SI enteroendocrine cells are traditionally considered as distinct cell types [e.g
. the I-cells (CCK), S-cells (Sct), K-cells (GIP), L-cells (GLP-1/PYY), and D-cells (Sst)] deriving from a common intestinal stem cell precursor (6
). Our data suggest, however, that these cell populations are less distinct than previously recognized. The extent of the similarities was evident in the global microarray expression profiles of FACS-purified L- and K-cells which showed, surprisingly, that upper SI L-cells were more similar to their neighboring K-cells than to L-cells from the colon, although the latter are classically considered as the same cell type.
In the upper and lower SI and colon, 75–80% of Venus-positive cells from GLU-Venus mice stained positively for GLP-1. Although the apparent lack of GLP-1 staining in 20–25% of Venus-positive cells could represent Venus fluorescence in a non-L-cell population, detection of GLP-1 by immunofluorescence is inevitably less than 100% efficient because of factors such as antibody affinity and the requirement for cell permeabilization. Indeed, varying affinities between antibodies against different hormones preclude exact comparisons between the percentages of cells stained using different antibodies. We also observed that although Venus fluorescence was found in about 80% of GLP-1-positive cells from GLU-Venus mice, it had a lower penetrance in GIP-Venus mice, being detectable in only approximately 30% of GIP-immunopositive cells. This mirrors the level of Venus fluorescence, which was considerably brighter in enteroendocrine cells from GLU-Venus than GIP-Venus mice, likely due to a higher transgene copy number in the former strain. It is therefore probable that our collected K-cell populations were dominated by cells with high GIP promoter activity, whereas the L-cell populations spanned a broader range of proglucagon promoter activity and might also have included some cells at an earlier stage of differentiation.
The finding that upper SI and, to a lesser extent, lower SI L-cells expressed messages for GIP, CCK, Sct, and Nts, in addition to GLP-1 and PYY, prompted experiments to determine the proportions of L-cells staining positive for different key hormones. Despite the transcriptomic similarities between upper SI K- and L-cells, fewer than 15% of upper SI L-cells contained GIP, or of upper SI K-cells contained GLP-1, as assessed by immunostaining and FACS analysis. Almost all upper SI L-cells, however, were strongly immunoreactive against CCK, accounting for about 50% of the total CCK-positive cell population. This suggests that most upper SI L-cells contain CCK, but that there is also a distinct, similar-sized pool of I-cells that are not GLP-1 positive. Whether the CCK-containing cell populations that are GLP-1 positive or GLP-1 negative differ markedly in their functional responsiveness and physiological role will be interesting topics for future evaluation. Although mRNA analysis indicates that SI L-cell populations express gcg, gip, sct, cck, nts, and pyy, our data do not enable us to distinguish how many different hormones can be produced by any single enteroendocrine cell.
FACS analysis of upper SI K-cells revealed that most are also CCK positive and that there is an overlap of approximately 5% with Sst-producing D-cells. The detection of iapp
message in upper SI K-cells is consistent with a previous report that IAPP was identified in cells containing Sst, CCK, or PYY in the rat small intestine (26
). Apart from a higher L-cell number and percentage of L-cells coexpressing PYY in the lower SI, FACS analysis revealed only minor differences in the number of L-cells coexpressing GIP and CCK along the length of the SI. Because the lower SI cell suspensions contained many Venus-negative cells that bound secondary antibodies even in the absence of a primary antibody, we were unable to determine whether there are ileal pools of PYY-, GIP-, or CCK-positive cells that do not also express proglucagon. The lower levels of GIP and CCK mRNA expression in L-cell populations from the lower SI suggest, however, that the content of these peptides per L-cell, although detectable by FACS analysis, is less in the distal compared with the proximal SI.
