Dietary short-chain fructooligosaccharides increase calbindin-D9k levels only in the large intestine in rats independent of dietary calcium deficiency or serum 1,25 dihydroxy vitamin D levels

Dietary short-chain fructooligosaccharides (Sc-FOS) increase mucosal calbindin-D9k (CaBP) levels in the large intestine whereas levels in the small intestine are decreased in rats. In the present study, we investigated the mechanism by which Sc-FOS induce this increase in CaBP in the large intestine by measuring intestinal CaBP levels in rats fed normal and calcium-deficient diets. Dietary groups included a calcium-containing (0.5%) diet with or without Sc-FOS (100 g/kg diet) and a calcium-deficient (abt. 0.01%) diet with or without Sc-FOS (100 g/kg diet). The rats were fed these diets for 10 days following which they were killed and the intestine removed for collection of the entire mucosa which was divided into four segments, i.e., proximal and distal segments of the small intestine, the cecum and the colorectum. Mucosal CaBP and plasma calcium (Ca), 1,25-dihydroxycholecalciferol (1,25(OH)2D3), 25-hydroxycholecalciferol (25(OH)D3), parathyroid hormone (PTH) and calcitonin levels were measured. Feeding of calcium deficient diet resulted in an increase in CaBP levels in the small intestine, but did not influence levels in the large intestine. Moreover, a significant positive correlation between plasma 1,25(OH)2D3 and CaBP levels in the case of both small intestinal segments (proximal, r = 0.77012, p < 0.00007; distal, r = 0.75056, p < 0.00014) was observed, but not in the case of the large intestinal segments. Sc-FOS increased CaBP levels in the large intestine. These results suggest that the large intestinal CaBP levels do not change in response to dietary calcium conditions and are not regulated by circulating 1,25(OH)2D3 indicating that the effect of Sc-FOS on CaBP levels in the large intestine is independent of the action of 1,25(OH)2D3.     

The effect of fructooligosaccharides was analyzed by cDNA expression arrays

We have already reported that indigestible fructooligosaccharides (FOS) increased calcium absorption in rat large intestines and that calbindin-D9k (CaBP), which is an intestine-specific calcium-binding protein, is involved in that increasing effect. In this study, not only the CaBP gene, every gene that changed expression profiles as the result of FOS feeding was identified by cDNA expression arrays. Sprague-Dawley male rats were fed an experimental diet containing 10% FOS for 10 d. To compare gene expression with rats fed a control diet, total mRNA was extracted from the colorectum and analyzed using a Rat cDNA Expression Arrays filter. This arrays filter contains probes of 588 genes, and 195 of them showed detectable changes in their expression by FOS feeding. There were six genes that increased their expression more than twice that of the control. Among them, genes related to the induction of cell growth such as Map kinase 1 and Max were included. Expressions that decreased to less than half were observed in 20 genes, such as somatostatin and prohibitin, which prohibit cell growth. These results are consistent with the other observation that FOS increases cell growth in the colorectum. This approach has revealed that cDNA array technology is an effective tool for nutritional sciences that involve the regulation of a large number of genes, especially for molecular mechanisms of regulation, by nutritional constituents.     

Dietary fructo-oligosaccharides improve insulin sensitivity along with the suppression of adipocytokine secretion from mesenteric fat cells in rats

Short-chain fructo-oligosaccharides (FOS) are known to have beneficial effects on health. However, the effects of FOS on insulin resistance have not been fully clarified. We observed the effects of FOS feeding on insulin sensitivity and adipocytokine release from abdominal adipocytes in weaning rats. Male Sprague-Dawley rats, 3 weeks old, were divided into three groups and fed a sucrose-based American Institute of Nutrition (AIN)-93 growth diet (control), the control diet containing 5?% FOS for 5 weeks (FOS-5wk) or the control diet for 2 weeks followed by the 5?% FOS diet for 3 weeks (FOS-3wk). Tail blood was collected after fasting for 9?h on day 33 of feeding, and glucose and insulin levels were measured. On the last day, rats were anaesthetised and killed after the collection of aortic blood. Small- and large-intestinal mesenteric fat tissues were immediately excised, and the release of adiponectin, leptin and TNF-α was evaluated from the subsequently isolated adipocytes. The weight of the large-intestinal mesenteric fat, fasting blood insulin level and homeostatic model assessment for insulin resistance decreased in a time-dependent manner, and were much lower in the FOS-5wk group than in the control group. These values were correlated with aortic blood leptin levels. The secretion rate of leptin from the isolated mesenteric adipocytes in the small intestine, but not in the large intestine, was lower in the FOS-fed groups than in the control group. In conclusion, FOS feeding improved insulin sensitivity accompanied by the reduction in large-intestinal fat mass and leptin secretion from the mesenteric adipocytes of the small intestine.     

