ISCs and intestinal homeostasis

The intestinal epithelium is a rapidly renewing tissue in which the intestinal epithelial cells turnover every 3–5 d. This rapid renewal is maintained by ISCs residing in the crypts of Lieberkühn that generate daughter cells to differentiate and migrate up towards the crypt–villus axis (Fig. 1A)^{(Reference Barker, van Es and Kuipers21)}. Two populations of ISCs have been identified in the mammalian intestine. One is the crypt base columnar cells, also called ‘activated ISCs’, which are intercalated with Paneth cells at the bottom of the crypt. The key marker for this population is Lgr5, a target gene of the Wnt signaling pathway^{(Reference Barker, van Es and Kuipers21)}. The discovery of Lgr5 as a specific marker for this compartment has promoted tremendous advances in the molecular characterisation of ISCs^{(Reference Barker, van Es and Kuipers21)}.The second ISC population is located at the fourth position above the base of the crypt and is also called ‘+4’ ISCs^{(Reference Hendry and Potten22)} (Fig. 1A). These ‘+4’ ISCs are a quiescent population of stem cells and are resistant to radiation-induced intestinal injury^{(Reference Yan, Chai and Li23)}. Hopx, Bmi1, mTert and Lrig1 have been identified as markers of these ‘+4’ ISCs^{(Reference Montgomery, Carlone and Richmond24–Reference Sangiorgi and Capecchi26)}.

Fig. 1. A model of intestinal stem cell-driven epithelial renewal during homeostasis and regeneration. (A) In the small intestine, Lgr5⁺ ISCs are located at the crypt base and divide to generate daughter cells and proliferating transit-amplifying (TA) cells, which then differentiate into the various mature cell types, including enterocytes, enteroendocrine cells, goblet cells, tuft cells and Paneth cells in the crypt and villi. (B) Acute injury that leads to the loss of the Lgr5⁺ ISCs triggers a regenerative response to restore intestinal epithelial renewal. Some stimulations, such as irradiation and DSS, induce Lgr5⁺ ISC apoptosis, but retain the damage-resistant ‘+4’ ISCs and the Paneth cell precursors. The ‘+4’ ISCs are activated upon injury and rapidly produce Lgr5⁺ ISCs or committed progenitor cells. Surviving non-stem cells from various lineages also retain stem cell potential and can dedifferentiate into Lgr5⁺ ISCs to restore intestinal epithelial renewal.

In the homeostatic small intestine, Lgr5⁺ ISCs continue to divide to generate their daughter ISCs and proliferative progeny cells known as transit amplifying (TA) cells (Fig. 1A)^{(Reference Clevers27)}. TA cells can continuously migrate toward the tip of the villi and differentiate into mature cell types that fulfil the various intestinal functions (Fig. 1A)^{(Reference Barker14,Reference Barker, Bartfeld and Clevers15)} . Enterocytes occupy most of the villi and are highly polarised with a dense apical brush border critical for nutrient absorption. Other cell types contain mucin-producing goblet cells, hormone-secreting enteroendocrine cells and tuft cells, which are involved in regulating immune response against pathogens^{(Reference von Moltke, Ji and Liang28)}. Paneth cells located at the crypt base not only provide niche factors for ISCs, but also secrete antibacterial agents to prevent pathogen infection^{(Reference Sato, van Es and Snippert29)}. Lgr5⁺ ISCs are responsible for intestinal epithelial renewal under normal physiological conditions, and the ‘+4’ ISCs are involved in intestinal regeneration following injury (Fig. 1B)^{(Reference Barker14)}. Acute injury leads to a reduction in the proliferating Lgr5⁺ ISC pool, but retains the damage-resistant ‘+4’ ISCs and the Paneth cell precursors^{(Reference Tian, Biehs and Warming30)}. The ‘+4’ ISCs are activated upon injury and rapidly produce Lgr5⁺ ISCs to restore the renewal of the intestinal epithelium (Fig. 1B)^{(Reference Tian, Biehs and Warming30)}. In addition, evidence has reported on the conversion of various cell types following damage to the stem cells, and this process is called dedifferentiation. These cell types, including enterocyte precursors, secretory progenitors and other TA cells, also retain stem cell potential and are consequently converted into Lgr5⁺ ISCs to restore intestinal epithelial renewal (Fig. 1B)^{(Reference Barker14)}. These data suggest that mature cell types within the intestinal epithelium can dedifferentiate and act as an alternative source of stem cells upon tissue damage.