Interestingly, the CgA antibody strongly stained a small population of cells in the upper SI but only weakly labeled the K- and L-cells in this region. The identity of the strongly positive CgA cells was not investigated here, but it was reported previously that the majority of enterochromaffin and gastrin cells are CgA positive, whereas CgA immunostaining was only variably detected in cells containing GIP, Sct, Nts, and PYY (27
). Although CgA is widely used as an indicator of enteroendocrine cells, our data therefore confirm that it is not a reliable marker of upper SI K- and L-cells.
We identified a number of TF specific to L- and K-cells, including factors previously implicated in enteroendocrine development, e.g.
Fev, Insm1, Isl1, NeuroD1, Nkx2.2, and Pax6 (7
), and/or pancreatic islet cell formation and function, e.g.
Arx, Glis3, Mlxipl, Myt1, Prox1, and Tle2 (31
). In addition, we found a number of TF not previously documented to play a role in enteroendocrine development, including Ankrd6, Bmi1, Dtx1, Egr2, Etv1, Fubp1, Grhl1, Ikzf4, Jazf1, Klf12, Nr4a2, St18, Tbx3, Zfp90, Zfp179, Zfp644, and Zfp667. The high expression of Mlxipl in colonic L-cells is interesting, because it encodes the carbohydrate response element binding protein, a known glucose-responsive regulator of metabolism (37
). This supports the idea that colonic L-cells are glucose sensitive, consistent with their expression of a number of genes involved in glucose metabolism (13
Upper SI L-cells were distinguishable from colonic L-cells by expression of several TF, including Dtx1, Egr2, and Tbx3, and from upper SI K-cells by expression of Etv1 and Prox1. Pax4 was higher in colonic L-cells than upper SI K-cells, consistent with the idea that this factor may contribute to repression of the GIP
). Also in agreement with a previous report that Pdx1 expression is required for coexpression of GIP in L-cells (38
), we detected Pdx1 in upper small intestinal L- and K-cells but not colonic L-cells. Interestingly, no TF were identified that were both specific to enteroendocrine cells and enriched in upper SI K-cells compared with upper SI or colonic L-cells. Etv1 appeared restricted to cell populations that produced gcg
(L- and α-cells), although is not a known regulator of gcg
gene expression or of enteroendocrine or pancreatic islet cell development. Jazf1, Glis3, and Prox1, which appeared here as enteroendocrine markers, have been reported as type 2 diabetes susceptibility genes (39
), so it will be interesting to determine whether human carriers of the susceptible genotypes have reduced incretin responses.
Profiles of TF expression in pancreatic α- and β-cells were consistent with previous observations in mice (42
) and largely mirrored a recent transcriptomic analysis of human α- and β-cells (44
). Thus, in mice, as in humans, Pdx1, Mafa, and Nkx6.1 were largely β-cell restricted, Mafb, Irx2, and Arx were largely α-cell restricted, and Nkx2.2 was found in both α- and β-cells. We found Mafb to be largely α-cell restricted consistent with previous data from mice, but differing from humans in whom Mafb was detected in both α- and β-cells. Our data also revealed that in mouse islets, Etv1 was strongly expressed in α-cells and Mlxipl was strongly expressed in β-cells. Contrary to the findings in human islets, we did not detect Hdac9 expression in either α- or β-cell populations (data not shown).
Cells coexpressing GLP-1 and GIP have been detected previously by immunostaining (45
) and reported to increase in number in subjects with diabetes (46
) or in the jejunum of rats that have undergone gastric bypass surgery (47
), suggesting that enteroendocrine cell development can be influenced by the metabolic and nutritional environment. GIP expression in the duodenum was also reported to vary according to dietary glucose availability (48
). Our data suggest that altered activity of very few TF could shift enteroendocrine cells from one phenotype to another. An interesting question for future research therefore will be to investigate the extent to which diet and metabolic disease influence the spectrum of gut endocrine cells, and whether shifts in the enteroendocrine cell population translate into alterations in feeding behavior or nutrient metabolism. The current interest in developing drugs targeting different intestinal endocrine cell populations, however, underlies the importance of an improved understanding of the interrelationships between these cell types.