The Effects of Overnight Fasting Duration on Glucose and Lipid Metabolism in a Sprague-Dawley Rat Model of Nonalcoholic Steatohepatitis with Advanced Fibrosis.

Nonalcoholic steatohepatitis (NASH) can progress to hepatic fibrosis, and is associated with cardiovascular and liver-related mortality. To understand the pathogenesis of NASH, reliable animal models of the disease are useful. In animal studies, the animals are usually fasted overnight before biospecimens are taken, but little is known about the effects of fasting. Here, we investigated the impact of overnight fasting for approximately 9 to 17 h on glucose and lipid metabolism in a Sprague-Dawley (SD) rat model of diet-induced moderate and advanced NASH in comparison to normal SD rats. Our results revealed that in the moderate NASH model rats, the fasting duration did not affect glucose and lipid metabolism, the histopathological findings, or the hepatic mRNA expression levels of genes related to lipid metabolism, cholesterol metabolism, inflammation, fibrosis, and oxidative stress. In contrast, in the normal rats, significant fasting time-dependent reductions were observed in the epididymal fat pad weight and the hepatic mRNA expression levels of adipose differentiation-related protein and heme oxygenase-1. Moreover, in the advanced NASH model rats, a significant fasting time-dependent reduction and increase were observed in the serum insulin level and mRNA expression level of alpha-smooth muscle actin, respectively. Our present results suggest that the influence of the overnight fasting duration differs among the healthy condition, moderate NASH, and advanced NASH statuses. Further studies are needed in humans to determine the appropriate overnight fasting duration for the accurate evaluation of glucose and lipid metabolism in NASH patients.

Influence of Fasting Time on Serum and Hepatic Lipid Profiles in a Sprague-Dawley Rat Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is associated with several cardiovascular risk factors, including atherogenic dyslipidemia. Recently, fasting prior to lipid profile evaluation has been thought to be unnecessary for most individuals. We investigated the impact of fasting for up to 9 h on the serum and hepatic lipid profiles in Sprague-Dawley (SD) rats of dietary-induced NASH model in comparison to SD rats fed a normal diet. In both groups, fasting affected the serum and hepatic triglyceride (TG), serum free fatty acid (FFA) and leptin levels, histopathologically assessed hepatocyte ballooning, and hepatic mRNA expression levels of several genes related to lipid metabolism. In contrast, the serum adiponectin and aminotransferase levels, serum and hepatic total cholesterol contents, and liver histopathological findings of hepatic steatosis, lobular inflammation and fibrosis were not influenced by fasting. A significant fasting time-dependent reduction was seen in the serum TG level only in the normal SD rats group. Regarding the hepatic TG level, a significant fasting time-dependent increase was seen only in the NASH model rat group. A significant fasting time-dependent reduction was also seen in the serum FFA level only in the NASH model rat group. Our present results indicate that excessive fasting can be avoided before blood or hepatic tissue sampling for the evaluation of several parameters in non-NASH and/or NASH model rats. Further investigations are needed in humans to determine whether excessive fasting before blood or hepatic tissue sampling can be avoided in both healthy individuals and NASH patients.

Dietary fat, cholesterol, and cholic acid affect the histopathologic severity of nonalcoholic steatohepatitis in Sprague-Dawley rats.

Understanding of the pathogenesis of nonalcoholic steatohepatitis (NASH)-associated fibrosis has been hampered by the lack of a comprehensive and physiological small animal model of NASH with fibrosis. Feeding a high-fat and high-cholesterol (HFC) diet supplemented with cholic acid to rats is known to replicate human NASH pathology, and it induces fibrosis earlier than with an HFC diet alone. In the present study, physiological and histopathological observations from 65 Sprague-Dawley (SD) rats fed an HFC diet with or without cholic acid for 9 or 18 weeks in our laboratory between January 2013 and February 2018 were retrospectively reviewed. The liver weight/body weight ratio at the end of the rearing period was higher in rats fed an HFC diet than in rats fed a normal diet in a cholesterol dose-, cholic acid dose-, or rearing period dependent manner. Dietary fat, cholesterol and/or cholic acid and rearing period affected the histopathologic severity of NASH. Overall, 56 (86.2%) of 65 SD rats fed an HFC diet for 9 or 18 weeks developed histopathologically proven NASH. It is noted that the SD rats fed an HFC diet supplemented with 2% (w/w) cholic acid for 18 weeks frequently developed advanced fibrosis, including cirrhosis. Thus, this diet-induced NASH rat model is likely to be a highly reproducible.