ISCs and intestinal diseases

Intestinal diseases are closely interconnected with the imbalance between self-renewal and differentiation of ISCs^{(Reference Sanders and Majumdar31)}. Lgr5⁺ ISCs not only act as stem cells for epithelial renewal, but also are identified as the cells of origin for a large proportion of CRCs^{(Reference Barker, Ridgway and van Es32)}. CRCs are the common intestinal diseases in humans and are mainly initiated by mutation of the adenomatous polyposis coli (APC) gene, resulting in nuclear accumulation of β-catenin^{(Reference Rhodes and Campbell33)}. Deletion of the APC gene in Lgr5⁺ ISCs leads to early tumorous lesions and metastasis, while APC deletion in TA cells does not form adenomas^{(Reference Barker, Ridgway and van Es32)}, suggesting that Lgr5⁺ ISCs accumulate some of the early mutations and may serve as early target cells for CRCs. In addition, a population of cells with similar properties of ISCs, such as multilineage differentiation and self-renewal, are found in CRCs and are called colon cancer stem cells (CSCs)^{(Reference Vermeulen, Todaro and Mello34)}. It is worth noting that the gene expression profiles of CSCs have high similarity with that of Lgr5⁺ ISCs^{(Reference Barker, Ridgway and van Es32)}, suggesting that CSCs may originate from Lgr5⁺ ISCs in human colon adenocarcinoma. Gene mutation results in an imbalance between self-renewal and differentiation of ISCs, which is an important determinant for the formation of CRCs. Excessive ISC self-renewal expands the stem cell pool and promotes the total number of target cells transformed into cancer, thereby increasing the risk of tumorigenesis. In contrast, decreased self-renewal or enhanced differentiation, reduces the ISC population and impairs intestinal epithelial regeneration. Intestinal diseases, in turn, affect the function of ISCs and their TA cells. For example, inflammatory bowel disease is characterised by acute and chronic inflammation of intestinal tissue regions, and is accompanied by a decline of Paneth cell number^{(Reference Khaloian and Rath35)}. The reduction of Paneth cell number damages the ISC niche, which reduces the expression of Lgr5 in crypt regions^{(Reference Khaloian and Rath35)}. Lgr5⁺ ISC loss has been found in the primary response to dextran sodium sulphate (DSS)-induced inflammation or high-dose γ-irradiation^{(Reference Schmitt, Schewe and Sacchetti36,Reference Yan, Chia and Li37)} . Recently, low density of Lgr5⁺ ISCs and low differentiation toward enteroendocrine cells have been reported in patients with irritable bowel syndrome^{(Reference El-Salhy38)}. These data indicate that abnormalities in ISCs are key factors in the occurrence of intestinal diseases. Therefore, the precise regulation of ISC self-renewal and differentiation could provide new therapeutic opportunities for treating intestinal diseases.