上皮钙通道基因在维生素D受体和Calbindin-D28k双基因敲除鼠中的变化

目的研究上皮钙通道TRPV5和TRPV6基因与维生素D受体(VDR)和钙结合基因Calbindin-D28k的关系,以及肾钙的重吸收减少是否与TRPV5和TRPV6基因表达减少有关。方法制备VDR和CaBP-D28k双基因敲除小鼠,普通和高钙食物喂养下,检测野生型鼠(WT),CaBP-D28(-/-),VDR(-/-),和VDR(-/-)/CaBP-D28k(-/-)鼠的体重、摄食量及血尿参数值,用实时RT-PCR的方法检测各种鼠肾脏TRPV5和TRPV6的mRNA水平。结果普通饮食下,双基因敲除小鼠尿钙的分泌和血清甲状旁腺激素水平更高。TRPV5和TRPV6两者的表达在CaBP-D28k敲除鼠中均无变化,而在VDR敲除鼠和双基因敲除鼠中下调的幅度相当。高钙饮食下,VDR及双基因敲除小鼠的血离子钙水平正常。所有小鼠这两个基因的表达普遍降低,而在VDR和双基因敲除鼠中的表达更低。结论这些结果证实了上皮钙通道基因受钙和维生素D的调节,CaBP-D28k的缺失对两者无影响。肾钙的重吸收减少与TRPV5和TRPV6基因表达无明显关系。     

小鼠Calbindin-D28k基因对钙代谢的影响

目的：探讨Calbindin-D28k对钙代谢的影响及作用。方法：制备维生素D受体（VDR）／Calbindin-D28k双基因剔除小鼠模型，常规及高钙饮食下，检测小鼠体重、摄食量、血尿参数值及甲状旁腺大小等。结果：常规饮食下，双基因剔除小鼠发育更迟缓，体重比VDR单基因剔除小鼠轻42％。尿钙的分泌更高，并发展为严重的继发性甲状旁腺功能亢进。高钙饮食下，VDR及双基因剔除小鼠的血钙离子水平恢复正常。结论：CaBP-D28k对钙代谢平衡起了重要的作用，它的作用大都被CaBP-D9k代偿。     

Calbindin-D28k对小鼠骨质生长的影响

目的 探讨小鼠肾脏Calbindin-D28k(CaBP-D28k)在钙代谢中的作用.方法 构建维生素D受体(Vitamin D Recep-tor,VDR)/Calbindin-D28k双基因剔除小鼠模型,在普通及高钙饮食下,检测小鼠体重、摄食量、血尿参数值、下肢骨的长度、密度以及胫骨切片组织学染色等.结果 普通饮食下,VDR(-/-)/CaBP-D28k(-/-)小鼠发育更迟缓,体重比VDR(-/-)小鼠轻42%,尿钙的分泌更高,发展为更严重的佝偻病骨表型,表现为骨密度较低和骨小梁区生长板变形更多.高钙饮食下,VDR(-/-)及VDR(-/-)/CaBP-D28k(-/-)小鼠的血钙离子正常,VDR(-/-)/CaBP-D28k(-/-)小鼠的骨骼异常未得到完全纠正.结论 CaBP-D28k对骨质的生长发育起了重要作用,它对钙代谢的作用大都可被CaBP-D9k补偿.     

Opposing effects of ethanol and nicotine on hippocampal calbindin-D28k expression.

Long-term ethanol exposure produces multiple neuroadaptations that likely contribute to dysregulation of Ca(2+) balance and neurotoxicity during ethanol withdrawal. Conversely, nicotine exposure may reduce the neurotoxic consequences of Ca(2+) dysregulation, putatively through up-regulation of the Ca(2+)-buffering protein calbindin-D(28k). The current studies were designed to examine the extent to which 10-day ethanol exposure and withdrawal altered calbindin-D(28k) expression in rat hippocampus. Further, in these studies, we examined the ability of nicotine, through action at alpha(7)(*)-bearing nicotinic acetylcholine receptors (nAChRs), to antagonize the effects of ethanol exposure on calbindin-D(28k) expression. Organotypic cultures of rat hippocampus were exposed to ethanol (50-100 mM) for 10 days. Additional cultures were exposed to 500 nM (-)-nicotine with or without the addition of 50 mM ethanol, 100 nM methyllycaconitine (an alpha(7)*-bearing nAChR antagonist), or both. Prolonged exposure to ethanol (>/=50 mM) produced significant reductions of calbindin-D(28k) immunolabeling in all regions of the hippocampal formation, even at nontoxic concentrations of ethanol. Calbindin-D(28k) expression levels returned to near-control levels after 72 h of withdrawal from 10-day ethanol exposure. Extended (-)-nicotine exposure produced significant elevations in calbindin-D(28k) expression levels that were prevented by methyllycaconitine co-exposure. Co-exposure of cultures to (-)-nicotine with ethanol resulted in an attenuation of ethanol-induced reductions in calbindin-D(28k) expression levels. These findings support the suggestion that long-term ethanol exposure reduces the neuronal capacity to buffer accumulated Ca(2+) in a reversible manner, an effect that likely contributes to withdrawal-induced neurotoxicity. Further, long-term exposure to (-)-nicotine enhances calbindin-D(28k) expression in an alpha(7)* nAChR-dependent manner and antagonizes the effects of ethanol on calbindin-D(28k) expression.     