Age-Related Alterations of Nonalcoholic Steatohepatitis in Sprague-Dawley Rats Fed a High-Fat and High-Cholesterol Diet.

Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), has a potentially progressive course that can lead to liver cirrhosis. Age is strongly associated with the development and progression of NAFLD/NASH, but the natural history of pediatric NAFLD is still not fully understood. Here, we evaluated the age-related alterations of NASH in 5-, 9- and 13-wk-old male Sprague-Dawley rats that were fed a high-fat and high-cholesterol diet (30% fat, 1.25% cholesterol and 0.5% sodium cholate, w/w) for 9 wk (6 rats/group). Our results showed that the cumulative energy intake, body weight gain and food efficacy during the 9-wk rearing period were highest in the youngest group and lowest in the oldest group. Serologically, almost all parameters including the serum triglyceride and total cholesterol were similar regardless of age. Histopathological findings, such as hepatic steatosis, lobular inflammation and hepatocyte ballooning, were also similar regardless of age, but hepatic fibrosis was more evident in the oldest group. Also, the mRNA expression levels of some fibrogenic, inflammatory, oxidative stress and cholesterol or lipid metabolism-related genes in the liver were highest in the oldest group and lowest in the youngest group, although the difference was not statistically significant. These results indicated that aging is likely associated with the development of NASH. Because the cumulative energy intake and daily food intake/body weight were not similar among groups in the present study, further studies designed with an equivalent daily food intake/body weight among groups are needed in order to interpret the exact nutritional effect.

Dietary ß-Conglycinin Modulates Insulin Sensitivity, Body Fat Mass, and Lipid Metabolism in Obese Otsuka Long-Evans Tokushima Fatty (OLETF) Rats.

The physiological effects of dietary ß-conglycinin (ß-CON), one of the major components of soy protein (SOY), were examined in an obese animal model. Prior studies show that ß-CON intake decreases plasma triglycerides and visceral adipose tissue weight, and increases plasma adiponectin in rodents. Since plasma adiponectin is known to affect both lipid and glucose metabolism, feeding a diet containing ß-CON could modulate insulin sensitivity. Therefore, we examined the effects of dietary ß-CON on insulin sensitivity and blood glucose levels, as well as lipid metabolism in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats (pre-symptomatic stage of type 2 diabetes mellitus). Male OLETF rats (6 weeks old) were fed diets containing 20% protein such as casein (CAS), CAS replaced with soy protein (SOY), or ß-CON at a proportion of 50% for 13 weeks. Fasting blood glucose levels were measured every 3 weeks, and an insulin tolerance test (ITT; 0.75 IU/kg body weight) was conducted at week 12. During the feeding period, fasting blood glucose was comparable among the groups. Insulin sensitivity measured by the ITT revealed that the SOY and ß-CON diets decreased blood glucose levels at 30 min after intraperitoneal insulin injection (vs. CAS diet). In addition, the ß-CON diet increased plasma adiponectin concentrations, hepatic gene expression of insulin receptor substrate (IRS) 2, and muscle gene expression of adiponectin receptor 1 (AdipoR1) and IRS1, and with a decrease in plasma insulin concentration. Finally, the ß-CON diet decreased the mesenteric adipose tissue weight and liver triglyceride concentration compared to the CAS diet. These results suggest that the metabolic effects of dietary ß-CON are mediated by increasing plasma adiponectin to increase insulin sensitivity and influence the hepatic lipid metabolism in obese OLETF rats.

A fermented mixed tea made with camellia (Camellia japonica) and third-crop green tea leaves prevents nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet.

