Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells

A panel of monoclonal antibodies to intestinal cell surface components has been used to compare the expression of differentiation-specific antigens in the epithelial cells of fetal, suckling, and adult rat small intestine. Indirect immunofluorescence staining, and immunopurification of detergent-solubilized membrane proteins, followed by single- and two-dimensional slab gel electrophoretic analysis, have demonstrated that fetal intestinal cells (at day 21 of gestation) express most differentiation-specific markers typical of adult absorptive villus cells. A marked heterogeneity in antigen expression was observed among different villus cell populations in suckling rat intestine, and three cell surface components were identified which are exclusively present during this period of intestinal development. Striking changes in the patterns of antigen expression in crypt and villus cells, and variations in the apparent isoelectric points for most luminal membrane components, were associated with the maturation of the intestinal mucosa at weaning. These changes could not be prematurely induced by cortisone injection in newborn rats, suggesting that factors other than glucocorticoids are responsible for the postnatal development of the intestinal epithelium. These results suggest that basic differences in biological properties and regulatory mechanisms exist among intestinal epithelial cells at different stages of pre- and postnatal maturation.     

Diet and metabolic development

For many years, investigators have been concerned with mechanisms that control and alter genetically regulated development. An intriguing aspect of these mechanisms is the ability of environmental factors to induce certain metabolic processes. Animal studies have shown that dietary manipulation of cholesterol and fatty acid metabolism during development can have persistent and permanent effects. In addition, there appears to be a critical period when changes in the diet can have lasting consequences. The changes in the control exerted by nutritional factors on metabolic development coincide with three phases of development: prenatal, suckling, and weaning. The effects of diet on cholesterol and fatty acid metabolism throughout these three phases of development will be addressed in this review.     

Developmental changes of gut microflora and enzyme activity in rat pups exposed to fat-rich diet

The aim of this study was to investigate the effect of a high-fat (HF)/energy diet on the intestinal microbiota, the alkaline phosphatase (AP) activity, and related parameters of growth and obesity during the suckling and weaning periods in male Sprague-Dawley rats. From birth, nutrition in suckling pups was manipulated by feeding rat dams either HF or a standard diet, and then after weaning, by exposure of experimental pups to the HF, and control rats to normal diet. On days 15, 20, 40 the numbers of 2 microbial groups, i.e., Bacteroides/Prevotella (BAC) and the Lactobacillus/Enterococcus (LAB) in the jejunum, were determined by fluorescent in situ hybridization technique, and the AP activity was assayed histochemically. During all investigated periods HF pups gained body fat more rapidly than control animals, but from weaning they displayed significantly stunted growth resulting in final body weight loss. Obesity in HF rats was also accompanied by higher LAB and lower numbers of BAC and with permanently higher AP activity. Correlation of these data showed significant negative correlation between LAB, AP, and weight gain and energy efficiency, and significant positive correlation of BAC and AP activity with body fat. These data support the concept that postnatal nutritional experience represents an important factor affecting the ontogeny of intestinal microbial communities and intestinal function. These acquired changes could be a component of regulatory mechanisms involved in adverse and/or positive consequences of HF diet for adiposity, body weight, and energy-balance control in later life.     

Development of insulin sensitivity in rat skeletal muscle. Studies of glucose transporter and insulin receptor mRNA levels.

Expression of GLUT-4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT-4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1-8 days and at 2-3-fold higher levels during and after weaning (18-40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT-4 abundance seen between 15 and 29 days in animals weaned on a high-carbohydrate diet.     

A methyl-deficient diet fed to rats during the pre- and peri-conception periods of development modifies the hepatic proteome in the adult offspring

A methyl-deficient diet (MD) lacking folic acid and the associated methyl donors choline and methionine, fed to the laboratory rat during the periods of oocyte and embryo development, has been shown to programme glucose metabolism in the offspring. The hepatic proteome of the male offspring of female rats fed MD diets for 3 weeks prior to mating and for the first 5 days of gestation has been examined by 2-dimensional gel electrophoresis. Three groups of differentially abundant proteins associated with energy metabolism, amino acid metabolism and antioxidant defence were identified in the soluble proteins extracted from the liver from the MD offspring at both 6 and 12 months of age. Altered mitochondrial activity in other programming models leads to a similar pattern of differential protein abundance. Two of the differentially abundant proteins were identified as GAPDH and PGK-1 by mass spectrometry. Western blotting showed that there were multiple isoforms of both proteins with similar molecular weights but different isoelectric points. The differentially abundant spots reduced in the MD offspring corresponded to minor isoforms of GAPDH and PGK-1. The levels of PPAR-alpha, SREBP and glucocorticoid receptor mRNAs associated with other models of prenatal programming were unchanged in the MD offspring. The data suggest that a diet deficient in folic acid and associated methyl donors fed during the peri-conception and early preimplantation periods of mammalian development affects mitochondrial function in the offspring and that the posttranslational modification of proteins may be important.     