mTOR in ISCs

mTORC1 is a vital regulator that integrates multiple upstream signals, such as growth factors and nutrients to control cellular and organismal growth, protein synthesis, mitochondrial biogenesis and autophagy^{(Reference Saxton and Sabatini40,Reference Zoncu, Efeyan and Sabatini41)} . The mTORC1 pathway has been reported to be involved in ISC proliferation. In general, mTORC1 is abundantly expressed in Lgr5⁺ ISCs but silenced in ‘+4’ ISCs or TA cells. Upon exposure to damage, mTORC1 is activated in ‘+4’ ISCs and further stimulates active Lgr5⁺ ISCs to proliferate and differentiate into secretory cell progenitors and enterocyte progenitors^{(Reference Kapuria, Karpac and Biteau42,Reference Kaur and Moreau43)} . Intestinal epithelium deletion of mTORC1 in mice results in atrophy of small intestinal villi and the defect of epithelial cells^{(Reference Sampson, Davis and Grogg44)}. Interestingly, the ‘+4’ ISC knockout of mTORC1 does not impact intestinal homeostasis under normal conditions, but impairs intestinal regeneration following injury^{(Reference Yousefi, Nakauka-Ddamba and Berry9)}. These data indicate that mTORC1 in ‘+4’ ISCs is mainly involved in the repair of damaged intestinal epithelium. Therefore, mTORC1 is required for intestinal epithelial regeneration, and elevated level of mTORC1 in ‘+4’ ISCs is beneficial for intestinal health. However, aging-induced intestinal epithelial dysfunction is positively correlated with mTORC1 activity in ISCs or TA cells^{(Reference Igarashi, Miura and Williams39,Reference He, Wu and Xiang45)} . mTORC1 is activated in Lgr5⁺ ISCs and Paneth cells during aging^{(Reference He, Wu and Xiang45,Reference Pentinmikko, Iqbal and Mana46)} . The activation of mTORC1 by tuberous sclerosis complex 1 deletion reduces organoid-forming capacity and the regenerative capacity of old intestinal epithelium^{(Reference Pentinmikko, Iqbal and Mana46)}. In contrast, inhibition of mTORC1 partially rescues the regenerative capacity of aged intestinal epithelium in mice, which is attributable to the effects on both Paneth cells and ISCs^{(Reference He, Wu and Xiang45,Reference Pentinmikko, Iqbal and Mana46)} . Mechanistically, aging-induced mTORC1 activates the MKK6-p38-p53 pathway, resulting in exhaustion of ISC and decrease of villus size and density^{(Reference He, Wu and Xiang45)}. These data suggest that appropriate inhibition of mTORC1 in Lgr5⁺ ISCs is beneficial for intestinal epithelial regeneration in the elderly.

Lkb1/AMPK in ISCs

Energy is an essential signal that influences ISC metabolism and homeostasis. AMPK, a master sensor of energy, is identified as a regulator of ISC proliferation^{(Reference Igarashi and Guarente11)}. AMPK can be activated in ISCs and crypts of mice in response to low-energy status, such as CR and fasting^{(Reference Igarashi and Guarente11)}. Inhibition of AMPK significantly inhibits the potential for high colony formation (proliferation) in ISCs of mice. On the contrary, activation of AMPK induces colony formation of ISCs^{(Reference Igarashi and Guarente11)}, suggesting that AMPK is required for ISC proliferation. AMPK regulation of ISC function mainly depends on its downstream molecule Sirt1, which activates mTORC1 signalling to induce ISC expansion^{(Reference Igarashi and Guarente11)}. The tumour suppressor protein Lkb1 is identified as an upstream molecule of AMPK to control ISC fate. Lkb1 phosphorylation in ISCs is also significantly increased in CR mice, suggesting that AMPK activation in ISCs is likely attributable to the activation of Lkb1^{(Reference Igarashi and Guarente11)}. Lkb1 is also involved in the regulation of ISC fate in an AMPK-independent manner. Lgr5⁺ ISC-specific Lkb1 deficiency in mice disrupts ISC homeostasis and promotes differentiation into secretory lineages by activation of Atoh1^{(Reference Gao, Yan and Tripathi47)}. Lkb1 knockdown inhibits AMPK activity, but depletion of AMPKα1 or α2 of the catalytic subunit of AMPK fails to induce Atoh1 expression^{(Reference Gao, Yan and Tripathi47)}, indicating that AMPK is not required for the regulation of ISC fate by Lkb–Atoh1 axis.

Sirt1 in ISCs

Sirt1, an NAD-dependent protein deacetylase, plays important roles in cellular processes, including cell proliferation, differentiation and metabolism^{(Reference Finkel, Deng and Mostoslavsky48)}. Recent evidence has implicated that Sirt1 acts as an important mediator in ISC proliferation in response to CR and aging^{(Reference Igarashi and Guarente11,Reference Igarashi, Miura and Williams39)} . Intestinal epithelium or Lgr5⁺ ISC deletion of Sirt1 in mice blocks long-term CR-induced expansion of crypts and ISCs^{(Reference Igarashi and Guarente11)}. In contrast, Sirt1 overexpression in the intestine of mice mimics the role of CR, indicating that Sirt1 is required for the response to CR in the intestine. The activation of Sirt1 mainly depends on its upstream molecule AMPK, which leads to synthesis of NAD⁺ by stimulating nicotinamide phosphoribosyl transferase (Nampt) in ISCs^{(Reference Brandauer, Vienberg and Andersen49)}. In addition, aging induces a decrease of Sirt1 level in ISCs and crypts, accompanied by a significant reduction in ISC number^{(Reference Igarashi, Miura and Williams39)}. The activation of Sirt1 by supplementation with an NAD⁺ precursor rescues the reduction in colony formation efficiency in crypt-derived organoids from old mice^{(Reference Igarashi, Miura and Williams39)}. More importantly, the activation of Sirt1 also inhibits the increase in susceptibility to DSS in old mice. Therefore, regulation of ISC function by the Sirt1 may contribute to aspects of intestinal regeneration and protect from intestinal aging.