BACKGROUND: Established treatments for non-alcoholic steatohepatitis (NASH) are few, thus it is imperative to develop novel dietary strategies that can prevent NASH. A fermented mixed tea (FMT) made with Camellia japonica (Japanese camellia) and third- crop green tea leaves by tea-rolling processing was reported to reduce body weight and adipose tissue weight in Sprague-Dawley (SD) rats. Because visceral fat is one of the most important factors for the development of hepatic steatosis, this FMT supplementation can be a candidate dietary strategy for the prevention of NASH. METHODS: Nine-week-old male SD rats were fed a high-fat and high-cholesterol (HFC) diets with or without FMT (camellia and third-crop green tea leaves at ratios of 1:5, 1:2 and 1:1) for 9 weeks (n=6-7/group). Histopathology, serology and expressions of fibrogenetic, proinflammatory, oxidative stress and lipid metabolism-related genes in the liver were evaluated. RESULTS: Histologically, HFC diet with FMT at a ratio of 1:5 dramatically reduced NASH progression (14%) compared to the HFC diet without FMT (100%). FMT at a ratio of 1:5 reduced hepatic steatosis due to the activation of microsomal triglyceride transfer protein, and FMT at a ratio of 1:2 reduced mRNA levels of some proinflammatory, lipid metabolism-related, fibrogenic and oxidative stress marker genes. CONCLUSIONS: Our data suggest that FMT at a ratio of 1:5 or 1:2 likely possesses a preventive effect on NASH progression.

A diet-induced Sprague-Dawley rat model of nonalcoholic steatohepatitis-related cirrhosis.

Certain modified diets containing saturated fatty acids, cholesterol or fructose lead to the development of nonalcoholic steatohepatitis (NASH)-related fibrosis in rodents; however, progression to cirrhosis is rare. Experimental liver cirrhosis models have relied on genetic manipulation or administration of hepatotoxins. This study aimed to establish a reliable dietary model of NASH-related cirrhosis in a relatively short period. Male Sprague-Dawley rats (9 weeks of age) were randomly assigned to normal, high-fat (HF), or two types (1.25% or 2.5% cholesterol) of high-fat and high-cholesterol (HFC) diets for 18 weeks. All HFC diets contained 2% cholic acid by weight. Histopathological analysis revealed that the HFC diets induced obvious hepatic steatosis, inflammation with hepatocyte ballooning and advanced fibrosis (stage 3-4) in all 12 rats at 27 weeks of age. In contrast, all five rats given the HF diet developed mild steatosis and inflammation without fibrosis. The amount of cholesterol in the liver and hepatocellular mitochondrial and microsomal fractions was significantly higher in rats fed the HFC diets than the normal or HF diets. The HFC diets significantly suppressed mRNA levels of microsomal triglyceride transfer protein, adenosine triphosphate binding cassette transporter G5, bile acid CoA: amino acid N-acyltransferase and bile salt export pump, as well as the enzymatic activity of carnitine palmitoyltransferase in the liver. In conclusion, the HFC diets induced liver cirrhosis in conjunction with hepatic features of NASH in Sprague-Dawley rats within 18 weeks, and altered gene expression and enzyme activity to accumulate lipid and bile acid in the liver.

High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague-Dawley rats.

AIM: The development of fibrosis is considered an important phase in the progress of non-alcoholic steatohepatitis (NASH) towards the end stage of liver disease, including cirrhosis. However, few small animal models can display NASH-associated fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis using genetically normal rats. METHODS: Nine-week-old male Sprague-Dawley rats were fed with normal, high-fat (HF), or two types of high-fat and high-cholesterol (HFC) diets for 9 weeks (n = 5 each). All HFC diets contained 1.25% or 2.5% cholesterol. RESULTS: The rats fed with the HF diet developed mild steatosis and inflammation without fibrosis at 18 weeks of age, whereas all rats given the HFC diet developed obvious steatosis and inflammation with hepatocyte ballooning and fibrosis. Two of five (40%) rats given the HFC diet containing 2.5% cholesterol progressed to liver cirrhosis. Hepatic total cholesterol levels were significantly higher in rats given the HFC, than the normal or HF diets. The HFC diet significantly and dose-dependently decreased microsomal triglyceride transfer protein expression. Cholesterol tended to suppress carnitine palmitoyltransferase activity and adenosine triphosphate-binding cassette transporter G5 expression. Adding cholesterol to the HF diet modified hepatic lipid metabolism at the molecular level. CONCLUSION: The HFC diet induced hepatic features of NASH and eventually progressed cirrhosis in Sprague-Dawley rats within 9 weeks.

