viability and increased apoptosis. However, combined pseudoislets exhibited enhanced resistance (1.2-1.7 fold increased LD50, 1.2-1.4 fold decreased apoptosis). Implantation of pseudoislets into streptozotocin-diabetic SCID mice precipitated cell masses containing immunoreactive insulin and GLP-1. Implantation of both pseudoislet types was associated with significant reductions in blood glucose, increased plasma insulin, greater bodyweight, decreased polydipsia and improved glucose tolerance. These changes greatly exaggerated in MIN6 pseudoislet recipients, with mice becoming severely hypoglycaemic. In contract, combined pseudoislet recipients achieved tempered restoration of normoglycaemia and exhibited increased plasma GLP-1, decreased plasma and pancreatic glucagon, increased pancreatic insulin and enhancements in islet ß:α cells and the ratio of Ki67: TUNEL positive ß-cells. MIN6 pseudoislet implantation increased islet ß:α cell ratio but did not affect ß-cell proliferation or hormone content. Our observations highlight the potential of combining insulin and GLP-1 cell therapy using heterotypic pseudoislets.

Alpha-cyclodextrin increases glucagon-like peptide-1 secretion in multiple models and improves metabolic status in mice.

Alpha-cyclodextrin (α-CD) is a non-absorbable and soluble fiber that causes weight loss. We studied whether this is due to an effect on GLP-1 secretion. In GLUTag cells, α-CD increased GLP-1 secretion up to 170% via adenylyl cyclase, phospholipase C, and L-type calcium channels dependent processes. In rat isolated colon perfusions, luminal α-CD increased GLP-1 secretion with 20%. In lean mice, once daily α-CD versus saline caused weight loss and lowered the peak in glucose after an oral glucose tolerance test (OGTT). In obese mice, α-CD added to high-fat diet caused weight loss similar to the control group (receiving cellulose). However, compared to cellulose, the α-CD group ate less. During an OGTT, no differences were observed in glucose, insulin and GLP-1. Thus, α-CD increases GLP-1 secretion in a dose-dependent manner and could be a safe and easy addition to food products to help reduce body weight.

ßHB inhibits glucose-induced GLP-1 secretion in GLUTag and human jejunal enteroids.

Ingestion of nutrients stimulates incretin secretion from enteroendocrine cells (EECs) of the epithelial layer of the gut. Glucagon-like peptide-1 (GLP-1) is one of these incretins that stimulate postprandial insulin release and signal satiety to the brain. Understanding the regulation of incretin secretion might open up new therapeutic options for obesity and type-2 diabetes mellitus. To investigate the inhibitory effect of the ketone body ß-hydroxybutyrate (ßHB) on glucose-induced GLP-1 secretion from EECs, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were stimulated with glucose to induce GLP-1 secretion. The effect of ßHB on GLP-1 secretion was studied using ELISA and ECLIA methods. GLUTag cells stimulated with glucose and ßHB were analysed using global proteomics focusing on cellular signalling pathways and the results were verified by Western blot. Results demonstrated ßHB had a significant inhibitory effect on glucose-induced GLP-1 secretion at a dose of 100 mM in GLUTag cells. In differentiated human jejunal enteroid monolayers, glucose-induced secretion of GLP-1 was inhibited at a much lower dose of 10 mM ßHB. The addition of ßHB to GLUTag cells resulted in decreased phosphorylation of kinase AKT and transcription factor STAT3 and also influenced the expressions of signalling molecule IRS-2, kinase DGKε and receptor FFAR3. In conclusion, ßHB displays an inhibitory effect on glucose-induced GLP-1 secretion in vitro in GLUTag cells and in differentiated human jejunal enteroid monolayers. This effect may be mediated through multiple downstream mediators of G-protein coupled receptor activation, such as PI3K signalling.

NMDA Receptor Antagonists Increase the Release of GLP-1 From Gut Endocrine Cells.

Type 2 diabetes mellitus (T2DM) remains one of the most pressing health issues facing modern society. Several antidiabetic drugs are currently in clinical use to treat hyperglycaemia, but there is a need for new treatments that effectively restore pancreatic islet function in patients. Recent studies reported that both murine and human pancreatic islets exhibit enhanced insulin release and ß-cell viability in response to N-methyl-D-aspartate (NMDA) receptor antagonists. Furthermore, oral administration of dextromethorphan, an over-the-counter NMDA receptor antagonist, to diabetic patients in a small clinical trial showed improved glucose tolerance and increased insulin release. However, the effects of NMDA receptor antagonists on the secretion of the incretin hormone GLP-1 was not tested, and nothing is known regarding how NMDA receptor antagonists may alter the secretion of gut hormones. This study demonstrates for the first time that, similar to ß-cells, the NMDA receptor antagonist MK-801 increases the release of GLP-1 from a murine L-cell enteroendocrine model cell line, GLUTag cells. Furthermore, we report the 3' mRNA expression profiling of GLUTag cells, with a specific focus on glutamate-activated receptors. We conclude that if NMDA receptor antagonists are to be pursued as an alternative, orally administered treatment for T2DM, it is essential that the effects of these drugs on the release of gut hormones, and specifically the incretin hormones, are fully investigated.

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.

Let-7e-5p Regulates GLP-1 Content and Basal Release From Enteroendocrine L Cells From DIO Male Mice.

