Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem cells.

Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFPLow), activatable reserve IESC and enteroendocrine cells (Sox9-EGFPHigh), Sox9-EGFPSublow progenitors, and Sox9-EGFPNegative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFPLow IESC and Sox9-EGFPHigh cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.

Obesity and intestinal epithelial deletion of the insulin receptor, but not the IGF 1 receptor, affect radiation-induced apoptosis in colon.

Current views suggest that apoptosis eliminates genetically damaged cells that may otherwise form tumors. Prior human studies link elevated insulin and reduced apoptosis to risk of colorectal adenomas. We hypothesized that hyperinsulinemia associated with obesity would lead to reduced colon epithelial cell (CEC) apoptosis after radiation and that this effect would be altered by deletion of the insulin-like growth factor (IGF) 1 receptor (IGF1R) or the insulin receptor (IR). Mice with villin-Cre-mediated IGF1R or IR deletion in CECs and floxed littermates were fed a high-fat diet to induce obesity and hyperinsulinemia or control low-fat chow. Mice were exposed to 5-Gy abdominal radiation to induce DNA damage and euthanized 4 h later for evaluation of apoptosis by localization of cleaved caspase-3. Obese mice exhibited decreased apoptosis of genetically damaged CECs. IGF1R deletion did not affect CEC apoptosis in lean or obese animals. In contrast, IR loss increased CEC apoptosis in both diet groups but did not prevent antiapoptotic effects of obesity. Levels of p53 protein were significantly reduced in CECs of obese mice with intact IR but increased in both lean and obese mice without IR. Levels of mRNAs encoding proapoptotic Perp and the cell cycle inhibitor Cdkn1b/p27 were reduced in CECs of obese mice and increased in lean mice lacking IR. Together, our studies provide novel evidence for antiapoptotic roles of obesity and IR, but not IGF1R, in colonic epithelium after DNA damage. However, neither IR nor IGF1R deletion prevented a reduction in radiation-induced CEC apoptosis during obesity and hyperinsulinemia.

IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations.

Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.

Deletion of intestinal epithelial insulin receptor attenuates high-fat diet-induced elevations in cholesterol and stem, enteroendocrine, and Paneth cell mRNAs.

The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IR(Δ/Δ)) and littermate controls with floxed, but intact, IR (IR(fl/fl)) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IR(Δ/Δ) mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IR(fl/fl) and VC-IR(Δ/Δ) mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IR(fl/fl) mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IR(Δ/Δ) mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs.

Reduced insulin-like growth factor I receptor and altered insulin receptor isoform mRNAs in normal mucosa predict colorectal adenoma risk.

BACKGROUND: Hyperinsulinemia resulting from obesity and insulin resistance is associated with increased risk of many cancers, but the biology underlying this risk is unclear. We hypothesized that increased mRNA levels of the insulin-like growth factor I receptor (IGFIR) versus the insulin receptor (IR) or elevated ratio of IR-A:IR-B isoforms in normal rectal mucosa would predict adenoma risk, particularly in individuals with high body mass index (BMI) or plasma insulin. METHODS: Biopsies from normal rectal mucosa were obtained from consenting patients undergoing routine colonoscopy at University of North Carolina Hospitals (Chapel Hill, NC). Subjects with colorectal adenomas were classified as cases (n = 100) and were matched to adenoma-free controls (n = 98) based on age, sex, and BMI. IGFIR and IR mRNA levels were assessed by qRT-PCR, and IR-A:IR-B mRNA ratios by standard PCR. Plasma insulin and crypt apoptosis were measured by ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. Logistic regression models examined relationships between receptor mRNAs, BMI, plasma insulin, and adenoma risk. RESULTS: Unexpectedly, cases were significantly more likely to have lower IGFIR mRNA levels than controls. No overall differences in total IR mRNA or IR-A:IR-B ratios were observed between cases and controls. Interestingly, in patients with high plasma insulin, increased IR-A:IR-B ratio was associated with increased likelihood of having adenomas. CONCLUSIONS: Our work shows novel findings that reduced IGFIR mRNA and, during high plasma insulin, increased IR-A:IR-B ratios in normal rectal mucosa are associated with colorectal adenoma risk. IMPACT: Our work provides evidence supporting a link between IGFIR and IR isoform expression levels and colorectal adenoma risk.

Insulin receptor isoform switching in intestinal stem cells, progenitors, differentiated lineages and tumors: evidence that IR-B limits proliferation.

Despite evidence for the impact of insulin on intestinal epithelial physiology and pathophysiology, the expression patterns, roles, and regulation of insulin receptor (IR) and IR isoforms in the intestinal epithelium are not well characterized. IR-A is thought to mediate the proliferative effects of insulin or insulin growth factors (IGFs) in fetal or cancer cells. IR-B is considered to be the metabolic receptor for insulin in specialized tissues. This study used a novel Sox9-EGFP reporter mouse that permits isolation of intestinal epithelial stem cells (IESCs), progenitors, enteroendocrine cells and differentiated lineages, the Apc(Min/+) mouse model of precancerous adenoma and normal human intestinal and colorectal cancer (CRC) cell lines. We tested the hypothesis that there is differential expression of IR-A or IR-B in stem and tumor cells versus differentiated intestinal epithelial cells (IECs) and that IR-B impacts cell proliferation. Our findings provide evidence that IR-B expression is significantly lower in highly proliferative IESCs and progenitor cells versus post-mitotic, differentiated IECs and in subconfluent and undifferentiated versus differentiated Caco-2 cells. IR-B is also reduced in Apc(Min/+) tumors and highly tumorigenic CRC cells. These differences in IR-B were accompanied by altered levels of mRNAs encoding muscleblind-like 2 (MBNL2), a known regulator of IR alternative splicing. Forced IR-B expression in subconfluent and undifferentiated Caco-2 cells reduced proliferation and increased biomarkers of differentiation. Our findings indicate that the impact of insulin on different cell types in the intestinal epithelium might differ depending on relative IR-B IR-A expression levels and provide new evidence for the roles of IR-B to limit proliferation of CRC cells.

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.