Induction of the BCMO1 gene during the suckling-weaning transition in rats is associated with histone H3 K4 methylation and subsequent coactivator binding and histone H3 acetylation to the gene.

The cells involved in nutrient absorption in the small intestine of rats undergo rapid maturation during the suckling-weaning transition period, i.e., 2-4 wk after birth. During this period, the serum thyroid hormone level is increased. However, the molecular mechanisms involved in the regulation of ß-carotene 15,15'-monooxygenase 1 (BCMO1) gene expression in the small intestine remain unknown. In this study, we found that jejunal ß-carotene 15,15' dioxygenase activity and the gene expression of BCMO1 were significantly increased during this transition period between days 13 and 27 after birth. A chromatin immunoprecipitation assay revealed that di- and tri-methylation of histone H3 at lysine 4 (K4) and the binding of thyroid hormone receptor (TR) α-1 binding on the promoter/enhancer and/or transcribed regions of the BCMO1 gene were enhanced from the earlier stage of weaning (i.e., 20 d after birth), prior to an enhancement of the acetylation of histone H3 and the binding of coactivator (SRC-1 and CBP) to the promoter/enhancer and/or transcribed regions of the BCMO1 gene, which was apparent at 27 d after birth. These results suggest that histone H3 K4 methylation and TRα-1 binding on the BCMO1 gene during the suckling-weaning transient period in rats predisposes to subsequent coactivator recruitment and histone H3 acetylation on the gene.

Changes in mucosal alpha-glucosidase activities along the jejunal-ileal axis by an Hm-HACS diet intake are associated with decreased lipogenic enzyme activity in epididymal adipose tissue.

Heat-moisture (hm)-high-amylose corn starch (HACS), which includes a larger amount of resistant starch than HACS or regular cornstarch (CS), is more indigestible in the small intestine than HACS or CS. An hm-HACS diet was also shown to ameliorate glucose intolerance and lipid abnormalities. This study examined the effects of feeding rats an hm-HACS diet for 14 days on the activities of mucosal alpha-glucosidase along the jejunal-ileal axis and lipogenic enzymes in epididymal adipose tissue. The contents in the lumen of the cecum were increased by feeding rats the HACS and hm-HACS diets, and the cecal weight was increased by the hm-HACS diet. The HACS diet reduced the activity of alpha-glucosidase in the upper jejunal mucosa, induced its activity in the upper ileal mucosa, reduced lipogenic enzyme activity in epididymal adipose tissue, and reduced serum triglyceride levels. These effects were more pronounced with the hm-HACS than with the HACS diet. These results suggest that feeding rats the hm-HACS diet reduced the activities of lipogenic enzymes in adipose tissue and alpha-glucosidase in the jejunal mucosa and induced the activity of alpha-glucosidase in the ileal mucosa compared with the HACS diet.

Feeding rats a high fat/carbohydrate ratio diet reduces jejunal S/I activity ratio and unsialylated galactose on glycosylated chain of S-I complex.

AIMS: We examined whether decreasing jejunal sucrase/isomaltase (S/I) activity ratio by feeding rats a high fat/carbohydrate ratio diet is regulated by changing glycosylated chains on the S-I complex. MAIN METHODS: Jejunal activities of sucrase, isomaltase and beta-1,4-galactosyltransferase were examined in rats fed a high fat/carbohydrate or a low fat/carbohydrate ratio diet. The amount of galactose and mannose in the glycosylated chain on the S-I complex in rats fed both diets was determined using RCA(120) and Con A lectins, respectively. The effects of reducing unsialylated galactose from the glycosylated chain on the S-I complex were assessed by determining sucrase activity in purified S-I complex treated with beta-galactosidase. KEY FINDINGS AND SIGNIFICANCE: Feeding rats a high fat/carbohydrate ratio diet reduced jejunal S/I activity ratio in mucosal homogenates and purified fractions. The level of unsialylated galactose in glycosylated chains on the S-I complex was reduced by feeding rats a high fat/carbohydrate ratio diet. The form with reduced levels of unsialylated galactose had lower sucrase activity than that with more unsialylated galactose. The reduction of galactose on the S-I complex by beta-galactosidase in vitro reduced sucrase activity. Feeding rats a high fat/carbohydrate ratio diet also reduced jejunal beta-1,4-galactosyltransferase activity. Taken together, decreasing the S/I activity ratio by feeding rats a high fat/carbohydrate diet is associated with the reduction of unsialylated galactose on the glycosylated chain of the S-I complex.

