Ketogenesis contributes to intestinal cell differentiation

Abstract

The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body β-hydroxybutyrate (βHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21^Waf1). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/βHB signaling in intestinal cells. In contrast, treatment of intestinal cells with βHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/βHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis.

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, p21^Waf1 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 p21^Waf1, CDX2, H3K9ac, KRT20 and β-actin. The images are representative of three independent experiments

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 p21^Waf1 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 p21^Waf1, CDX2, villin, HMGCS2 and β-actin. The images are representative of three independent experiments

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), p21^Waf1 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 p21^Waf1, CDX2, H3K9ac, MUC2, KRT20 and β-actin. The images are representative of three independent experiments

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), p21^Waf1 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 p21^Waf1, CDX2, MUC2, LYZ, H3K9ac and β-actin. The images are representative of three independent experiments

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. p21^Waf1, CDX2, H3K9ac, HMGCS2, Flag-tagged HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21^Waf1, 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) p21^Waf1, CDX2, H3K9ac, HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21^Waf1, 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 p21^Waf1 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

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, p21^Waf1, 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

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