Enteroendocrine Cell Models: General Introduction

Part IV Enteroendocrine Cell Models: General Introduction During and after a meal, ingested nutrients modulate the release of a variety of hormones, many of which serve roles to enhance nutrient digestion. A number of these gut hormones, most notably cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), are key players in infl uencing eating behaviour and food intake, largely through induction of satiety (Begg and Woods 2013 ). Some of these hormones (glucose-dependent insulinotropic peptide (GIP) and GLP-1)) are also incretins, enhancing glucose-dependent insulin secretion. Identifying food components that alter the gastrointestinal hormonal milieu may aid in our ‘battle of the bulge’ as well as in the treatment of related metabolic diseases (Bruen et al. 2012 ). Mammalian enteroendocrine cell lines provide the starting framework to measure gut hormonal responses to food. In vitro models of enteroendocrine cell lines have proven diffi cult to develop, primarily because enteroendocrine cells are sparsely dispersed throughout the gastro-intestinal tract and are co-localized with abundant enterocytes. However, several groups have successfully established enteroendocrine cell models that are available for in vitro screening bioassays (Table 1 ). The NCI-H716 cell line is the only human enteroendocrine cell line available and it provides a unique L-cell model to screen food components for their ability to modulate secretion of the incretin hormone, GLP-1. This cell line was originally derived from cells present in the ascites fl uid of a male 33 year old patient with a poorly differentiated adenocarcinoma of the cecum (Park et al. 1987 ). When propagated in vitro, these cells have been described to exhibit endocrine differentiation including the expression of secretory granules and chromogranin A (de Bruine et al. 1993 ). However, they do require attachment matrices (de Bruine et al. 1993 ; Anini and Brubaker 2003 ) which could limit their ease of use in high throughput assays. The NCI-H716 cells express both GLP-1 and PYY but not CCK (Reimer et al. 2001 ; Jang et al. 2007 ). GLP-1 expression in these cells is mostly homogenous but PYY expression is much less uniform, with only ~30 % of cells containing PYY immunoreactivity (Jang et al. 2007 ). The NCI- H716 cell line is discussed in more detail in Chap. 20 . 208 Part IV Enteroendocrine Cell Models: General Introduction c lab c c lab c cult to transfect (University of California, L-cell models can be pro- ailable (Anini and Brubaker cult to transfect cult to culture; small number of Heterogeneous cell population; so may murine- and tumour-derived not recapitulate primary human L-cells Murine- and tumour-derived so may Murine- and tumour-derived not recapitulate human L-cells; requires basement membrane to attach to some surfaces Possibly heterogeneous as not single-cell cloned; is tumour-derived so may not recapitulate primary human L-cell; requires basement membrane to attach culture plate Heterogeneous cell population; obtained from fetal rats and may therefore not be representative of the adult human L-cell; small number of L-cells precludes cell-specifi western techniques (i.e. RT-PCR, blot); diffi Diffi L-cells precludes cell-specifi western techniques (i.e. RT-PCR, blot); may be diffi ed mice can be used as Representative of proximal L-cell; all lab techniques possible; secretes several gut hormones Representative of distal L-cell; subcloned homogenous cells; all lab techniques possible; easy to transfect Representative of distal L-cell; all lab techniques possible; easy to transfect Primary cells derived from normal intestine Primary cells derived from normal intestine; genetically- modifi tissue source; can be used to study single cells if derived from L-cell reporter mice 1 %) 1 %) ≅ ≅ Poorly differentiated; secretes CCK, GLP-1, pancreatic PYY, GIP, polypeptide, neurotensin, GLP-2 and oxyntomodulin Single-cell subcloned population of L-cells; secretes GLP-1 and CCK Heterogeneous population of intestinal cells (L-cells Undifferentiated Undifferentiated tumour cell line with endocrine morphology; secretes and mucin GLP-1, PYY Heterogeneous population of intestinal cells (L-cells ) ) 1990 ) 1994 1987 ) proglucagon-SV40 Large T T proglucagon-SV40 Large antigen transgenic mice (Drucker et al. combined from fetal rat (Park et al. from proinsulin-SV40 Large T T from proinsulin-SV40 Large antigen/proinsulin-polyoma x antigen transgenic T Small mice (Rindi et al. colon from adult mouse 2008 Human Spontaneous colorectal tumour Enteroendocrine cell models ; Reimann et al. GLUTag GLUTag Mouse Large bowel tumour in FRIC Rat Small and large intestines Model NCI- Species H716 Cell source Characteristics Advantages Disadvantages STC-1 Mouse Duodenal secretin tumour cells AMIC Mouse Duodenum, jejunum, ileum or Table Table 1 2003 The NCI-H716 cells are available from American Type Culture Collection (ATCC). STC-1 cells are available from Dr. D. Hanahan STC-1 cells