Higher expression of jejunal LPH gene in rats fed the high-carbohydrate/low-fat diet compared with those fed the low-carbohydrate/high-fat diet is associated with in vitro binding of Cdx-2 in nuclear proteins to its promoter regions.

It has been previously demonstrated that the expression of lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) genes are higher in rats fed a high-carbohydrate/low-fat (HCT) diet than in those fed a low-carbohydrate/high-fat (LCT) diet. In the present study, using a nuclear run-on assay we clearly show that higher expression of LPH and SI genes in jejunum of rats fed the HCT diet compared with those fed a LCT diet was regulated at the transcription levels. DNase I foot printing analysis of the 5' flanking region of the rat LPH gene demonstrated that by incubating the jejunal nuclear extract the protected region was conserved as the same sequence as the homeodomain protein-binding element designated as CE-LPH1. UV-cross linking and electromobility shift assay in vitro clearly showed that Cdx-2 was including proteins bound to CE-LPH1. Moreover, in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1, a cis-element identified as the binding element of Cdx-2 on the SI gene, in jejunal nuclear extracts of rats fed a HCT diet were greater than those fed a LCT diet. These results suggest that in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1 in jejunal nuclear extracts is associated with the higher expression of the LPH and SI genes in rats fed the HCT diet compared with those fed a LCT diet.

Distribution and dietary induction of cellular retinol-binding protein type II along the villus-crypt axis of the rat jejunum.

Cellular retinol-binding protein type II (CRBPII) is exclusively expressed in the small intestinal absorptive cells. We previously reported that dietary fat induces CRBPII expression within 12 h of fat intake. To examine at which locus of the villus-crypt axis this response to dietary fat occurs, 6-wk-old rats were fed a low-fat diet (7% energy) for 7 d, and then given free access to a high-fat diet (70% energy) for the subsequent 12, 24 or 48 h. Cryostat sectioning of jejunal segments followed by RNA blot hybridization of the transcripts revealed that CRBPII mRNA was expressed maximally in the lower villus, and the immunoreactive protein of CRBPII was expressed maximally in the mid-villus. Feeding the high-fat diet caused a pronounced increase in CRBPII mRNA level from the lower- to middle-villus within 12 h. These results suggest that the CRBPII gene is maximally expressed in the lower villus, and that dietary fat causes an enhancement of CRBPII gene expression in the villus cells.

Fatty acids in component of milk enhance the expression of the cAMP-response-element-binding-protein-binding protein (CBP)/p300 gene in developing rats.

Fatty acids in milk are thought to play an important role in intestinal maturation and gene expression in the rat small intestine during the suckling-weaning period. In the present study, we determined the jejunal mRNA level of the cAMP-response-element-binding-protein-binding protein (CBP)/p300, which is one of the chromatin remodelling factors and regulates histone acetylation, during the postnatal period in rats. The mRNA level of CBP/p300 was high during the suckling and middle of the weaning period (day 5 to 20) and then declined sharply to a low level at the end of the weaning period and after weaning. In situ hybridisation also showed that CBP/p300 mRNA levels in the villus as well as the basal membrane clearly decreased after weaning. Rat pups at age 17 d, weaned to a high-fat diet, showed higher levels of CBP/p300 mRNA than those weaned to a low-fat diet. Oral administration of caprylic acid, oleic acid and linoleic acid, which are major fatty acid components in milk, induced jejunal CBP/p300 gene expression. The present results suggest that fatty acids in components of milk enhance expression of the CBP/p300 genes in the small intestine.

Developmental changes in gene expressions of beta-carotene cleavage enzyme and retinoic acid synthesizing enzymes in the chick duodenum.