Caloric restriction

CR is defined as energy intake which is 60–80 % of normal caloric intake, without causing malnutrition^{(Reference Johnson, Rabinovitch and Kaeberlein50)}. CR has been reported to maintain intestinal epithelial homeostasis through regulating ISC proliferation (Fig. 2A)^{(Reference Mihaylova, Cheng and Cao10)}. The number of ISCs is significantly increased in CR-treated mice^{(Reference Yilmaz, Katajisto and Lamming51)}. Interestingly, co-cultivation of Paneth cells from CR mice and ISCs from ad libitum mice also promotes ISC proliferation^{(Reference Yilmaz, Katajisto and Lamming51)}. This indicates that regulation of Paneth cell niche by CR controls ISC function. Mechanistically, CR inhibits mTORC1 signalling in Paneth cells, and further increases the level of cyclic ADP ribose (cADPR)^{(Reference Yilmaz, Katajisto and Lamming51)}. Paneth cell-derived cADPR induces the activation of mTORC1 in ISCs via AMPK–Nampt-Sirt1 axis, which promotes ISC proliferation (Fig. 2A)^{(Reference Igarashi and Guarente11)}. CR also increases the pool of ‘+4’ ISCs and has a beneficial effect on the regenerative capacity of intestinal epithelium in response to damage. As opposed to Lgr5⁺ ISCs, CR downregulates mTORC1 signalling in ‘+4’ ISCs^{(Reference Yousefi, Nakauka-Ddamba and Berry9)}. This seems to be a paradox between the increase in ‘+4’ ISC number and the decrease in mTORC1 signalling, because mTORC1 signalling activation promotes cell proliferation. In fact, ISCs with high expression of mTORC1 are more sensitive to undergo apoptosis, and few mTORC1-high expressed cells remain present in ‘+4’ ISCs upon CR^{(Reference Yousefi, Nakauka-Ddamba and Berry9)}. Upon exposure to radiation injury, more mTORC1-low expressed ISCs are activated to contribute to epithelial regenerative capacity^{(Reference Yousefi, Nakauka-Ddamba and Berry9)}. Due to the unique roles of mTORC1 in Lgr5⁺ ISCs and ‘+4’ ISCs, regulation of intestinal epithelial regeneration with mTORC1 as a target will face a difficult challenge.

Fig. 2. Mechanisms of the effects of dietary patterns on ISC function. Dietary patterns, including caloric restriction, fasting, high-fat diet, ketogenic diet and high-carbohydrate diet, control ISC homeostasis through nutrient-sensing pathways. (A) Caloric restriction inhibits mTORC1 signalling in Paneth cells, and further increases the level of cyclic ADP ribose (cADPR). Paneth cell-derived cADPR induces the activation of mTORC1 in ISCs via the AMPK–Sirt1 axis and increases Lgr5⁺ ISC number, thereby promoting intestinal regeneration. (B) Fasting induces PPAR-δ-mediated fatty acid β-oxidation and improves ISC number, and prevents aging induced-intestinal injury. Fasting also inhibits phosphatase and tensin homolog (PTEN), a phosphatase that negatively regulates PI3K-AKT-mTOR signalling, and increases ‘+4’ ISC number and contributes to intestinal regeneration. (C) High-fat diet increases the number and self-renewal of ISCs, enables ISCs to become niche independent, and confers stemness to non-Lgr5⁺ progenitors through PPAR-δ signalling, which contributes to intestinal tumorigenesis. (D) Ketogenic diet improves ketone bodies levels in Lgr5⁺ ISCs, leading to higher Notch activity and ISC number, and promotes post-injury regeneration; compared with ketogenic diet, high-carbohydrate diet has opposite effects.

