Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells

Kuhre, Rune Ehrenreich; Albrechtsen, Nicolai J. Wewer; Deacon, Carolyn F.; Balk-Møller, Emilie; Rehfeld, Jens F.; Reimann, Frank; Gribble, Fiona M.; Holst, Jens J.

doi:10.1530/jme-15-0293

Cited by 88 publications

(75 citation statements)

References 56 publications

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“…Several reports have suggested that, in addition to being derived from endocrine cells in the intestinal mucosa, both GLP‐1 and GIP may be produced locally within the pancreas, although in our laboratory we have never been able to identify GIP in murine or human pancreas extracts from healthy individuals (Holst and Hartmann, unpublished studies). Proglucagon is normally processed by prohormone convertase (PC) 1/3 in the intestinal L‐cell to produce GLP‐1 (and GLP‐2), while in the pancreatic α‐cells PC‐2 action produces glucagon . However, PC1/3 expression and fully processed GLP‐1 have been identified in rodent and human α‐cells, and islets in culture .…”

Section: Mechanism Of Actionmentioning

confidence: 84%

Do we know the true mechanism of action of the DPP‐4 inhibitors?

Andersen

Deacon

Holst

2017

Diabetes Obesity Metabolism

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The prevalence of type 2 diabetes is increasing, which is alarming because of its serious complications. Anti-diabetic treatment aims to control glucose homeostasis as tightly as possible in order to reduce these complications. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a recent addition to the anti-diabetic treatment modalities, and have become widely accepted because of their good efficacy, their benign side-effect profile and their low hypoglycaemia risk. The actions of DPP-4 inhibitors are not direct, but rather are mediated indirectly through preservation of the substrates they protect from degradation. The two incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, are known substrates, but other incretin-independent mechanisms may also be involved. It seems likely therefore that the mechanisms of action of DPP-4 inhibitors are more complex than originally thought, and may involve several substrates and encompass local paracrine, systemic endocrine and neural pathways, which are discussed here. and, thereby, to enhance its antihyperglycaemic actions. 5,6 GLP-1 is a gut hormone, released in response to digestion and absorption of food in the small intestine, which is responsible for an important part of postprandial insulin secretion. 7 This, the so-called incretin effect, is markedly reduced in patients with type 2 diabetes (T2D), which is believed to contribute to the inferior glucose tolerance seen after food ingestion in these individuals. 7 An additional incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), is also degraded by DPP-4, 2,4,8 but given that the insulinotropic, and hence glucose-lowering, effect of GIP is almost entirely absent in patients with T2D, 9 it was originally thought that anti-hyperglycaemic actions of DPP-4 inhibitors were mediated entirely by GLP-1 escaping DPP-4 degradation. Clinical studies have consistently shown that DPP-4 inhibitors increase the circulating concentrations of intact endogenous GLP-1 (and GIP) by about 2-to 4-fold. 10,11 Although considerably less than the 10-fold increase predicted from the observation that only about 10% of exogenously administered GLP-1 survives in the intact form, 12 this is nevertheless sufficient to cause a relative (in relation to plasma glucose levels) increase in insulin and reduction in glucagon concentrations. 11,13Accordingly, the mechanism by which DPP-4 inhibitors improve glycaemic control was believed to be simply due to their ability to enhance the endocrine actions of GLP-1. However, evidence now suggests that this is unlikely to be the only mechanism, and that other pathways and mediators may be involved.

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Section: Mechanism Of Actionmentioning

confidence: 84%

Do we know the true mechanism of action of the DPP‐4 inhibitors?

Andersen

Deacon

Holst

2017

Diabetes Obesity Metabolism

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“…NCI-H716 cells are widely used in the study of GLP-1 secretion,17 and the presence of TGR5 receptor in NCI-H716 cells has been characterized 10. Therefore, we used NCI-H716 cells to investigate the effect of betulinic acid on GLP-1 secretion in vitro.…”

Section: Resultsmentioning

confidence: 99%

Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

Lo¹,

Cheng²,

Li³

et al. 2016

DDDT

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BackgroundG-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported.MethodsTransfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits.ResultsBetulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time.ConclusionSimilar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future.

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“…90080 and 90060; Zaandam, Netherlands). For chromatography, 4 ml of a single pool of plasma from mice (n ϭ 20) and rats (n ϭ 27) were centrifuged (1,650 g, 4°C, 10 min), and the supernatant was fractionated by gel filtration as described previously (4,11,15).…”

Section: Biochemical Measurementsmentioning

confidence: 99%

Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes

Albrechtsen

Kuhre

Windeløv

et al. 2016

American Journal of Physiology-Endocrinology and Metabolism

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Wewer Albrechtsen NJ, Kuhre RE, Windeløv JA, Ørgaard A, Deacon CF, Kissow H, Hartmann B, Holst JJ. Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes. Am J Physiol Endocrinol Metab 311: E302-E309, 2016. First published May 31, 2016 doi:10.1152/ajpendo.00119.2016.-Glucagon is a metabolically important hormone, but many aspects of its physiology remain obscure, because glucagon secretion is difficult to measure in mice and rats due to methodological inadequacies. Here, we introduce and validate a low-volume, enzyme-linked immunosorbent glucagon assay according to current analytical guidelines, including tests of sensitivity, specificity, and accuracy, and compare it, using the BlandAltman algorithm and size-exclusion chromatography, with three other widely cited assays. After demonstrating adequate performance of the assay, we measured glucagon secretion in response to intravenous glucose and arginine in anesthetized mice (isoflurane) and rats (Hypnorm/midazolam). Glucose caused a long-lasting suppression to very low values (1-2 pmol/l) within 2 min in both species. Arginine stimulated secretion 8-to 10-fold in both species, peaking at 1-2 min and returning to basal levels at 6 min (mice) and 12 min (rats). D-Mannitol (osmotic control) was without effect. Ketamine/xylazine anesthesia in mice strongly attenuated (P Ͻ 0.01) ␣-cell responses. Chromatography of pooled plasma samples confirmed the accuracy of the assay. In conclusion, dynamic analysis of glucagon secretion in rats and mice with the novel accurate sandwich enzyme-linked immunosorbent assay revealed extremely rapid and short-lived responses to arginine and rapid and profound suppression by glucose.␣-cell; islet biology; immunoassay; enzyme-linked immunosorbent assay GLUCAGON REGULATES BLOOD GLUCOSE by stimulating hepatic glucose production (3). Importantly, elevated fasting and postprandial glucagon levels in patients with type 2 diabetes contribute to the hyperglycemia of these patients as demonstrated clearly using specific antagonists of the glucagon receptor (13). However, regulation of glucagon secretion has only been incompletely characterized, due partly to technical limitations with regard to its quantification (volume requirements, inadequate specificity, and accuracy) using current immune-based detection methods: radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) (7,8,16,18,22).Here, we introduce a novel low-volume sandwich ELISA that exclusively detects fully processed pancreatic glucagon, corresponding to proglucagon-(33-61) (see Fig. 1), and use it to characterize the dynamics of glucagon secretion in mice and rats in response to intravenous injections of L-arginine, D-glucose, and D-mannitol. By extensive assay validation, we conclude that the applied assay is reliable for measurements of glucagon in small plasma volumes. Because of this technical advance, we were able to make reliable real-time estimates of glucagon secretion (in vivo) in mice and rats. Our...

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Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells

Cited by 88 publications

References 56 publications

Do we know the true mechanism of action of the DPP‐4 inhibitors?

Do we know the true mechanism of action of the DPP‐4 inhibitors?

Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes

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