In the gastrointestinal GI tract CART expression has been de

In the gastrointestinal (GI) tract CART expression has been described in the enteric nervous system in rats (Ekblad et al., 2003), pigs (Wierup et al., 2007), sheep (Arciszewski et al., 2009), guinea pigs (Ellis and Mawe, 2003) and humans (Kasacka et al., 2012). Less is known about CART expression in enteroendocrine cells. In sheep, a small population of CART immunoreactive (IR) endocrine Marizomib was detected in the abomasum part of the stomach (Arciszewski et al., 2009). In rats, CART-expressing endocrine cells were confined to the antrum and duodenum. The majority of these cells were identical to gastrin-producing G-cells, but a duodenal subpopulation remains unidentified (Ekblad et al., 2003). Although CART is produced by human neuroendocrine GI tumors (Landerholm et al., 2011) there is to this date no information on CART expression in enteroendocrine cells in the normal human GI-tract.
Materials and methods
Results
Discussion We found expression of CART in subpopulations of human K- and L-cells in the human upper small intestine. L-cells are most numerous in the distal parts of the gut (Baggio and Drucker, 2007), but it is well established that L-cells are present also in the upper small intestine (Theodorakis et al., 2006). The present study was limited to duodenal and jejunal specimens, and further studies are needed to understand if CART has a similar expression pattern also in the lower segments of the gut. Nevertheless, confocal imaging localized CART to the secretory granules in both cell types. This suggests, but does not prove, that CART is co-secreted with GIP and GLP-1. CART plasma levels were unaffected by an oral glucose load in humans. However, we observed a modest increase in CART plasma levels 60 min after ingestion of a mixed-meal. Plasma CART levels were previously reported to be unaffected by food intake (Bech et al., 2008). Plasma CART concentrations were in the same range as in our present study (Bech et al., 2008). It should be mentioned that CART was measured in the systemic circulation and we cannot exclude a more pronounced postprandial response of CART in the veins draining the intestine. CART-silencing in GLUTag cells caused decreased GLP-1 secretion in response to glucose, whereas exogenous CART treatment did not have any effect on GLP-1 secretion. Although the exact mechanisms behind the effect are difficult to dissect due to the lack of identified CART receptor(s) (Rogge et al., 2008; Vicentic et al., 2006), our data imply that endogenous L-cell CART exerts a stimulatory action on GLP-1 secretion. The present GLP-1 data are reminiscent of those obtained for insulin in beta-cells, in which silencing of CART caused reduced insulin secretion and production (Shcherbina et al., 2017). The observed reduction in insulin secretion is likely related to reduced transcription of genes encoding proteins with important roles in the exocytotic machinery, as well as a network of beta-cell transcription factors. Thus, although further studies are needed to understand the mechanisms behind the effect of CART KD on GLP-1 secretion, it is tempting to speculate that CART has similar roles also in GLUTag cells used in the present study. In line with the effect on GLP-1 secretion, silencing of CART caused reduced GLP-1 mRNA expression, as well as protein content. In clonal INS-1 (832/13) beta-cells CART KD resulted in decreased expression of the transcription factors Isl1 and Tcf7l2, both of which have been shown to regulate GLP-1 transcription in enteroendocrine cells (Jin, 2008; Shao et al., 2013; Yi et al., 2005; Terry et al., 2014). If CART KD has the same effect in GLUTag cells, this could be a potential explanation for the observed reduction in GLP-1 mRNA expression. The effect of CART on incretin secretion in response to an oral glucose load was studied in vivo in mice. In accordance with the effect of CART KD, administration of CART increased the secretion of GLP-1, but also of GIP. In mammals GIP is mainly expressed in the enteroendocrine K-cells (Baggio and Drucker, 2007). In mice and humans, also the pancreatic alpha-cells have been reported to express GIP (Fujita et al., 2010; Prasadan et al., 2011). Intestinal proGIP is mostly processed by PC1/3 to yield GIP1-42, whereas in islet alpha-cells PC2 cleavage of proGIP yields the shorter form GIP1-30 (Fujita et al., 2010). Importantly, the islet processing form of GIP is not detected by the GIP ELISA used here. Therefore, we conclude that CART increased GIP secretion specifically from the K-cells. Although GIP mRNA expression levels do not necessarily need to be linked to the levels of hormone secretion (Baggio and Drucker, 2007), it should be noted that, silencing of CART in STC-1 cells resulted in increased expression of GIP mRNA, suggesting that endogenous K-cell CART is an inhibitor of GIP transcription. This may be a consequence of the differences in promotor architecture and binding sites for the transcription factors and factors regulating GIP and GLP-1 genes (Baggio and Drucker, 2007). Alternatively, the stimulatory effect of CART on GIP expression is indirect as STC-1 cells express multiple peptides, including glucagon, somatostatin, CCK and amylin, that could in turn mediate the effect on GIP expression (Rindi et al., 1990; Boylan et al., 1997).