Fasting

Fasting, which is also called food deprivation, has been shown to be effective in increasing lifespan and promoting tissue regeneration by improving adult stem cell function in diverse tissues^{(Reference Cheng, Adams and Perin52,Reference Longo and Mattson53)} . Growing evidence has shown that fasting promotes intestinal epithelial regenerative capacity after damage through the preservation of ISC function^{(Reference Rangan, Choi and Wei7,Reference Mihaylova, Cheng and Cao10,Reference Tinkum, Stemler and White12,Reference Richmond, Shah and Deary13,Reference de la Cruz Bonilla, Stemler and Jeter-Jones54)} . Fasting for 48 h inhibits phosphatase and tensin homolog, a phosphatase that negatively regulates PI3K–AKT–mTOR signalling, and increases ‘+4’ ISC number and contributes to intestinal regeneration (Fig. 2B)^{(Reference Richmond, Shah and Deary13)}. A fasting-mimicking diet (50 % of standard daily calorie intake on day 1, and 10 % of standard daily calorie intake on days 2–4) ameliorates DSS-induced inflammatory bowel disease by promoting ISC activity^{(Reference Rangan, Choi and Wei7)}. Tinkum et al. reported that 24 h fasting protects against small-intestinal injury by boosting the regenerative potential of ISCs undergoing dose-intensive chemotherapy^{(Reference Tinkum, Stemler and White12)}. Similarly, 24 h fasting not only increases surviving crypt number, but also promotes the organoid-forming capacity in ISCs and crypts^{(Reference Mihaylova, Cheng and Cao10)}. Different with the CR phenotype^{(Reference Igarashi and Guarente11,Reference Yilmaz, Katajisto and Lamming51)} , co-culture of ISCs with fasting Paneth cells does not increase the ability of organoid formation, suggesting that fasting regulates ISC function directly. In addition, fasting also induces PPAR-δ-mediated fatty acid β-oxidation and reverses the age-dependent decline of ISC number and function, and improves intestinal repair after injury in the elderly (Fig. 2B)^{(Reference Mihaylova, Cheng and Cao10)}. Collectively, the beneficial effect of CR and fasting on intestinal function has been extensively confirmed in animal models. Further clinical trials are needed to evaluate their roles in human intestinal health.

High-fat diet

Excess caloric intake induces obesity and is related to cancer incidence in many tissues, especially in the gastrointestinal tract. Long-term HFD (60 % fat, 9–14 months) feeding mildly reduces intestinal villi height and villus enterocyte numbers, and increases crypt depth in mice^{(Reference Beyaz, Mana and Roper3,Reference Andres, Santoro and Mah55)} . However, short-term HFD (45–60 % fat, 8–20 weeks) feeding increases both villi height and crypt density^{(Reference Mah, Van Landeghem and Gavin56–Reference Baldassano, Amato and Cappello58)}. This observation indicates that short-term HFD feeding may not be long enough to induce the effects on intestinal morphology observed with longer HFD feeding. Consistently, both long-term and short-term HFD feeding increases the number and proliferation of ISCs and progenitor cells and reduces Paneth cell number, which contributes to intestinal tumour formation (Fig. 2C)^{(Reference Beyaz, Mana and Roper3,Reference Andres, Santoro and Mah55–Reference Mao, Hu and Xiao57)} . Ex vivo experiments have confirmed that ISCs and crypts from HFD-fed mice exhibit fewer crypt budding (a process of both ISC self-renewal and differentiation) domains and reduce organoid-forming ability^{(Reference Beyaz, Mana and Roper3,Reference Mah, Van Landeghem and Gavin56)} . Reciprocally, CR also increases the number of ISCs, but decreases the risk of intestinal tumour formation^{(Reference Mihaylova, Sabatini and Yilmaz59)}. The mechanistic differences underlying altered ISC function in the two dietary patterns may be the main reason for this discrepancy. Mechanistically, HFD-induced PPAR-δ activation not only increases ISC number via the Wnt/β-catenin pathway, but also enhances the ISC-like progenitor population to induce intestinal tumorigenesis^{(Reference Beyaz, Mana and Roper3)}. HFD-induced obesity also enhances plasma insulin and insulin-like growth factor 1 (IGF-1) levels to impact on ISC hyper-proliferation^{(Reference Mah, Van Landeghem and Gavin56)}. Andres et al. have reported that deletion of insulin receptor in intestinal epithelial cell can block the HFD-induced increase in ISC marker expression^{(Reference Andres, Santoro and Mah55)}. These data suggest that insulin signalling is required for HFD-induced ISC proliferation. Therefore, reducing fat intake may be a potential intervention strategy to prevent intestinal tumorigenesis in humans.