Alternative perspective on intestinal calcium absorption: proposed complementary actions of Ca(v)1.3 and TRPV6

Transcellular models of dietary Ca(2+) absorption by the intestine assign essential roles to TRPV6 and calbindin-D(9K) . However, studies with gene-knockout mice challenge this view. Something fundamental is missing. The L-type channel Ca(v) 1.3 is located in the apical membrane from the duodenum to the ileum. In perfused rat jejunum in vivo and in Caco-2 cells, Ca(v) 1.3 mediates sodium glucose transporter 1 (SGLT1)-dependent and prolactin-induced active, transcellular Ca(2+) absorption, respectively. TRPV6 is activated by hyperpolarization and is vitamin D dependent; in contrast, Ca(v) 1.3 is activated by depolarization and is independent of calbindin-D(9K) and vitamin D. This review considers evidence supporting the idea that Ca(v) 1.3 and TRPV6 have complementary roles in the regulation of intestinal Ca(2+) absorption as depolarization and repolarization of the apical membrane occur during and between digestive periods, respectively, and as chyme moves from one intestinal segment to another and food transit times increase. Reassessment of current arguments for paracellular flow reveals that key phenomena have alternative explanations within the integrated Ca(v) 1.3/TRPV6 view of transcellular Ca(2+) absorption.     

Influence of fructooligosaccharides on Peyer's patch lymphocyte numbers in healthy and endotoxemic mice

OBJECTIVE: The purpose of this study was to determine whether fructooligosaccharides (FOS) exert an immunomodulating effect on Peyer's patches (PP), the main inductive site of the intestinal immune system. We investigated the effects of FOS in healthy and endotoxemic animals. METHODS: Six-week-old female Balb/c mice were fed a control diet or a diet supplemented with 10% FOS over a period of 16 d. To induce endotoxemia, mice were challenged intraperitoneally with lipopolysaccharide (LPS) on day 15. PP were excised from mice, and lymphocyte subpopulations (B lymphocytes, T lymphocytes, CD4(+) cells, and CD8(+) cells) were determined by flow cytometry. RESULTS: The FOS-enriched diet increased the total cell yield in healthy and endotoxemic mice (P < 0.001). Similarly, B lymphocytes were increased in both groups (P < 0.001). In contrast, T lymphocytes were unaltered in healthy mice but increased in LPS-challenged mice after FOS enrichment (P < 0.001). In endotoxemic mice but not in control animals, the increase of CD4(+) cells (P < 0.001) was more pronounced than that of CD8(+) cells (P < 0.001), thus increasing the CD4:CD8 ratio (P < 0.01). CONCLUSION: FOS showed an immunostimulating effect on PP lymphocytes under healthy and endotoxemic conditions. Thus it can be concluded that FOS administration affects not only the large intestine but also the main inductive part of the mucosal immune system in the small intestine.     

Stimulation of butyrate production in the large intestine of weaning piglets by dietary fructooligosaccharides and its influence on the histological variables of the large intestinal mucosa

Fructooligosaccharides (FOS) reach the large intestine and are fermented into short-chain fatty acids (SCFA), lactate, and carbon dioxide. As the major energy source for the epithelial cells of the large intestine, n-butyrate stimulates the proliferation of cells as well as mineral and water absorption from the lumen. We examined the effect of dietary FOS supplementation on luminal SCFA production and its influence on the morphometrical variables of mucosa of the large intestine in commercially available pigs. Six weaning piglets were used. After 7 d of adaptation, three pigs were given a test diet containing FOS (10%) ad libitum for 10 d. The other three remained on the basal diet and were used as controls. At the end of the experiment, their large intestines were removed, and the cecum, gyri centripetales, gyri centrifugales, and rectum were separated. The contents of each portion were collected and measured for SCFA concentration, pH, and moisture. A micrometer was used to measure the crypt depth. The numbers of epithelial and mitotic cells in the crypt columns were also counted. The concentration of SCFA was significantly higher in piglets fed FOS than in the controls. The concentration of n-butyrate was markedly stimulated by FOS. The number of epithelial. mitotic, and mucin-containing cells was higher in piglets fed FOS than in the controls. Accordingly, the crypt depth was larger in the FOS-fed piglets. The luminal n-butyrate concentration showed a significantly positive correlation with the crypt depth and the number of epithelial, mitotic, and mucin-containing cells.     