Characterization of enteroendocrine L cells in diabetes is critical for better understanding of the role of glucagon-like peptide-1 (GLP-1) in physiology and diabetes. We studied L-cell transcriptome changes including microRNA (miRNA) dysregulation in obesity and diabetes. We evaluated the regulation of miRNAs through microarray analyses on sorted enteroendocrine L cells from control and obese glucose-intolerant (I-HFD) and hyperglycemic (H-HFD) mice after 16 weeks of respectively low-fat diet (LFD) or high-fat diet (HFD) feeding. The identified altered miRNAs were studied in vitro using the mouse GLUTag cell line to investigate their regulation and potential biological functions. We identified that let-7e-5p, miR-126a-3p, and miR-125a-5p were differentially regulated in L cells of obese HFD mice compared with control LFD mice. While downregulation of let-7e-5p expression was observed in both I-HFD and H-HFD mice, levels of miR-126a-3p increased and of miR-125a-5p decreased significantly only in I-HFD mice compared with controls. Using miRNA inhibitors and mimics we observed that modulation of let-7e-5p expression affected specifically GLP-1 cellular content and basal release, whereas Gcg gene expression and acute GLP-1 secretion and cell proliferation were not affected. In addition, palmitate treatment resulted in a decrease of let-7e-5p expression along with an increase in GLP-1 content and release, suggesting that palmitate acts on GLP-1 through let-7e-5p. By contrast, modulation of miR-125a-5p and miR-126a-3p in the same conditions did not affect content or secretion of GLP-1. We conclude that decrease of let-7e-5p expression in response to palmitate may constitute a compensatory mechanism contributing to maintaining constant glycemia in obese mice.

Casein materials show different digestion patterns using an in vitro gastrointestinal model and different release of glucagon-like peptide-1 by enteroendocrine GLUTag cells.

Physicochemical properties of casein (CN) materials manufactured using different processes are well studied; however, data on their bioaccessibility or bioactivity are limited. We compared the digestion patterns and glucagon-like peptide-1 (GLP-1)-releasing activities of micellar CN concentrate (MCC) and sodium caseinate (SCN). MCC and SCN mixed with whey protein isolate (SCN + WPI) were subjected to in vitro gastrointestinal digestion; the digestibility of MCC was higher than that of SCN + WPI, and both CN materials showed different patterns of peptides released after in vitro digestion. A comparison of GLP-1-releasing activities showed that MCC induced GLP-1 secretion to a greater extent than SCN + WPI. Candidate peptides identified from CN digesta were chemically synthesized to test their GLP-1-releasing activity. GPVRGPFPIIV identified only in the MCC digesta, could stimulate GLP-1 release. In conclusion, the digestion patterns and GLP-1-releasing activity of CN materials depend on the production process.

Pancreatic-derived factor impaired glucagon-like Peptide-1 production from GLUTag enterendorine L-cell line and intestines.

PURPOSE: Pancreatic-derived factor (PANDER) is a pancreatic islet-specific cytokine that co-secretes with insulin. However, its biological function remains largely unknown. We have recently shown that the intestine might be its novel target tissue. The aim of this study was to clarify whether PANDER impacts the production of glucagon-like peptide-1 (GLP-1). METHODS: We treated GLUTag cells from the mouse intestine L cell line with recombinant PANDER protein and hepatic overexpression of PANDER in an obese murine model. RESULTS: In GLUTag cells, PANDER exposure led to decreased proglucagon gene mRNA expression and GLP-1 secretion without affecting cell viability or caspase-3 activation. Overexpression of PANDER in mice induced glucose intolerance and impaired glucose-stimulated GLP-1 secretion Moreover, PANDER blocked insulin-induced GLP-1 secretion by inhibiting the insulin signalling-Wnt pathway and directly inhibited the cAMP/PKA pathway. CONCLUSIONS: Our findings indicate that intestinal L cells are responsive to PANDER, and elevated PANDER levels impair GLP-1 production in vitro and in vivo.

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.

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors.

Oleic acid stimulates glucagon-like peptide-1 release from enteroendocrine cells by modulating cell respiration and glycolysis.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) is a potent satiating and incretin hormone released by enteroendocrine L-cells in response to eating. Dietary fat, in particular monounsaturated fatty acids, such as oleic acid (OA), potently stimulates GLP-1 secretion from L-cells. It is, however, unclear whether the intracellular metabolic handling of OA is involved in this effect. METHODS: First we determined the optimal medium for the bioenergetics measurements. Then we examined the effect of OA on the metabolism of the immortalized enteroendocrine GLUTag cell model and assessed GLP-1 release in parallel. We measured oxygen consumption rate and extracellular acidification rate in response to OA and to different metabolic inhibitors with the Seahorse extracellular flux analyzer. RESULTS: OA increased cellular respiration and potently stimulated GLP-1 release. The fatty acid oxidation inhibitor etomoxir did neither reduce OA-induced respiration nor affect the OA-induced GLP-1 release. In contrast, inhibition of the respiratory chain or of downstream steps of aerobic glycolysis reduced the OA-induced GLP-1 release, and an inhibition of the first step of glycolysis by addition of 2-deoxy-d-glucose even abolished it. CONCLUSION: These findings indicate that an indirect stimulation of glycolysis is crucial for the OA-induced release of GLP-1.

Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress.

OBJECTS: Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a high-fat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model. METHODS: To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate. RESULTS: Palmitate, but not oleate caused ER stress and decreased the protein levels of prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells. CONCLUSIONS/INTERPRETATION: These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production.