Ketogenic diet

Ketogenic diet is characterised by high fat, moderate protein and low carbohydrate, which dramatically elevates circulating ketone body levels^{(Reference Newman, Covarrubias and Zhao60)}. Recent evidence implicates an emerging role for ketone bodies in regulating Lgr5⁺ ISC function and intestinal epithelial homeostasis (Fig. 2D)^{(Reference Cheng, Biton and Haber8,Reference Gebert, Cheng and Kirkpatrick61)} . Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme for ketone bodies synthesis, is selectively expressed in ISCs^{(Reference Cheng, Biton and Haber8,Reference Newman and Verdin62)} . Deficiency of Hmgcs2 in Lgr5⁺ ISCs damages ISC stemness and induces their differentiation into Paneth and goblet cells by increasing HDAC-mediated suppression of the Notch signalling^{(Reference Cheng, Biton and Haber8)}. The ketogenic diet increases ketone body concentrations both systemically and within ISCs and boosts ISC number and function to improve intestinal regenerative capacity^{(Reference Cheng, Biton and Haber8)}. Wang et al. have reported that a ketogenic diet significantly increases Hmgcs2 expression and promotes intestinal epithelial cell differentiation to maintain intestinal homeostasis^{(Reference Wang, Zhou and Rychahou63)}. Therefore, ketogenic diet may act as a dietary intervention for treating intestinal diseases.

High-carbohydrate diet

High-carbohydrate-based dietary models such as the high-sugar diet and the Western diet have deleterious consequences for metabolic health. Excessive high-sugar diet intake increases blood glucose, insulin and triacylglycerol levels, which contributes to the prevalence of obesity and type 2 diabetes^{(Reference Palmeira, Varela and Rolo64)}. Recent studies have shown that high-carbohydrate diet damages intestinal epithelial regenerative capacity through the regulation of ISC function. A glucose-supplemented diet (13 % glucose in drinking-water) diminishes the self-renewal capacity of ISCs and promotes their differentiation towards Paneth and goblet cells^{(Reference Cheng, Biton and Haber8)}. These processes suppress intestinal ketogenesis and ISC stemness, and damage post-injury regeneration^{(Reference Cheng, Biton and Haber8)}. In Drosophila, a high-sugar diet promotes ISC differentiation through up-regulation of the JNK pathway and down-regulation of the JAK/STAT pathway, and induces disruption of intestinal homeostasis^{(Reference Zhang, Jin and Li65)}. Together, these data suggest high-carbohydrate diet dampens the number and regenerative capacity of ISCs, and is harmful for intestinal homeostasis.

Amino acids

Amino acids are essential nutrients for maintaining intestinal epithelial homeostasis and preventing intestinal diseases. Methionine is an important essential amino acid that promotes ISC self-renewal (Table 1)^{(Reference Saito, Iwatsuki and Hanyu18,Reference Obata, Tsuda-Sakurai and Yamazaki66)} . Ex vivo experiment has observed that methionine deprivation leads to a decrease in ISC proliferation, and enhances ISC differentiation toward secretory cells, including goblet, enteroendocrine and Paneth cells^{(Reference Saito, Iwatsuki and Hanyu18)}. Evidence also confirmed that depletion of dietary methionine inhibits ISC proliferation in Drosophila ^{(Reference Obata, Tsuda-Sakurai and Yamazaki66)}. S-adenosylmethionine, a universal methyl donor from methionine metabolism, is a critical metabolite for maintaining ISC proliferation via the regulation of methyltransferases^{(Reference Obata, Tsuda-Sakurai and Yamazaki66,Reference Gibbons, Owens and Fearon67)} . Arginine is also proven to be an indispensable nutrient for ISC proliferation and intestinal epithelial renewal (Table 1)^{(Reference Hou, Dong and Yu68)}. Arginine deficiency inhibits mouse organoid survival and growth and is accompanied by the loss of Paneth cells and Lgr5⁺ ISCs. The potential mechanism is that arginine can induce Wnt3a production by Paneth cells, which provides the niche factors for ISC self-renewal^{(Reference Hou, Dong and Yu68)}. Glutamine and glutamate have also been reported to impact ISC self-renewal and differentiation (Table 1)^{(Reference Deng, Gerencser and Jasper69–Reference Zhu, Qin and Gao71)}. Medium supplemented with glutamine significantly promotes crypt expansion via the activation of mTORC1 signalling in mouse jejunal organoids^{(Reference Moore, Guedes and Costa70)}. Glutamine deficiency inhibits organoid formation and induces crypt atrophy^{(Reference Moore, Guedes and Costa70)}. Additionally, dietary glutamate enhances organoid formation and budding efficiency as well as ISC proliferation in pig and Drosophila ^{(Reference Deng, Gerencser and Jasper69,Reference Zhu, Qin and Gao71)} . We recently found that aspartate can alleviate DSS-induced intestinal epithelial damage by activating ‘+4’ ISCs, while reducing the number of Lgr5⁺ ISCs (unpublished results). However, the exact mechanisms underlying the autonomous regulation of ISC function by aspartate remain to be identified.