Competitive regulation of human intestinal ß-carotene 15,15'-monooxygenase 1 (BCMO1) gene expression by hepatocyte nuclear factor (HNF)-1α and HNF-4α.

AIM: Among the pro-vitamin A carotenoids, ß-carotene is an excellent source of vitamin A. ß-Carotene 15,15'-monooxygenase 1 (BCMO1) is a critical enzyme involved in the conversion of ß-carotene into vitamin A (retinal) in the small intestine of many vertebrates. In the present study, we investigated the regulation of human BCMO1 gene expression using human intestinal Caco-2 BBe cells. MAIN METHODS: We performed electrophoretic mobility shift assays and chromatin immunoprecipitation assays to investigate the binding properties of hepatocyte nuclear factor (HNF)-1α and HNF-4α to the proximal promoter of the human BCMO1 gene. Caco-2 BBe cells were also transfected with HNF-1α and HNF-4α siRNAs, and BCMO1 gene expression levels and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and luciferase reporter assays, respectively. KEY FINDINGS: We identified overlapping binding sites for HNF-1α and HNF-4α in the human BCMO1 gene proximal promoter. Endogenous nuclear HNF-1α and HNF-4α proteins competitively bound these sites in Caco-2 BBe cells. BCMO1 gene expression levels and promoter activity were significantly decreased in HNF-1α siRNA-transfected Caco-2 BBe cells. In contrast, HNF-4α siRNA-transfected cells exhibited a significant increase in BCMO1 gene expression and promoter activity. Mutation of these overlapping binding sites dramatically decreased BCMO1 promoter activity. SIGNIFICANCE: Our study indicates that the competitive actions of HNF-1α and HNF-4α on their overlapping binding sites in the human BCMO1 gene promoter oppositely regulate BCMO1 gene expression in the human small intestine.

Hypolipidemic potential of squid homogenate irrespective of a relatively high content of cholesterol.

BACKGROUND: Our previous study has shown that regardless of a relatively high amount of cholesterol, squid homogenate lowers serum and hepatic cholesterol in animals. Since this work, we have developed a new method to inhibit autolysis of squid proteins with sodium citrate. This study aims to investigate how squid homogenate prepared with sodium citrate affects lipid metabolism in Sprague-Dawley rats at the molecular level. METHODS: We prepared squid homogenate with sodium citrate to inhibit autolysis of squid protein. In Experiment 1 (Exp. 1), rats were given a cholesterol-free control diet or a squid diet, with squid homogenate added at the level of 5% as dietary protein for 4 weeks. Blood, the liver and adipose tissue were taken after 6 hours fasting. Serum and hepatic lipids and activities of enzymes related to lipid metabolism were measured. In Experiment 2 (Exp. 2), the above-mentioned diets had cholesterol added at the level of 0.1% and given to rats. Lipid parameters, enzyme activities, and gene expression of proteins involved in lipid metabolism in the liver and the small intestine were determined. In addition, feces were collected for two days at the end of Exp. 2 to measure fecal excretion of steroids. RESULTS: In Exp.1, serum triglyceride and cholesterol were ~50% and ~20% lower, respectively, in the squid diet-fed rats than in the control diet-fed animals while hepatic cholesterol was ~290% higher in the squid diet-fed rats. When cholesterol was included into the diets (Exp. 2), serum lipids were significantly lower in the squid group while no difference of hepatic lipid was seen between two groups. Activities of hepatic lipogenic enzymes were significantly lower in rats on the squid diet while the enzyme responsible for fatty acid oxidation was not modified (Expt. 1 and 2). Hepatic level of mRNA of microsomal triglyceride transfer protein was significantly lower in the squid group. In the small intestine, the squid diet exhibited significantly lower gene expression of proteins involved in fatty acid transport and cholesterol absorption. Fecal secretion of acidic steroids, but not neutral steroids, was higher in rats fed the squid diet than in those fed the control diet. CONCLUSION: These results imply that newly-developed squid homogenate has hypolipidemic potential primarily through decreased absorption of bile acids in the small intestine and suppressed lipogenesis in the liver.

Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein α in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.

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 α-glucosidase activities along the jejunal-ileal axis of normal rats by the α-glucosidase inhibitor miglitol.

