Analysis of amino acid profiles of blood over time and biomarkers associated with non-alcoholic steatohepatitis in STAM mice.

The changes in free amino acid (AA) levels in blood during the progression from non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) are unclear. We investigated serum AA levels, along with biochemical and histological events, in a mouse model of NASH. We induced NASH in male C57BL/6J mice with a streptozotocin injection and high-fat diet after 4 weeks of age (STAM group). We chronologically (6, 8, 10, 12, and 16 weeks, n=4-12 mice/group) evaluated the progression from steatohepatitis to HCC by biochemical and histological analyses. The serum AA levels were determined using an AA analyzer. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in the STAM group than in the normal group (non-NASH-induced mice). Histological analysis revealed that STAM mice had fatty liver, NASH, and fibrosis at 6, 8, and 10 weeks, respectively. Moreover, the mice exhibited fibrosis and HCC at 16 weeks. The serum branched-chain AA levels were higher in the STAM group than in the normal group, especially at 8 and 10 weeks. The Fischer ratio decreased at 16 weeks in the STAM group, with increasing aromatic AA levels. These results suggested that this model sequentially depicts the development of fatty liver, NASH, cirrhosis, HCC, and AA metabolism disorders within a short experimental period. Additionally, serum amyloid A was suggested to be a useful inflammation biomarker associated with NASH. We believe that the STAM model will be useful for studying AA metabolism and/or pharmacological effects in NASH.

Daily Yogurt Consumption Improves Glucose Metabolism and Insulin Sensitivity in Young Nondiabetic Japanese Subjects with Type-2 Diabetes Risk Alleles.

This study investigated whether the association between postprandial plasma glucose (PPG) is affected by five type 2 diabetes mellitus (T2DM) susceptibility genes, and whether four weeks of yogurt consumption would affect these responses. We performed a single-arm intervention study in young nondiabetic Japanese participants, who consumed 150 g yogurt daily for four weeks, after which a rice test meal containing 50 g carbohydrate was administered. PPG and postprandial serum insulin (PSI) were measured between 0 and 120 mins at baseline and after the intervention. Genetic risk was evaluated by weighted genetic risk score (GRS) according to published methodology, and participants were assigned to one of two groups (n = 17: L-GRS group and n = 15: H-GRS group) according to the median of weighted GRS. At baseline, the H-GRS group had higher glucose area under the curve0⁻120 min after intake of the test meal than the L-GRS group (2175 ± 248 mg/dL.min vs. 1348 ± 199 mg/dL.min, p < 0.001), but there were no significant differences after the yogurt intervention. However, there was an improvement in PSI in the H-GRS group compared with baseline. These results suggest that habitual yogurt consumption may improve glucose and insulin responses in nondiabetic subjects who have genetically higher PPG.

The effect of fast eating on the thermic effect of food in young Japanese women.

The relationship between eating speed and the thermic effect of food (TEF) remains unclear. We investigated the difference in the TEF when meals containing the same amount of energy were eaten in 5 min (fast eating) or 15 min (regular eating). Subjects were nine non-obese young women. Following a 350 kcal (1464 kJ) meal, energy expenditure and autonomic nervous system activity were measured. The frequency of mastication was also calculated. The TEF for the 15-min period after the start of eating with fast eating was significantly lower than with regular eating (p < 0.01). There was a significant positive correlation between the low-frequency/high-frequency ratio and TEF at 5-min intervals up to 20 min after the start of eating and between total mastication frequency and TEF during ingestion. Fast eating may reduce the TEF, potentially because a decrease in mastication frequency decreases sympathetic nervous system activity.

Evaluation of postprandial glucose excursion using a novel minimally invasive glucose area-under-the-curve monitoring system.

OBJECTIVE: To develop a minimally invasive interstitial fluid extraction technology (MIET) to monitor postprandial glucose area under the curve (AUC) without blood sampling, we evaluated the accuracy of glucose AUC measured by MIET and compared with that by blood sampling after food intake. METHODS: Interstitial fluid glucose AUC (IG-AUC) following consumption of 6 different types of foods was measured by MIET. MIET consisted of stamping microneedle arrays, placing hydrogel patches on the areas, and calculating IG-AUC based on glucose levels in the hydrogels. Glycemic index (GI) was determined using IG-AUC and reference AUC measured by blood sampling. RESULTS: IG-AUC strongly correlated with reference AUC (R = 0.91), and GI determined using IG-AUC showed good correlation with that determined by reference AUC (R = 0.88). CONCLUSIONS: IG-AUC obtained by MIET can accurately predict the postprandial glucose excursion without blood sampling. In addition, feasibility of GI measurement by MIET was confirmed.

Norlichexanthone isolated from fungus P16 promotes the secretion and expression of adiponectin in cultured ST-13 adipocytes.