Table 1. Effects of nutrients on ISC self-renewal and differentiation

Fatty acids

The effects of the HFD pattern on villus height and crypt depth suggest that fatty acids may play a crucial role in ISC function. Fatty acids, especially long-chain fatty acids, have been reported to influence ISC proliferation and organoid formation (Table 1). Treatment of mouse organoids with palmitate and/or oleic acid boost Lgr5⁺ ISC number and give rise to more secondary organoids^{(Reference Beyaz, Mana and Roper3,Reference Mihaylova, Cheng and Cao10)} . Consistent with HFD studies, β-oxidation controls fatty-acid-driven ISC self-renewal^{(Reference Beyaz, Mana and Roper3)}. In metabolic flux studies, ¹³C₁₆-palmitate is efficiently incorporated into citrate and α-ketoglutarate in crypts, with over 20 % of carbons labelled with ¹³C^{(Reference Mihaylova, Cheng and Cao10,Reference Stine, Sakers and TeSlaa72)} . These data suggest that palmitate is oxidised to acetyl-CoA and enters into the TCA cycle for combustion. Indeed, acetate, an alternate source of acetyl-CoA, completely rescues the growth and budding defects and significantly increases organoid formation by inhibition of β-oxidation^{(Reference Stine, Sakers and TeSlaa72,Reference Chen, Vasoya and Toke73)} . However, acetate does not affect the growth, budding or passaging capacity of common organoids, suggesting that acetate is required for ISC self-renewal only when acetyl-CoA concentration is reduced. Recent study has reported that arachidonic acid promotes the proliferation of ‘+4’ ISCs by activating Wnt signalling, and facilitates small-intestinal regeneration (Table 1)^{(Reference Wang, Lin and Sheng16)}. In summary, fatty acids may serve as nutrient substrates to promote ISC proliferation.

Vitamins

Vitamin D, a highly pleiotropic hormone, is an essential nutrient that maintains Lgr5⁺ ISC proliferation (Table 1). Deficiency of dietary vitamin D₃ reduces Lgr5⁺ ISC number and their proliferation activity in mice^{(Reference Peregrina, Houston and Daroqui19)}. In addition, a low level of dietary vitamin D₃ increases enterocyte number, and decreases the number of secretory cells accompanied by a prominent increase in ectopic expression of Paneth cell markers^{(Reference Peregrina, Houston and Daroqui19)}. More importantly, long-term feeding of a low-vitamin D₃ diet induces sporadic intestinal tumour formation in mice, but the pro-tumour effects can be prevented by supplementing dietary vitamin D₃ ^{(Reference Peregrina, Houston and Daroqui19)}. ISC-specific knockout of the vitamin D receptor also decreases the proliferation ability of Lgr5⁺ ISCs at the bottom of the crypt^{(Reference Peregrina, Houston and Daroqui19)}. Activation of the vitamin D receptor downregulates the β-catenin target genes c-MYC and PPAR-δ, which may mediate the anti-oncogenic effects of vitamin D signalling^{(Reference Pálmer, González-Sancho and Espada74)}. Collectively, vitamin D is indispensable for maintaining proliferative ability of Lgr5⁺ ISCs via the vitamin D receptor signalling pathway. Vitamin A also has been reported to maintain intestinal epithelial renewal and development (Table 1). Dietary supplementation of vitamin A promotes villus height and crypt depth and Lgr5 expression in the jejunum mucosa of piglets^{(Reference Wang, Li and Wang75)}. Treatment with vitamin A metabolites retinol and retinoic acid significantly reduces the budding rate and budding number of organoids cultured from piglet small intestine^{(Reference Wang, Li and Wang75)}. Moreover, the mRNA expression of stem cell markers is increased and chromogranin A and muc2 expression is decreased, suggesting that vitamin A may increase ISC self-renewal at expense of differentiation^{(Reference Wang, Li and Wang75)}.