Miglitol, an α-glucosidase inhibitor that inhibits postprandial hyperglycemia by delaying carbohydrate digestion and absorption along the jejunal-ileal axis, has recently been approved for use in patients with type 2 diabetes mellitus. Miglitol treatment may lead to increased α-glucosidase activities toward the ileum because carbohydrate flow toward the ileum increases. However, it is not yet known if miglitol treatment alters the α-glucosidase activities along the jejunal-ileal axis. In this study, we examined the effects of miglitol supplementation for 3 or 7 days on α-glucosidase activities along the jejunal-ileal axis of Wistar rats. Supplementation with miglitol for 3 or 7 days in rats increased tissue weights of the lower jejunum and ileum, but did not alter tissue weights of the upper jejunum and cecum or the contents of the cecum. Furthermore, supplementation with miglitol for 7 days reduced the activities of isomaltase and maltase in the upper jejunum and increased the activities of sucrase, isomaltase, and maltase in the lower jejunum and ileum. These results suggest that the delay in carbohydrate digestion and absorption along the jejunal-ileal axis by miglitol supplementation in rats is associated with increased α-glucosidase activities toward the ileum.

Regulation of intestinal Cl-/HCO3- exchanger SLC26A3 by intracellular pH.

SLC26A3, a Cl(-)/HCO(3)(-) exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na(+)/H(+) exchanger and Cl(-)/HCO(3)(-) exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pH(i)). We therefore investigated whether SLC26A3 is regulated by pH(i). We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy-N-ethylquinolinium iodide (diH-MEQ). pH(i) was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl(-)/HCO(3)(-) and Cl(-)/NO(3)(-) exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl(-)/HCO(3)(-) exchange was steeply inhibited with pH(i) decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl(-)/NO(3)(-) exchange was essentially insensitive to pH(i) within physiological ranges. We also characterized the pH(i) dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pH(i) sensitivity. These results suggest that Cl(-)/HCO(3)(-) exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na(+)/H(+) exchanger 3 (NHE3) through the alteration of pH(i) under physiological conditions.

Hepatocyte nuclear factor-4alpha regulates human cellular retinol-binding protein type II gene expression in intestinal cells.

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4alpha expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4alpha significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4alpha protein levels by HNF-4alpha small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4alpha protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4alpha protein levels, HNF-4alpha protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4alpha is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4alpha in the regulation of human intestinal vitamin A absorption and metabolism.

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.

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells.

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRalpha1 and TRbeta1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T3 treatment in medium supplemented with fetal bovine serum. Furthermore, T3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T3 is an important hormone for the regulation of vitamin A and beta-carotene metabolism-related gene expression in human small intestinal cells.

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.

Effects of miglitol, an alpha-glucosidase inhibitor, on glycaemic status and histopathological changes in islets in non-obese, non-insulin-dependent diabetic Goto-Kakizaki rats.

Miglitol, a 1-deoxynojirimycin derivative, is an alpha-glucosidase inhibitor. In the present study, the effects of acute (single-dose) and chronic (8-week) oral administration of miglitol in Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes, were investigated. Dose-dependent decreases in incremental blood glucose concentrations integrated over a period of 2 h (deltaAUC0-2 h) for values of blood glucose after sucrose-loading in miglitol-treated GK rats were observed following an acute oral administration of miglitol (1, 3 or 10 mg/kg body weight). At 10 mg/kg, the deltaAUC0-2 h of blood glucose was decreased by 45 % compared with the control group. Following the oral administration of miglitol in a dietary mixture (10 mg, 20 mg or 40 mg miglitol/100 g control diet) for 8 weeks, the ratio of HbA1c at 8 weeks compared with 0 weeks in GK rats treated with 40 mg miglitol/100 g control diet miglitol was significantly decreased compared with control GK rats without changes in body weight. In oral glucose tolerance testing, miglitol caused a slight decrease in the deltaAUC0-2 h of plasma glucose concentration. In addition, miglitol treatment slightly inhibited the reduction in beta-cell mass, and lessened the irregular contours and fibrosis of the islets in GK rats. These results indicate that miglitol ameliorates the hyperglycaemic state of GK rats and the impaired function of the pancreatic islets, as well as preventing the degeneration of islets in GK rats.