are available from Dr. Culture Collection (ATCC). Type American The NCI-H716 cells are available from primary Additionally, Toronto). Daniel J. Drucker (University of cells can be requested from Dr. San Francisco, CA), and GLUTag duced in-house. Fetal rat intestinal culture (FRIC) protocols and adult mouse (AMIC) are widely av Part IV Enteroendocrine Cell Models: General Introduction 209 To study GLP-1 secretion, researchers also routinely use the alternative, albeit non-human, cell lines; STC-1 and GLUTag, which were developed from murine genetically-induced proximal and distal intestinal tumours, respectively. The STC-1 (secretin tumour cell) cell line is a heterogeneous enteroendocrine cell line which expresses several satiety and incretin hormones, including CCK, GIP, PYY, pancreatic polypeptide, neurotensin and the proglucagon-derived peptides: GLP-1, glucagon- like peptide-2 (GLP-2) and oxyntomodulin (Rindi et al. 1990 ). The pluri- hormonal nature of these cells appears to be consistent with recent fi ndings on the primary murine duodenal L-cell (Habib et al. 2012 , 2013 ). Molecular mechanisms underlying stimulus-hormone secretion coupling have been well defi ned in the STC-1 cell line. Primarily because of its ability to secrete several satiety hormones, it remains a popular choice for primary screening platforms, as detailed in the accompanying chapter. In contrast to STC-1 cells, the GLUTag (proglucagon SV40-large T antigen) cell line appears quite differentiated and demonstrates a stable pattern of proglucagon gene expression over a 4–8 week period (Drucker et al. 1994 ). However, unlike the NCI-H716 and STC-1 cells, the GLUTag cells express GLP-1 and CCK, but not PYY (Drucker et al. 1994 ). The absence of PYY is somewhat surprising given the colonic origin of the GLUTag cell line and the fact that most colonic L-cells express this hormone in vivo (Habib et al. 2013 ). Notwithstanding, GLUTag cells recapitulate the responsiveness of both of the other cell lines as well as of primary murine L-cells and fetal rat intestinal cultures, secreting multiple fully-processed glucagon- like peptides in response to known physiological and pharmacological secreta- gogues (Lim et al. 2009 ). For use in high throughput assays, it is worth noting that GLUTag cells require a matrix for proper attachment to multi-well plates. Further details regarding the GLUTag cell line can be found in the accompanying chapter. It is important to note that species-specifi c differences in nutrient sensitivity in vivo do exist and non-human cell lines may therefore not be appropriate beyond primary screens (Brubaker 1991 ; Brubaker et al. 1998 ; Habib et al. 2013 ). For example, while GIP is an effective stimulator of GLP-1 release in canine and rodent L-cells (Brubaker 1991 ; Brubaker et al. 1998 ) it has little effect on GLP-1 secretion in human L-cells in vivo (Kreymann et al. 1987 ). Moreover, it has been demon- strated that the transcription factors and promoter regions important for human proglucagon gene expression are distinct from those utilised by the rat proglucagon gene (Nian et al. 1999 ). Findings made in any cell line may not completely recapitulate those made in vivo, due to the extremely high likelihood that the L-cell con- stantly integrates multiple signals, from luminal, endocrine and neural inputs. It is thus imperative that all fi ndings made in vitro ultimately be confi rmed in the in vivo setting. With these limitations in mind, the enteroendocrine cell models still provide a set of powerful tools that enable a detailed examination of hormonal responses to food bioactives. 210 Part IV Enteroendocrine Cell Models: General Introduction References Anini Y, Brubaker PL (2003) Role of leptin in the regulation of glucagon-like peptide-1 secretion. Diabetes 52(2):252–259 Begg DP, Woods SC (2013) The endocrinology of food intake. Nat Rev Endocrinol 9(10): 584–597 Brubaker PL (1991) Regulation of intestinal proglucagon-derived peptide secretion by intestinal regulatory peptides. Endocrinology 128(6):3175–3182 Brubaker PL, Schloos J, Drucker DJ (1998) Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line. Endocrinology 139(10):4108–4114. doi: 10.1210/endo.139.10.6228 Bruen CM, O’Halloran F, Cashman KD, Giblin L (2012) The effects of food components on hormonal signalling in gastrointestinal enteroendocrine cells. Food Funct 3(11):1131–1143 de Bruine AP, Dinjens WN, van der Linden EP, Pijls MM, Moerkerk PT, Bosman FT (1993) Extracellular matrix components induce endocrine differentiation in vitro in NCI-H716 cells. Am J Pathol 142(3):773–782 Drucker DJ, Jin T, Asa SL, Young TA, Brubaker PL (1994) Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Mol Endocrinol 8(12):1646–1655 Habib AM, Richards P, Cairns LS, Rogers GJ, Bannon CA, Parker HE, Morley TC, Yeo GS, Reimann F, Gribble FM (2012) Overlap of endocrine hormone expression in the mouse intestine revealed by transcriptional profi ling and fl ow cytometry.

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