Intestinal cell differentiation is characterized by increased ketogenesis

To determine whether proliferating and differentiated cells demonstrate distinct metabolic patterns, we used 13C-glucose and 13C-glutamine stable isotope resolved metabolomics (SIRMs) to map metabolic changes in cultured Caco-2 cells as they undergo differentiation.20 Differentiation of Caco-2 cells elicited pronounced changes in central metabolism involving both glucose and glutamine utilization (Supplementary Figure 1). Analysis of the media showed that, upon differentiation, there was a marked decrease in glucose consumption and lactic fermentation. In addition, we observed a decrease in consumption of amino acids, particularly glutamine and valine. This is consistent with an overall decrease in metabolic activity as a consequence of differentiation.

Analysis of the intracellular metabolites showed that the decreased steady-state levels of intracellular metabolites, such as citrate and malate, were noted with differentiation (Supplementary Figure 1). Interestingly, increased amounts of total and of 13C-enriched βHB were noted in the differentiated cells grown in the presence of [U-13C]-glucose. We observed an increase of both m2 and m4 βHB (Supplementary Figure 1, insert) consistent with condensation of 13C-enriched acetyl CoA (AcCoA) with either unlabeled AcCoA or glucose-derived 13C-enriched AcCoA. Furthermore, βHB was not present in significant amounts in the media, suggesting that it is not exported out of the cell, nor was there obvious cell death. These novel findings demonstrate that glucose-derived carbon has entered the pathway acetyl CoA→AcAc→βHB in the differentiated cells and an enhanced ketogenesis occurs with intestinal cell differentiation. In contrast, there was insignificant incorporation of glutamine-derived 13C into βHB (data not shown).

βHB induces enterocyte differentiation in Caco-2 cells

Caco-2 cells can differentiate into an enterocyte-like phenotype, either with treatment by HDAC inhibitors or spontaneously with overconfluence, characterized by a polarized monolayer and the expression of cytokeratin 20 (KRT20) and the brush-border enzymes such as sucrase-isomaltase (SI).21, 22

To determine whether βHB has a role in human intestinal cell differentiation, Caco-2 cells were treated with βHB for 48 h and the mRNA levels of the enterocyte markers SI and KRT20 were determined. As shown in Figure 1a, treatment of Caco-2 cells with βHB-induced SI and KRT20 mRNA expression, suggesting that βHB increases enterocyte differentiation. p21Waf1 inhibits intestinal cell growth and induces differentiation;23 Caudal-related homeobox transcription factor 2 (CDX2) is an intestine-specific transcription factor regulating homeostasis of the continuously renewing intestinal epithelium.24 We found that treatment with βHB also increased p21Waf1 and CDX2 mRNA (Figure 1a) and protein expression (Figure 1b). Furthermore, HDAC inhibition by βHB was shown by the increased acetylation of histone H3 lysine 9 (H3K9ac) (Figure 1b). Consistent with the increased mRNA expression, treatment with βHB increased KRT20 protein expression in Caco-2 cells (Figure 1b). These results indicate that βHB, which is increased with intestinal cell differentiation, acts as an endogenous inhibitor of HDACs inducing intestinal cell differentiation.

Figure 1 βHB increases differentiation in Caco-2 cells. (a) Caco-2 cells were treated with βHB (10 mM) for 48 h. Total RNA was extracted, and SI, KRT20, p21Waf1 and CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus control). Data are from one of three independent experiments with similar results. (b) Caco-2 cells were treated with βHB at various dosages for 48 h. Cells were lysed and western blot analysis was performed using antibodies against p21Waf1, CDX2, H3K9ac, KRT20 and β-actin. The images are representative of three independent experiments Full size image

Knockdown of the ketone biosynthetic enzyme HMGCS2 inhibits enterocyte differentiation in Caco-2 cells

We have shown that treatment with βHB induces enterocytic differentiation in Caco-2 cells and that synthesis of βHB is increased in differentiated Caco-2 cells. βHB synthesis is dependent on the activity of mitochondrial HMGCS2.25 To determine the role of HMGCS2 in intestinal cell differentiation, we transfected pre-confluent Caco-2 cells with shRNA directed against HMGCS2 mRNA to determine whether loss of HMGCS2 can attenuate the differentiated phenotype associated with post-confluence. Caco-2 cell lines with stable HMGCS2 knockdown were cultured and harvested at different time points: pre-confluent (pre) or 3, 6 and 12 days post-confluent. As shown in Figure 2, spontaneous Caco-2 differentiation was shown by the increased mRNA expression of SI, KRT20 and p21Waf1 as determined by real-time RT-PCR (Figure 2a), and increased protein expression of p21Waf1, CDX2 and villin as determined by western blotting (Figure 2b); these increases were significantly attenuated by knockdown of HMGCS2, suggesting that HMGCS2 is required for Caco-2 spontaneous differentiation. In agreement with the increase of βHB, expression of HMGCS2 protein is markedly increased with spontaneous Caco-2 cell differentiation (Figure 2b).