Bile acids

Bile acids are synthesised from cholesterol by hepatocytes and participate in emulsification and absorption of dietary lipids^{(Reference Wahlström, Sayin and Marschall76)}. Bile acids are recently attracting extensive attention as a vital modulator of intestinal health and disease^{(Reference Hegyi, Maléth and Walters77)}. Bile acids promote intestinal epithelial regeneration via promoting ISC self-renewal in response to injury (Table 1)^{(Reference Sorrentino, Perino and Yildiz17)}. Mechanistically, bile acids can activate G-protein-coupled bile acid receptor 1, promoting expansion of the Lgr5⁺ ISC pool by the activation of SRC/YAP signalling^{(Reference Sorrentino, Perino and Yildiz17)}. Interestingly, evidence has revealed that bile acids act as potent factors for CRC owing to the promotion of CSC number^{(Reference Farhana, Nangia-Makker and Arbit78)}. HFD induces the increase of intestinal bile acids to promote CSC proliferation, thereby contributing to the initiation of CRC^{(Reference Fu, Coulter and Yoshihara79)}. Secondary bile acids, including deoxycholic acid and lithocholic acid, also induce the expansion of CSCs via Wnt/β-catenin signalling, and thus lead to CRC formation^{(Reference Farhana, Nangia-Makker and Arbit78)}. In short, bile acids likely act as a double-edged sword for intestinal health, and not only promote intestinal epithelial regeneration but also induce CRC formation via different mechanisms.

Gut microbial metabolites

The interactions between microbial metabolites and ISC homeostasis have been widely studied in recent years. Probiotics, including Bifidobacterium and Lactobacillus spp., have the promotive roles in ISC proliferation and organoid formation either under physiological or pathological conditions^{(Reference Lee, Kim and Kim20,Reference Hou, Ye and Liu80,Reference Wu, Xie and Miao81)} . The effects of intestinal microbiota on ISC function depend on their microbial metabolites. These metabolites include lactate, short-chain fatty acids and tryptophan metabolites (Table 1). Lactate is sensed by its receptor GPR81 on Paneth and stromal cells to promote Lgr5⁺ ISC proliferation by activating the Wnt3/β-catenin signalling pathway^{(Reference Lee, Kim and Kim20)}. Butyrate inhibits the proliferation of colonic ISCs by inhibiting HDAC activity^{(Reference Kaiko, Ryu and Koues82)}. However, butyrate does not affect the Lgr5⁺ ISC pool and budding efficiency in small intestinal organoids^{(Reference Yin, Farin and van Es83)}. The unique role of butyrate highlights its important position in maintaining colonic epithelial homeostasis. Tryptophan metabolites, including indole, indole-3-carbinol and indole-3-aldehyde, impact ISC proliferation via aryl hydrocarbon receptor or pregnane X receptor signalling. For example, dietary supplementation with indole-3-carbinol restricts Lgr5⁺ ISC proliferation via suppression of Wnt3/β-catenin signalling and prevents tumorigenesis^{(Reference Metidji, Omenetti and Crotta84,Reference Park, Lee and Lee85)} . Indole-3-aldehyde increases the number of ISCs via activation of STAT3 phosphorylation, and promotes post-injury regeneration^{(Reference Hou, Ye and Liu80)}. At present, the mechanisms by which microbial metabolites regulate ISC homeostasis are still poorly understood because of the complexity of microbiota. Future studies revealing mechanistic insights on regulation of ISC function by microbial metabolites will hopefully provide novel strategies to combat intestinal diseases.