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (BCMO1; EC 1.13.11.21). We previously reported that chick duodenal BCMO1 activity increased abruptly just after hatching. In this study, we further investigated mechanisms and physiological roles of the postnatal induction of BCMO1 expression in the chick duodenum. We showed that BCMO1 mRNA levels increased in the chick duodenum during postnatal period after hatching, but remain unchanged in the chick liver throughout the perinatal period. Serum hydrocortisone (HC) levels were also increased after hatching. Moreover, HC-administered chicks showed an enhancement of duodenal BCMO1 mRNA during the perinatal period. We further analyzed the developmental gene expression patterns of three types of retinoic acid (RA) synthesizing enzymes in the chick duodenum. Among them, retinal dehydrogenase 1 (RALDH1) mRNA levels in the chick duodenum increased during the postnatal period, indicating a similar developmental expression pattern to that of BCMO1. These results suggest that the postnatal induction of BCMO1 gene expression in the chick duodenum may be caused by the elevation of serum HC levels and may contribute to the RALDH1-mediated RA synthetic pathway.

Possible role of fatty acids in milk as the regulator of the expression of cytosolic binding proteins for fatty acids and vitamin A through PPARalpha in developing rats.

Fatty acids in milk are thought to play an important role in intestinal maturation and gene expression in the postnatal small intestine. In this study, we determined the jejunal mRNA levels, in rats, of peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARdelta which are nuclear receptors for fatty acids. We also measured expression of their target genes during the postnatal period, namely liver type fatty acid-binding protein (L-FABP) and cellular retinol-binding protein, type II (CRBPII). The mRNA levels of PPARalpha, L-FABP and CRBPII, but not PPARdelta, gradually increased during the suckling period and then sharply declined to a low level at the end of the weaning period. Rat pups at 17 d of age, weaned to a high-fat diet, showed significantly greater mRNA levels of PPARalpha, L-FABP and CRBPII than those weaned to a low-fat diet. Oral administration of PPARalpha ligand, WY14,643 during four consecutive days of the weanling period caused a parallel increase in the mRNA levels of PPARalpha, L-FABP and CRBPII genes. Furthermore, caprylic acid and oleic acid, which are major components of fatty acids in milk, induced jejunal PPARalpha, L-FABP and CRBPII gene expression. Our results suggest that fatty acids in milk may play a pivotal role in maintaining an enhanced level of expression of L-FABP and CRBPII genes in the small intestine, presumably by acting as inducers of PPARalpha gene expression.

Selectivity of fatty acid ligands for PPARalpha which correlates both with binding to cis-element and DNA binding-independent transactivity in Caco-2 cells.

It is thought that peroxisome proliferator-activated receptor alpha (PPARalpha) is a major regulator for fatty acid metabolism. Long-chain fatty acids have been shown to induce expression of the genes related to fatty acid metabolism through PPARalpha. However, it is unclear whether the intensity of PPARalpha activation is different among various fatty acids. In this study, we compared various fatty acids in the capability of PPARalpha activation by differential protease sensitivity assay (DPSA), electrophoretic mobility shift assay and GAL4-PPAR chimera reporter assay in intestinal cell line, Caco-2. DPSA revealed that polyunsaturated fatty acids of 18 to 20 carbon groups with 3-5 double bonds strongly induced a PPARalpha conformational change. The ligand-induced changes in the sensitivity to protease corresponded to the enhancement of the binding of PPARalpha-RXRalpha heterodimer to the PPAR-response element (PPRE). The GAL4-PPAR chimera reporter assay revealed that the DNA binding-independent transactivity of PPARalpha was induced by various fatty acids with a wide spectrum of intensity which correlated with the conformational change of PPARalpha. These results suggest that PPARalpha has greater selectivity to certain types of polyunsaturated fatty acids, and that the ligand-induced conformational change of PPARalpha leads to parallel increases in both DNA binding to the PPAR-response element and the DNA binding-independent transactivity.

Diet-related variation in cellular retinol-binding protein type II gene expression in rat jejunum.

Cellular retinol-binding protein type II (CRBPII) is involved in the transport of vitamin A and its metabolism in the small intestine. In the present study, we demonstrated diet-related variations in CRBPII expression in rat jejunum. The CRBPII protein and mRNA levels increased in parallel after the start of feeding period regardless of whether the feeding period was restricted to the hours of darkness or of light. In addition, this variation was observed in the rats fed high-fat diet or low-fat diets, but not in those fed a fat-free diet or in fasted rats. A similar diet-induced variation was seen in the mRNA of liver-type fatty acid-binding protein in rat jejunum. In the transient transfection experiment, unsaturated fatty acid increased rat CRBPII gene promoter activity via the PPARalpha/retinoid X receptor-alpha heterodimer. Taken together, these results suggest that the diet-related variation in CRBPII expression in rat jejunum may be brought about by the transcriptional induction of CRBPII gene expression mainly triggered by dietary fatty acids.