The beneficial effect of the sap of Acer mono in an animal with low-calcium diet-induced osteoporosis-like symptoms

The sap of Acer mono has been called 'bone-benefit-water' in Korea because of its mineral and sugar content. In particular, the calcium concentration of the sap of A. mono is 37.5 times higher than commercial spring water. In the current study, we examined whether A. mono sap could improve or prevent osteoporosis-like symptoms in a mouse model. Male mice (3 weeks old) were fed a low-calcium diet supplemented with 25, 50 or 100 % A. mono sap, commercial spring water or a high calcium-containing solution as a beverage for 7 weeks. There were no differences in weekly weight gain and food intake among all the groups. Mice that were given a low-calcium diet supplemented with commercial spring water developed osteoporosis-like symptoms. To assess the effect of sap on osteoporosis-like symptoms, we examined serum calcium concentration, and femur density and length, and carried out a histological examination. Serum calcium levels were significantly lower in mice that received a low-calcium diet supplemented with commercial spring water (the negative control group), and in the 25 % sap group compared to mice fed a normal diet, but were normal in the 50 and 100 % sap and high-calcium solution groups. Femur density and length were significantly reduced in the negative control and 25 % sap groups. These results indicate that a 50 % sap solution can mitigate osteoporosis-like symptoms induced by a low-calcium diet. We also examined the regulation of expression of calcium-processing genes in the duodenum and kidney. Duodenal TRPV6 and renal calbindin-D9k were up-regulated dose-dependently by sap, and the levels of these factors were higher than those attained in the spring water-treated control. The results demonstrate that the sap of A. mono ameliorates the low bone density induced by a low-calcium diet, most likely by increasing calcium ion absorption.     

Dose-response assessment of the anti-cancer efficacy of soy isoflavones in dimethylhydrazine-treated rats fed 6% fructooligosaccharide

We investigated the combinatorial effects of different doses of dietary soy isoflavones (SI) and fructooligosaccharide (FOS) in a rat model of colon cancer. We hypothesized that increased bioavailability of SI metabolites due to dietary FOS may increase production of bioactive equol and affect colon carcinogenesis in a dose-dependent manner. Sprague-Dawley male rats were injected with 12-dimethylhydrazine (DMH) and were provided experimental diets that contained 0, 10, 50, 150, or 500 mg SI per kg of diet and 6% FOS for 12 weeks. The number of aberrant crypt foci (ACF) and the expression of cyclooxygenase-2 (COX-2) in colonic tissues were significantly decreased in the 6% FOS-fed groups compared to the control group. Gut transit time and fecal pH were significantly lower, and fecal concentrations of bifidobacteria were increased with 6% FOS. However, dietary SI supplementation in combination with 6% dietary FOS did not affect ACF formation or COX-2 expression. Plasma equol concentrations were dose-dependently increased by supplementation of SI up to 500 mg/kg of diet. In conclusion, SI supplementation up to 500 mg/kg of diet appeared to have no additive beneficial effects in rats with chemically-induced colon cancer that were fed 6% FOS, although plasma equol was dose-dependently increased.     

Fructooligosaccharide and soy isoflavone suppress colonic aberrant crypt foci and cyclooxygenase-2 expression in dimethylhydrazine-treated rats

This study investigated the inhibitory effects of soy isoflavones and fructooligosaccharide (FOS) on colon carcinogenesis. Sprague-Dawley male rats were injected with 1,2-dimethylhydrazine (DMH) and given experimental diets that contained 0%, 3%, 6%, or 9% FOS with or without soy isoflavones (1,000 mg/kg of diet). After 12 weeks, colonic aberrant crypt foci (ACF) formation, cyclooxygenase-2 (COX-2) expression, and fecal bile acid profiles were determined. The numbers of ACF, the numbers of ACF containing four or more crypts per focus of colonic mucosa, and the levels of COX-2 protein in the colonic epithelial tissues were significantly decreased in a dose-dependent manner in the FOS-fed, DMH-treated rats (P < .001), as compared to the DMH-treated control rats. Soy isoflavones significantly decreased the number of ACF with four or more aberrant crypts per focus (P < .001) and the amount of COX-2 protein (P < .01), independently of the effect of the oligosaccharide. The highest suppression of ACF formation was obtained with soy isoflavones combined with >or=6% FOS. No significant relationship was found between the dosage of FOS or soy isoflavones and the concentration of fecal secondary bile acid. We conclude that the combination of FOS and soy isoflavones inhibits colonic ACF formation and reduces COX-2 expression in DMH-treated rats.     