Figure 2 Knockdown of HMGCS2 attenuates spontaneous differentiation of Caco-2 cells. Caco-2 cells, stably transfected with control or HMGCS2 shRNA, were incubated 3, 6 and 12 days after confluency to differentiation. (a) Total RNA was extracted, and SI, KRT20 and p21Waf1 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus pre-confluent; #P<0.01 versus control shRNA). Data are from one of three independent experiments with similar results. (b) Cells were lysed and western blot analysis was performed using antibodies against p21Waf1, CDX2, villin, HMGCS2 and β-actin. The images are representative of three independent experiments Full size image

HMGCS2/βHB contributes to the induction of goblet and Paneth cell marker expression

Treatment with butyrate, an HDAC inhibitor, induces differentiation as noted by the increased expression of intestinal alkaline phosphatase (IAP), an enterocyte differentiation marker, and Mucin2 (MUC2), a goblet cell differentiation marker in LS174T cells.26, 27 We have shown that βHB inhibits HDAC in Caco-2 cells. To determine whether βHB also increases goblet cell differentiation, we treated LS174T cells with βHB. As shown in Figure 3, treatment of LS174T with 10 mM βHB increased not only the expression of KRT20 and IAP but also increased the expression of MUC2 and p21Waf1 and CDX2 (Figures 3a and b). The inhibition of HDAC by βHB was demonstrated by increased expression of H3K9ac (Figure 3b).

Figure 3 βHB increases the expression of enterocyte and goblet cell markers in LS174T cells. LS174T cells were treated with βHB (10 mM) for 48 h. Total RNA was extracted, and IAP (enterocyte marker) and KRT20 (enterocyte marker), MUC2 (goblet cell marker), p21Waf1 and CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus control). Data are from one of three independent experiments with similar results. (b) LS174T cells were treated with βHB at various dosages for 48 h. Cells were lysed and western blot analysis was performed using antibodies against p21Waf1, CDX2, H3K9ac, MUC2, KRT20 and β-actin. The images are representative of three independent experiments Full size image

To determine whether βHB also contributes to Paneth cell differentiation, we next treated HT29 cells with βHB. HT29 cells produce IAP, and treatment with butyrate increases expression of IAP in HT29 cells.21 In addition, HT29 cells produce MUC29 and lysozyme (LYZ) (a differentiation marker of Paneth cells).28 Treatment of HT29 cells with βHB increased the mRNA (Figure 4a) and/or protein levels (Figure 4b) of IAP, MUC2, LYZ and p21Waf1 and CDX2 and protein levels of H3K9ac. Taken together, these novel results suggest that βHB is an endogenous inhibitor of HDACs and an inducer of intestinal cell differentiation.

Figure 4 βHB induces the expression of enterocyte, goblet and Paneth cell markers in HT29 cells. HT29 cells were treated with βHB (10 mM) for 48 h. Total RNA was extracted, and MUC2 (goblet cell marker), IAP (enterocyte marker), LYZ (Paneth cell marker), p21Waf1 and CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus control). Data are from one of at least three independent experiments with similar results. (b) HT29 cells were treated with βHB at various dosages for 48 h. Cells were lysed and western blot analysis was performed using antibodies against p21Waf1, CDX2, MUC2, LYZ, H3K9ac and β-actin. The images are representative of three independent experiments Full size image

To test the effect of βHB on cell proliferation and apoptosis along with the increased differentiation, LS174T and HT29 cells were treated with βHB and cell numbers were counted. βHB treatment inhibited HT29 and LS174T cell proliferation (Supplementary Figures 2A and B). There was no apparent increase in apoptosis (as determined by caspase-3 cleavage and DNA fragmentation) after treatment with βHB at same dosages for 48 h (data not shown).

To delineate the role of HMGCS2 in the regulation of intestinal cell differentiation more precisely, we overexpressed HMGCS2 in Caco-2 and LS174T cells. Overexpression of HMGCS2 increased expression of p21Waf1, CDX2 and H3K9ac in Caco-2 (Figure 5a) and LS174T cells (Figures 5b and c). These results further indicate that, similar to our findings by treatment of intestinal cells with βHB, HMGCS2 contributes to the differentiation process.