Developmental changes of the expression of the genes regulated by retinoic acid in the small intestine of rats.

Retinoic acid (RA) serves as a hormone-like nutrient and it plays pivotal roles in cellular differentiation and proliferation in various tissues including the small intestine. In this study, we aimed to explore a possible role of RA signaling in the developing rat small intestine of perinatal (embryonic and newborn) and suckling-weaning transition period, and we investigated the changes in the expression of several genes regulated by RA. Northern blot analysis showed that both retinal dehydrogenase 1 (RALDH1) and retinal dehydrogenase 2 (RALDH2) mRNA levels were higher in 19-day fetal (2 days before birth) small intestine and then declined after birth. Retinoid X receptor alpha (RXRalpha) mRNA and retinoic acid receptor alpha (RARalpha) mRNA levels in the small intestine showed high levels in perinatal period compared with suckling-weaning transition period. RA-target genes such as retinoic acid receptor beta (RARbeta) and cellular retinol-binding protein, type II (CRBPII) mRNA levels were significantly increased in the perinatal small intestine. Furthermore, mRNA levels of hepatocyte nuclear factor-4 (HNF-4), which is one of the possible RA-target gene and a transcription factor regulating CRBPII gene expression, was also increased in the perinatal small intestine. These results suggest that the possible perinatal RA production by RALDHs might regulate various RA-target genes including CRBPII and RARalpha through RXRalpha or HNF-4 in the small intestine.

Postnatal changes in gene expression of retinal dehydrogenase and retinoid receptors in liver of rats.

Retinoic acid (RA) plays important roles in cellular differentiation and proliferation in various tissues including the liver. To explore a possible role of RA in the postnatal development of hepatic function, we analyzed RA-generation enzyme activity and the RA-related hepatic gene expressions in the suckling and weaning rats. At 5 days after birth, retinal dehydrogenase (RALDH) activity in the liver was relatively high. Its activity decreased by 70% until day 17, and then it gradually increased to a high level by the completion of weaning period. Northern blot analysis showed that RALDH2 mRNA levels decreased in the suckling period, whereas RALDH1 mRNA levels increased in the weaning period. Retinoid X receptor alpha (RXRalpha) mRNA levels increased in the suckling period and attained to a higher level at 17 days after birth. Retinoic acid receptor alpha (RARalpha) mRNA level showed only a slight and temporary increase on day 13. The mRNA levels of hepatocyte nuclear factors (HNF-4 and HNF-1alpha) exhibited parallel increases around suckling-weaning period, and the transcript levels of albumin, a typical target gene of the hepatocyte nuclear factors, increased during the suckling-weaning transition period. Electrophoretic mobility shift assay using a putative nuclear receptor-binding element on rat HNF-1 alpha gene revealed that HNF-4 homodimer, but not RXRalpha homodimer, bound to this element. These results suggest that postnatal expressions of hepatocyte-specific genes might be up-regulated by retinoid receptors, which may be related with the alterations of RALDH expression during postnatal development in the liver.

Major intestinal coactivator p300 strongly activates peroxisome proliferator-activated receptor in intestinal cell line, Caco-2.

We have previously reported that several genes related to intestinal fatty acid and vitamin A metabolism are coordinately regulated by peroxisome proliferator-activated receptor (PPAR) [Arch. Biochem. Biophys. 389 (2001) 41; Biochim. Biophys. Acta 1531 (2001) 68]. In this study, we demonstrated that PPAR alpha and PPAR delta interacted with endogenous coactivators in intestinal cell line, Caco-2 in a ligand specific manner. We isolated rat cDNA clones encoding the nuclear receptor interaction domains of the two transcriptional coactivators, CREB-binding protein (CBP) and p300. Expression level of CBP mRNA was relatively low in the small intestine, while p300 mRNA was ubiquitously expressed in various tissues including the small intestine in the rat. Southern blot analysis revealed that these coactivators were encoded by different genes. Mammalian two-hybrid assays in Caco-2 cells revealed that p300 interacted with PPAR alpha or PPAR delta in the presence of their specific ligands more efficiently than CBP did. These results suggest that the major intestinal coactivator, p300 strongly interacts with PPAR alpha and PPAR delta.