Kupffer cell activity is involved in the hepatoprotective effect of dietary oligofructose in rats with endotoxic shock

In the present study, we tested the hypothesis that dietary oligofructose (FOS) can modulate both the response to an endotoxic shock induced by lipopolysaccharide (LPS) administration and the activity of resident hepatic macrophages, i.e., Kupffer cells. Male Wistar rats (n = 5-9 per group) were fed a standard diet or a diet supplemented with 10 g/100 g FOS for 3 wk. LPS (10 mg/kg) or saline were injected i.p. after dietary treatment. After LPS injection, serum levels of tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine, and prostaglandin E(2) (PGE(2)), an immunosuppressive mediator, were higher in FOS-treated rats than in control rats. Alanine aminotransferase (ALT) activity was approximately 50% lower than in controls 24 h after LPS administration in FOS-treated rats, suggesting less hepatic injury; this was confirmed through histological analysis. FOS treatment increased the number of large phagocytic Kupffer cells, as assessed by histological examination of the liver after colloidal carbon injection into the portal vein. Precision-cut liver slices (PCLS) from FOS-treated rats released more TNF-alpha and PGE(2) into the incubation medium than PCLS from control rats, independently of LPS challenge in vitro. This would suggest that the higher Kupffer cell phagocytic activity and secretion capacity due to FOS supplementation improve LPS clearance in liver tissue and reduce hepatocyte alterations. This study supports the hypothesis that oligofructose might decrease liver tissue injury after endotoxic shock and sepsis.     

Enhancement effects of vitamin K1 (phylloquinone) or vitamin K2 (menaquinone-4) on intestinal alkaline phosphatase activity in rats

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters into inorganic phosphoric acid and alcohol at a high optimal pH, and is thought to play an important role in phosphate metabolism. Intestinal ALP, located at the brush border of intestinal epithelial cells, is known to be affected by several kinds of nutrients, but little is known about the physiological function of intestinal ALP Vitamin K is an essential cofactor for the post-translational carboxylation of glutamate residues into gamma-carboxy glutamate (Gla). Recently, novel functions of vitamin K have been clarified, but no data exist on the relation between vitamin K and intestinal ALP. The aim of this study was to examine the effects of both vitamin Ks (K1: phylloquinone, and K2: menaquinone) on ALP activity. Sprague-Dawley rats (6-wk-old) were divided into three groups: a control, phylloquinone (PK: 600 mg/kg diet), or menaquinone-4 (MK-4: 600 mg/kg diet) diet group. After 3 mo of feeding, we measured intestinal ALP activity by dividing it into five segments. In each segment, both PK and MK-4 increased intestinal ALP activity. The levels of intestinal ALP activity in the duodenum and proximal jejunum from the PK group were significantly higher than in the control group (p < 0.05). Moreover, the levels of intestinal ALP activity from the proximal jejunum and distal ileum of the intestine in the MK group were significantly higher than in the control group (p < 0.05). In this study, we clarified for the first time that both vitamin K1 and K2 as nutritional factors enhance intestinal ALP activity.     

EphA2与E—cadherin在大肠癌组织中的表达

目的探讨EphA2和E—cadhefin在大肠癌组织中的表达及其临床意义。方法采用免疫组织化学S-P方法检测67例大肠癌和28例正常大肠黏膜组织中EphA2和E—cadherin的表达,并分析其与临床病理因素的关系。结果大肠癌组织中EphA2阳性和E-cadhefin阴性表达均显著高于正常大肠黏膜组织（P〈0．01）。EphA2阳性和E-cadhefin阴性表达与大肠癌分化程度、浸润深度和淋巴结转移差异有统计学意义（P〈0．01或P〈0．05）,而与大肠癌大体类型差异无统计学意叉（P〉0．05）。EphA2与E-cadherin表达呈负相关（r=-0．45,P〈0．05）。结论EphA2与E—cadhefin异常表达可能共同参与大肠癌的发生、发展与转移,EphA2可与E-cadhefin结合作为判断大肠癌恶性程度和预后的指标。