Figure 5 HMGCS2 contributes to intestinal differentiation. (a) Caco-2 cells were transfected with empty vector (control) or transfected with Flag-HMGCS2 constructs. After 48 h, cells were lysed and extracted for protein. p21Waf1, CDX2, H3K9ac, HMGCS2, Flag-tagged HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus control vector). (b and c) LS174T cells were infected with a recombinant adenovirus encoding the human HMGCS2 or vector control encoding GFP. After 48 h, cells were lysed and extracted for RNA and protein. (b) p21Waf1, CDX2, H3K9ac, HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). (c) CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). Data are from one of three independent experiments with similar results. Overexpression of HMGCS2 inhibits HDAC and increases p21Waf1 and CDX2 expression in Caco-2 and LS174T cells. (d) Immunohistochemical analysis of HMGCS2 protein expression in normal human small intestine. Human normal small intestine sections were fixed and stained with primary anti-human HMGCS2 antibody. HMGCS2 is specifically expressed in the more differentiated region (i.e., villus; arrows). Scale bars=50 μm. The images are representative of five cases Full size image

Finally, to correlate the expression pattern of HMGCS2 protein in the human intestine, sections of adjacent normal human small bowel were obtained from five adult patients following intestinal resection for GI pathology (Supplementary Table 1). Intense staining for HMGCS2 was localized to the most differentiated region of the intestine (i.e., villus), which correlates with our in vitro results by linking increased HMGCS2 expression with the most highly differentiated region of the intestinal mucosa (Figure 5d).

Feeding a ketogenic diet to mice enhances intestinal differentiation

We next determined whether the increased ketogenesis enhances the differentiation in the epithelium of mouse intestine. Mice fed with ketogenic diets demonstrate elevated HMGCS2/βHB levels in tissues including the intestine.29, 30 1,3-Butanediol, a ketone body precursor, is metabolized by alcohol dehydrogenase and aldehyde dehydrogenases to βHB.31 We fed mice with a ketogenic diet (normal chow diet mixed with 1,3-butanediol ketone diesters)30 for 14 days to increase ketogenesis; the intestinal tissues were then harvested for analysis. As shown in Figure 6a, mice fed with the ketogenic diet demonstrated inhibition of HDAC as noted by the increased expression of H3K9ac. Importantly, mice fed with the ketogenic diet showed increased intestinal expression of MUC2 and p21Waf1 protein, suggesting that ketogenesis contributes to intestinal cell differentiation. Along with the increased differentiation, mTOR signaling was inhibited as shown by the decreased expression of p-S6 (Figure 6a). Collectively, these results suggest a cross-talk between ketogenesis and mTOR signaling during intestinal cell differentiation. Mice fed with the ketogenic diet increased expression of HMGCS2 in conjunction with decreased mTOR signaling in the small intestine (Figure 6a). As inhibition of mTOR has been shown to increase the expression of HMGCS2 mRNA and ketogenesis,32 feeding a ketogenic diet may increase the expression of HMGCS2 through the inhibition of mTOR signaling in intestinal cells. We did not find increased HMGCS2 expression in the colonic mucosa of mice fed with the ketogenic diet compared with mice fed a normal control chow (Supplementary Figure 3A). As the level of HMGCS2 is much higher in colon mucosa than that in small bowel mucosa (Supplementary Figure 3B), it may be difficult to significantly increase the expression of basal HMGCS2 in the colonic epithelium.

Figure 6 Enhanced intestinal cell differentiation by ketone diet. Mice were fed with normal chow (n=5) or a ketogenic diet (n=5) for 14 days. (a) Small intestinal mucosal protein lysates were extracted for western blot detection of H3K9ac, MUC2, p21Waf1, p-S6, S6, HMGCS2 and β-actin protein expression. Each well represents a different mouse from the relevant group. (b) Representative Fast Red staining of the small intestine revealed an increase in IAP expression. (c) Representative AB staining of the small intestine revealed an increase in mucinous goblet cells in ketogenic diet-fed mice compared with control mice (arrows). (d) Quantification of AB-positive cells in control and ketogenic diet-fed mice. (n=15 (3 crypts per mouse)), data represent mean±S.D.; *P<0.01 versus control diet). (e) Representative IHC staining of the small intestine for LYZ showed the increase in Paneth cells (arrows) in ketogenic diet-fed mice compared with control mice. (f) Quantification of LYZ-positive cells in control and ketogenic diet-fed mice. (n=15 (3 crypts per mouse), data represent mean±S.D.; *P<0.01 versus control diet). (g) Representative IHC staining (arrows) for CDX2 demonstrated increased expression in the intestinal epithelium of ketogenic diet-fed mice compared with control mice. Scale bars=50 μm Full size image