Ontogenic and nutritional modifications in the intestinal fucosylation process at the weaning period. Influence of dietary fibers

In the rat small intestine, the glycosylation changes which normally take place at the weaning period are characterized by a shift from sialylation of fucosylation. The introduction of dietary fibers at weaning is one of the more striking nutritional modification so that some authors have suggested that the presence of fibers and the development of colonic fermentation might be important for the development of the small intestine, as for the colon. In order to define the respective contribution of ontogenic and nutritional factors to the intestinal glycosylation changes at this period, some aspects of the intestinal glycosylation were studied in five groups of rats (16-day-old suckling rats, prolonged nursing 23-day-old rats, 23-day-old rats weaned at day 19 with either a fiber-free, a cellulose or a pectin diet). Intestinal glycoproteins of suckling rats are characterized by a low fucose content and a high proportion of mannose. The amounts of the neutral sugars (fucose, mannose and galactose), expressed either per gram of intestine or for one intestine, are alwars higher in the fiber-fed groups than in the prolonged-nursing group or the group fed the fiber-free diet. Activities which promote fucosylation process (GDP-fucose production and fucosyltransferase activities) and those which are opposed to fucosylation (endogenous inhibitor of fucosyltransferase and GDP-fucose pyrophosphatase) are strongly modified in opposite ways at day 23 as compared to day 16. These modifications depend on the age of the animal (ontogenic factors) with additional modifications induced by the dietary factors. In particular, similar sugar contents and patterns are obtained with cellulose and pectin diests though the enzymatic activities of the fucosylation pathway are very different. No correlation was found between the caecal content of short chain fatty acids and any of the parameters under study. Thus, dietary fibers induce metabolic changes in the small intestine glycosylation in short-term experiments independently of colonic fermentation. Besides, these results point out that the consideration of fucosyl-transferase activities alone are not sufficient to predict glycoprotein fucose content and that other regulatory sites are involved. Dietary manipulations at the weaning period could represent a good model for the study of glycosylation regulation.     

Amino acids modulates the intestinal proteome associated with immune and stress response in weaning pig

The objective of this study was to investigate the effects of free amino acids supplementation to protein restricted diet on the intestinal morphology and proteome composition in weaning pigs. Weanling piglets were randomly fed one of the three diets including a corn-soybean based control diet and two lower protein diets with or without free amino acids supplementation for 2 weeks. The jejunum samples of piglets were collected for morphology and proteome analysis. Compared with the control diet, the protein restricted diet had a significant lower average daily gain and higher feed conversion rate. Free amino acids supplementation to the protein restricted diet significantly improved average daily gain and higher feed conversion rate, compared with the protein restricted diet. The villous height in pigs fed the protein restricted diet was lower than that of the control and free amino acids diet. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 16 differentially expressed protein spots in the jejunum of the weaning piglet. These proteins were related to stress and immune response, the metabolism of carbohydrates and lipids, and tissue structure. Based on the proteome and ELISA analysis, free amino acids diet significantly down-regulated the jejunal expression of stress protein heat shock 60 kDa protein. Our results indicated that amino acids supplementation to the protein restricted diet could enhance weight gain and feed efficiency in weanling pigs through improving intestinal nutrient absorption and transportation, gut health, and mucosal immunity.     

Corticosteroid regulation of colonic ion transport during postnatal development: methods for corticosteroid analysis

Many mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinal tract that undergoes abrupt transitions coinciding with birth and weaning. This review deals with the maturation of ion transport functions in colon, the intestinal segment that plays an important role in sodium and potassium absorption and secretion. The purpose of the present review is to summarize the mechanism of sodium and potassium transport pathways and show how these transport processes change postnatally and how hormones, particularly corticosteroids, modify the pattern of development. Finally we describe some of the ways, how to analyze corticosteroid metabolism in target tissue.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

Intestinal epithelial cell proteome in IL-10 deficient mice and IL-10 receptor reconstituted epithelial cells: impact on chronic inflammation

The interaction of nonpathogenic enteric bacteria with intestinal epithelial cells (IEC) in the absence of host-derived Interleukin 10 (IL-10) may contribute to the development of chronic inflammation. Functional proteome analysis of primary IEC from Enterococcus faecalis-monoassociated WT and IL-10-/- mice as well as IL-10 receptor reconstituted IEC revealed expression changes of proteins that are involved in endoplasmic reticulum stress, energy metabolism, and apoptosis, suggesting a protective role for IL-10 at the epithelial cell level.     

