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Intestinal adaptations increase basolateral intestinal glucose uptake and glycolysis in a Cystic Fibrosis mouse model

ABSTRACT: The intestinal manifestations of Cystic Fibrosis (CF) continue to be a source of morbidity despite major advances in CF therapies over the past several years. CF intestinal disease may include obstruction at birth or throughout the lifespan, dysmotility, constipation, small intestinal bacterial overgrowth, and abdominal pain and discomfort. People with CF also have a five to ten-fold increased risk of developing gastrointestinal cancers earlier in life, the reason for which remains largely unknown. The mechanisms underlying the intestinal manifestations of CF are not fully understood. Impaired chloride and bicarbonate ion transport due to absent or dysfunctional CFTR results in dehydrated, viscous mucus that coats the intestinal lumen and altered pH. These changes are thought to create an environment that hinders digestive enzymatic and immune function to promote dysbiosis, dysmotility, and obstruction. The other major organs of digestion, including the pancreas and hepatobiliary system, are also affected in CF, which can result in a variety of symptoms mainly related to fat malabsorption, in addition to various hepatic and biliary pathologies. Most of what is known about the CF intestines has been garnered from studies on the luminal function pertaining to digestion, absorption, mucus accumulation, and bacterial dysbiosis, but little has been described regarding the serosal side. In the process of evaluating systemic carbohydrate utilization in CF mice, we identified the intestine as a key site of increased glucose uptake from circulation in CF. Consequently, we employed a series of complementary in vivo and ex vivo, transcriptional and functional approaches to characterize intestinal glucose demand and metabolism in a mouse model of CF to gain new insights into mechanisms of intestinal disease. We show elevated GLUT1 (Slc2a1) expression in both CF intestinal tissue and isolated crypts, suggesting GLUT1 is responsible for the increased intestinal glucose uptake from the circulation in CF mice. We also identified concomitant increases in glycolysis, in addition to evidence of a proliferative adaptive response in the CF intestine. Taken together, these results are indicative of an increased intestinal glucose demand in CF to maintain intestinal homeostasis and offer a novel approach to understanding CF intestinal disease that may result in new markers of disease status and potentially new critical insight into cancer risk in people with CF. Our findings emphasize a continued need to investigate how the interplay of numerous digestive and absorptive defects in CF contribute to disease pathogenesis to develop more effective therapies for the gastrointestinal manifestations of CF.

ORGANISM(S): Mus musculus

PROVIDER: GSE291307 | GEO | 2025/12/10

REPOSITORIES: GEO

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Project description:Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., {alpha}-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Total RNA was extracted from pooled small intestines of three WT and three CF mice using Tri-Reagent (Molecular Research Center, Cincinnati, OH), and poly(A) RNA was purified by using the MicroPoly(A) mRNA purification kit (Ambion, Austin, TX). The WT and CF poly(A) RNA samples were sent to IncyteGenomics (St. Louis, MO) where they were labeled with cyanine 3 (Cy3) and Cy5, respectively, and hybridized with the UniGEM1.31 array representing 9,570 known genes and expressed sequence tags.

Project description:Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype. Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels. Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. For each group (control wild type, lubiprostone-treated wild type, contol Cftr null, and lubiprostone-treated cftr null), equal amounts of total RNA extracted from the entire small intestine were pooled for analysis.

Project description:In this study we compared the intestinal mucus phenotype of Clca1-deficient (Clca1-/-) mice and wild-type (WT) controls. The human CLCA1 (chloride channel regulator, calcium-activated) and its murine ortholog mouse CLCA1 (formerly known as CLCA3) are secreted proteins which have been linked to human respiratory diseases such as asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) as well as to the corresponding murine models of these diseases. Common, disease severity-related features are goblet cell hyperplasia, mucus overproduction and disturbed clearance. Neutralization of mouse CLCA1 ameliorates the symptoms in the asthmatic mouse model and restoration of the decreased mouse CLCA1 protein level in a CF mouse model ameliorates the intestinal CF lesions, pointing toward these proteins being potential therapeutic targets. The expression CLCA1 can be induced in airways but this protein is normally expressed and secreted by goblet cells of the intestinal tract in both human and mouse together with the intestinal MUC2 mucin. This makes CLCA1 a major constituent of the extracellular intestinal mucus in which the MUC2 mucin is the main structural component. To study the possible further effects of murine CLCA1 deficiency on Muc2 and on other important components of the mucus, such as Agr2, Fcgbp, Klk and Zg16, a shotgun label-free proteomics study was performed which compared their relative abundance in the mucus layer, showing a tight and significant correlation between WT and Clca1-/- mucus proteins (Pearson r = 0.931, r2 = 0.868, p < 0.0001, based on 809 valid pairs). No significant differences in known mucus protein expression were observed. However, the abundance of CLCA1 in the mucus together with the domain structure suggests a structural role and other methods to reveal the function of these domains might shed light on the overall function in mucus. Because of its abundance in the mucus and the well preserved structure thought a long evolution together with mucus we still speculate that CLCA1 was developed to protect the expressing organism, although the threat is elusive.

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Intestinal adaptations increase basolateral intestinal glucose uptake and glycolysis in a Cystic Fibrosis mouse model

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