We next determined the effect of increased ketogenesis on intestinal cell differentiation. In mice fed with the ketogenic diet, the intestine appeared normal by histology (Supplementary Figure 4A). Fast Red staining revealed a marked increase in IAP activity in the small bowel of mice fed with a ketogenic diet (Figure 6b), demonstrating increased enterocyte differentiation. MUC2 expression was markedly increased in the small bowel of mice fed the ketogenic diet as noted by Alcian blue (AB) staining and IHC (Figures 6c, d and Supplementary Figure 4B). Moreover, staining the intestinal sections from ketogenic diet-fed mice for LYZ revealed an obvious increase in Paneth cells (Figures 6e and f). In agreement with the increased CDX2 expression mediated by treatment with βHB or overexpression of HMGCS2, increased expression of CDX2 was detected in the small bowel of mice fed the ketogenic diet (Figure 6g), demonstrating a ketogenesis-dependent regulation of CDX2. Therefore, results from our in vitro and in vivo studies show that ketogenesis is required for intestinal cell differentiation. Consistent with the decreased phosphorylation of S6 and increased expression of HMGCS2 (Figure 6a), decreased staining for p-S6 and increased staining for HMGCS2 was also noted in mice fed with a ketogenic diet compared with control mice (Supplementary Figures 4C and D). These results indicate that increased ketogenesis inhibits mTORC1 signaling in the intestinal epithelium.

Cross-talk between mTOR and HMGCS2/βHB in intestinal cells

Previously, we showed that decreased mTOR activity is associated with differentiation.33 Moreover, we showed that knockdown or inhibition of mTOR increases, whereas activation of mTOR reduces, the levels of intestinal differentiation markers.9, 33, 34 In this study, we showed that the expression of HMGCS2 is increased in differentiated cells (Figure 5d). To determine whether mTOR regulates HMGCS2 expression in intestinal cells, HT29 cells were transfected with mTOR siRNA or non-targeting control (NTC). As shown in Figure 7a, knockdown of mTOR increased HMGCS2 expression. To next determine whether mTOR inhibition increases HMGCS2 in vivo, mice were treated with rapamycin (4 mg/kg, i.p., daily for 6 days) and mucosal proteins were extracted from small bowel for analysis of HMGCS2 expression. As shown in Figure 7b, administration of rapamycin inhibited mTOR signaling as noted by the decreased expression of p-S6. Importantly, rapamycin markedly increased HMGCS2 protein expression in intestinal epithelium, demonstrating mTORC1 regulation of HMGCS2 expression in intestinal cells. In agreement with the increased HMGCS2 expression, mice treated with rapamycin showed increased differentiation in intestinal cells (Supplementary Figure 5), which is consistent with our previous findings showing that inhibition of mTOR by rapamycin increases differentiation in mouse intestinal epithelium.34

Figure 7 Cross-talk between mTORC1 and HMGCS2/βHB signaling. (a) HT29 cells were transfected with NTC or mTOR siRNA. After incubation for 48 h, cells were lysed and western blot analysis was performed using antibodies against HMGCS2, mTOR and β-actin. Knockdown of mTOR increased HMGCS2 expression in HT29 cells. The images are representative of three independent experiments. HMGCS2 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus NTC siRNA). (b) Mouse small intestinal mucosal protein extracted from mice treated without (control, n=3) or with rapamycin (n=3) for 6 days. Western blotting was performed for the expression of the indicated proteins. Each well represents a different mouse from the relevant group. Treatment with rapamycin significantly increased HMGCS2 expression in intestinal epithelium. (c) HT29 cells were treated with βHB at various dosages for 48 h. Cells were lysed and western blot analysis was performed using antibodies against p-S6 and S6. Treatment with βHB inhibited mTOR signaling as shown by the decreased phosphorylation of S6. The images are representative of three independent experiments. (d) LS174T cells were infected with a recombinant adenovirus encoding the human HMGCS2 or vector control encoding GFP. After 48 h, cells were lysed and western blot performed for the detection of the indicated proteins. Overexpression of HMGCS2 inhibited mTOR signaling as shown by the decreased expression of p-S6. p-S6 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to total S6. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). The images are representative of three independent experiments. (e) mTOR/HMGCS2/βHB pathway model. Inhibition of mTORC1 increases ketogenesis and contributes to intestinal cell differentiation. In contrast, increase in ketogenesis inhibits mTOR signaling and induces differentiation. mTORC1 acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis Full size image

Finally, to determine whether the endogenous HDAC inhibitor βHB inhibits mTOR, HT29 cells were treated with βHB for 48 h. As shown in Figure 7c, βHB inhibited mTOR signaling as noted by the dose-dependent decrease in the expression of p-S6. βHB-induced inhibition of mTOR signaling was also noted in FHs 74 Int human small intestinal epithelial cells (Supplementary Figure 6). As a result of mTOR inhibition, treatment with βHB increased HMGCS2 in FHs 74 Int cells (Supplementary Figure 6). Moreover, overexpression of HMGCS2 inhibited mTOR signaling in LS174T cells (Figure 7d). Taken together, our results identify a potential cross-talk between ketogenesis and mTOR signaling, which contributes to the process of intestinal cell differentiation.