Isobaric labeling-based quantitative proteomics of FACS-purified immune cells and epithelial cells from the intestine of Crohn's disease patients reveals proteome changes of potential importance in disease pathogenesis

Crohn's disease (CD) is a chronic condition characterized by recurrent flares of inflammation in the gastrointestinal tract. Disease etiology is poorly understood and is characterized by dysregulated immune activation that progressively destroys intestinal tissue. Key cellular compartments in disease pathogenesis are the intestinal epithelial layer and its underlying lamina propria. While the epithelium contains predominantly epithelial cells, the lamina propria is enriched in immune cells. Deciphering proteome changes in different cell populations is important to understand CD pathogenesis. Here, using isobaric labeling-based quantitative proteomics, we perform an exploratory study to analyze in-depth proteome changes in epithelial cells, immune cells and stromal cells in CD patients compared to controls using cells purified by FACS. Our study revealed increased proteins associated with neutrophil degranulation and mitochondrial metabolism in immune cells of CD intestinal mucosa. We also found upregulation of proteins involved in glycosylation and secretory pathways in epithelial cells of CD patients, while proteins involved in mitochondrial metabolism were reduced. The distinct alterations in protein levels in immune- versus epithelial cells underscores the utility of proteome analysis of defined cell types. Moreover, our workflow allowing concomitant assessment of cell-type specific changes on an individual basis enables deeper insight into disease pathogenesis.     

Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats

This study was designed to monitor the developmental changes in insulinemia and lipogenic enzyme activities in both inguinal adipose tissue and liver during suckling (7, 9, 14, and 17 days of age) and weaning (22 and 30 days of age) on to either a low-fat or a high-fat diet in lean (Fa/fa) and obese (fa/fa) rats. Tissues were removed through surgery and genotypes were retrospectively determined. During suckling, there was no difference in liver enzyme activities between the two groups. In contrast, adipose tissue fatty acid synthetase was increased by 50% and citrate cleavage enzyme and malic enzyme by 30% by 9 days of age. By 17 days of age, there was a threefold elevation in these enzyme activities and 6-phosphogluconic dehydrogenase and a twofold increase in glucose-6-phosphate dehydrogenase per inguinal fat pad in fa/fa versus Fa/fa. Consistent with these results, fat pad weight was increased by 20%, 50%, and 100% at 9, 14, and 17 days of age, respectively, in obese as compared to lean pups. However only by 17 days of age could a slight but significant increase in insulin level be detected in obese pups. Enlargement of inguinal fat pad accelerated after weaning on to a low-fat diet and still more after weaning on to a high-fat diet. Weaning on to a low-fat diet elicited an induction of hepatic lipogenic enzymes two or three times greater in fa/fa than in lean pups, while weaning on to a high-fat diet blunted the differences between genotypes. The lipogenic enzyme activities displayed per total inguinal fat were three to ten times greater in obese than in lean pups, regardless of the diet. However, adipose tissue lipogenic enzyme activities were much lower after weaning on to a high-fat than on to a low-fat diet in obese pups. The high-fat diet was as effective as the low-fat diet in triggering hyperinsulinemia in obese pups. The increased adipose tissue capacity for lipogenesis, starting during the suckling period, could play an important etiologic role in the development and maintenance of obesity in the Zucker rat.-Bazin, R., and M. Lavau. Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats.     

Proteomic analysis of glutamine-treated human intestinal epithelial HCT-8 cells under basal and inflammatory conditions

Glutamine (Gln) promotes intestinal growth and maintains gut structure and function, especially in situations of injury and during inflammation. Several mechanisms could contribute to Gln protective effects on gut. Proteomics enable us to characterize differentially expressed proteins in tissues in response to modifications of the biological or nutritional environment. Gln effects on the human intestinal epithelial HCT-8 cell line proteome were assessed under basal and proinflammatory conditions. The 2-DE gels were obtained and compared. Proteins were identified by MS and using databases. About 1200 spots were detected in both 2- and 10-mM Gln concentrations. Under basal conditions, 24 proteins were differentially expressed in response to Gln. Half of these proteins were implicated in protein biosynthesis or proteolysis and 20% in membrane trafficking. Under proinflammatory conditions, 27 proteins were up- or down-regulated by Gln 10 mM. From these proteins, 40% were involved in protein biosynthesis or proteolysis, 16% in membrane trafficking, 8% in cell cycle and apoptosis mechanisms and 8% in nucleic acid metabolism. This study provides the first holistic picture of proteome modulation by Gln in a human enterocytic cell line under basal and proinflammatory conditions, and supports further evaluation of nutritional modulation of intestinal proteome in humans.     

Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets.     

Effect of controlled feeding on the isoenzymes of pyruvate kinase, hexokinase and aldolase in liver during development

Controlled feeding schedules have been used to maintain rats during the first thirty days following birth to investigate the role of food in determining the course of liver development. Use of the ‘8 + 16’ feeding schedule and a new ‘16 + 8’ suckling schedule allowed rats to be reared through the neonatal period without access to solid food until the day they were weaned. Food-independent increases in Pyruvate kinase type I isoenzyme, glucokinase and aldolase B take place in the late suckling period. Thus it appears that the change in the nutritional status of the animal at weaning is not a major factor in liver development.     

Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.     

Glutamine regulates the human epithelial intestinal HCT-8 cell proteome under apoptotic conditions

Glutamine plays a key role in the metabolism of rapidly dividing cells, including enterocytes and lymphocytes, which may contribute to its beneficial clinical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apoptosis. In T cells, glutamine up-regulates antiapoptotic proteins and down-regulates proapoptotic proteins. In gut mucosa, glutamine prevents apoptosis in rat epithelial cell lines, whereas glutamine starvation induces apoptosis through caspase activation. Finally glutamine specifically prevents tumor necrosis factor-alpha-related apoptosis in the human intestinal cell line HT-29. Comparative functional proteomics enables the characterization of each differentially expressed protein in intestinal cells in response to modifications of nutritional environment. The influence of glutamine on intestinal proteome expression in apoptotic conditions has not been studied and evaluated. This comparative proteomics study was performed in the human epithelial intestinal cell line HCT-8 under experimental apoptotic conditions to investigate the influence of glutamine on protein expression during apoptosis. The pharmaconutritional effects of glutamine were determined under 2 mm (physiological concentration) and 10 mm (pharmaconutritional concentration) conditions. About 1,800 protein spots were revealed in both conditions. Comparative assessments indicated that 28 proteins were differentially expressed significantly (i.e. at least 2-fold modulated and Student's t test with p 相似文献   

Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine: I. Evidence of a developmental shift from terminal sialylation to fucosylation

We present evidence of a change from sialylation to fucosylation of intestinal microvillus membrane oligosaccharides during postnatal development in the rat. The initial high sialic acid to fucose molar ratio in native and delipidated membranes was completely reversed after weaning. The specific binding of 125I-labeled wheat germ agglutinin to neuraminidase-sensitive sites in the native and delipidated membranes decreased markedly from early suckling to weaning ages. The binding of 125I-labeled Ulex europeus agglutinin I showed an age-related pattern opposite to that of wheat germ agglutinin. The changes in membrane reactivities to these lectins were entirely consistent with the existence of a developmentally-controlled shift from terminal sialyl to fucosyl substitutions among various glycoconjugate classes. This could play a key role on the functional transformation experienced by the intestinal epithelium of suckling rats.     

The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities

An autosomal recessive mutation, termed fatty liver dystrophy (fld), can be identified in neonatal mice by their enlarged and fatty liver (Sweet, H. O., Birkenmeier, E. H., and Davisson, M. T. (1988) Mouse News Letter 81, 69). We have examined the underlying metabolic abnormalities in fld/fld mice from postnatal days 3-40. Serum and hepatic triglyceride levels were elevated 5-fold in suckling fld/fld mice compared to their +/? littermates but abruptly resolved at the suckling/weaning transition. Blot hybridization analysis of liver and intestinal RNAs revealed a liver-specific increase in apolipoprotein (apo) A-IV and C-II mRNA concentrations (100- and 6-fold, respectively) that was limited to the suckling and early weaning stages in fld/fld mice. Resolution of these differences during the weaning period could not be delayed by prolonging suckling to the 20th postnatal day nor could the mutant phenotype be elicited in young adult animals with a high fat diet. Lipoprotein lipase (LPL) activity was reduced 16-fold in the white adipose tissue of fld/fld mice until the onset of weaning. Heart activity was decreased less than 2-fold, but there were no deficits in brown adipose tissue or liver. Hepatic lipase (HL) mRNA levels and activity were significantly reduced in fld/fld livers and sera, respectively, during the suckling period. Mapping studies show the fld locus to be distinct from loci encoding LPL, HL, and apoA-IV, and those responsible for the combined lipase deficiencies in cld/cld and W/Wv mice. These data suggest that the fld mutation is associated with developmentally programmed tissue-specific defects in the neonatal expression of LPL and HL activities and provide evidence for a new regulatory locus which affects these lipase activities. This mutation could serve as a useful model for (i) analyzing the homeostatic mechanisms controlling lipid metabolism in newborn mice and (ii) understanding and treating certain inborn errors in human triglyceride metabolism.