Adiponectin, an adipose-derived protein, shows insulin-sensitizing, anti-diabetic and anti-atherogenic activities, which implies that the protein represents a potential target to improve lifestyle-related diseases like type 2 diabetes. Based on our hypothesis that agents that cause adipocyte differentiation could also act as adiponectin secretion enhancers, we screened butanol extracts of 96 fungus culture extracts for their differentiation-inducing activity in ST-13 preadipocytes. We found that the butanol extract of a fungus P16 culture extract possessed such an activity, and isolated norlichexanthone as an active compound through activity-guided fractionation. Oil red O staining showed that norlichexanthone induced adipogenesis in ST-13 cells. Its differentiation-inducing activity was supported by the observation that norlichexanthone dose-dependently increased the mRNA expression of fatty acid-binding protein and peroxisome proliferator activated receptor γ (PPARγ), markers of adipocyte differentiation. Western blot analysis demonstrated that the compound enhanced the secretion of adiponectin protein in a dose-dependent manner. An increase in mRNA expression of adiponectin was also observed in the norlichexanthone-treated ST-13 cells. Actinomycin D treatment blocked the enhancement of adiponectin mRNA expression by norlichexanthone, suggesting that it is the result of increased transcription. A luciferase reporter assay indicated that norlichexanthone was unlikely to be an agonist of PPARγ, implying that its action of mechanism might differ from those of thiazolidinediones which upregulate adiponectin expression via activation of PPARγ. These findings suggest the possibility that norlichexanthone has the potential to treat and/or prevent lifestyle-related diseases, including metabolic syndrome, type 2 diabetes, atherosclerosis and cardiovascular diseases.

Possible regulatory factors for intra-abdominal fat mass in a rat model of Parkinson's disease.

OBJECTIVE: Patients with Parkinson's disease (PD) lose body weight primarily due to decreased body fat mass. The purpose of this study was to elucidate possible factors related to reduction in the intra-abdominal fat mass of 6-hydroxydopamine (6-OHDA)-treated rats, which are frequently used as an animal model for PD. METHODS: Sham-operated (NPD: n = 4) and unilaterally 6-OHDA-injected (PD: n = 4) 14-wk-old male Sprague-Dawley rats were fed a relatively high-fat diet for 2 wk, during which food intake and body weight were measured. After the 2-wk feeding period, intra-abdominal fat was dissected out and weighed. Carbohydrate and fat absorption-related gene expressions in the jejunum and serum insulin and glucose concentrations were analyzed. RESULTS: Although final body weights did not differ, total intra-abdominal fat weight, expressed relative to body weight, was significantly lower in the PD group than in the NPD group (P < 0.05). There were no significant differences between the two groups in the mRNA expression of carbohydrate and fat digestion/absorption-related genes in the jejunum, or in fat absorption efficacy assessed by fecal fat excretion. However, PD rats showed significantly lower serum insulin and higher glucose concentrations than NPD rats (P < 0.05). CONCLUSION: PD model rats displayed loss of intra-abdominal fat, similar to the progressive loss of fat in PD patients. Our results provide preliminary evidence that reduced lipogenesis due to lower insulin levels, rather than impaired digestion/absorption, might have been involved in this decrease in intra-abdominal fat mass.

Nobiletin, a citrus polymethoxyflavonoid, suppresses multiple angiogenesis-related endothelial cell functions and angiogenesis in vivo.

Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration-dependent inhibitory effects on multiple functions of angiogenesis-related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro-matrix metalloproteinase-2 (proMMP-2) production and MMP-2 mRNA expression in ECGS-stimulated HUVEC. Nobiletin also downregulated cell-associated plasminogen activator (PA) activity and urokinase-type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis-related signaling molecules, such as extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase, and transcriptional factors (c-Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID(50) value being 10µg (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions.

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.

Identification of nobiletin, a polymethoxyflavonoid, as an enhancer of adiponectin secretion.

Adiponectin, an adipocyte-derived protein with insulin-sensitizing, anti-diabetic and anti-atherogenic activities, is known to be induced during adipocyte differentiation. Nobiletin, a citrus polymethoxy flavonoid, was found to induce the differentiation of ST-13 preadipocytes into mature adipocytes and enhance the production of adiponectin protein at a concentration of 10 microM.

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.

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.

The critical period for thyroid hormone responsiveness through thyroid hormone receptor isoform alpha in the postnatal small intestine.

During second and third weeks after birth in rats, serum thyroid hormone level is elevated. In this study, we investigated the jejunal expression of thyroid hormone receptor (TR) alpha in developing rats. The TRalpha-1 mRNA level and TRalpha-1/TRalpha-2 mRNA ratio increased two-fold from 5 to 13 days after birth. This high level of TRalpha-1 mRNA was maintained until 20 days and then decreased to the basal level by the end of weaning period at 27 days; however, the level of TRalpha-2 mRNA remained unchanged throughout the developmental period. The increase in the TRalpha-1/TRalpha-2 mRNA ratio from 5 to 13 days was accompanied by an initial rise in the levels of mRNA for hexose transporters in the jejunum. Administration of T(3) during the suckling period (8-13 days) caused a 50% increase in the TRalpha-1/TRalpha-2 mRNA ratio, while administration of T(3) on days 12-17 and days 16-21, but not on days 22-27, caused a two to four-fold increase in the levels of mRNA for hexose transporters. These results suggest that a transient variation in the TRalpha-1/TRalpha-2 expression ratio is closely related to the critical period of thyroid hormone responsiveness for hexose transporters expression in the developing rat jejunum.

Dipalmitoylation of radicicol results in improved efficacy against tumor growth and angiogenesis in vivo.

Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.

The possible roles of homeobox protein, Cdx-2 for the expression of LPH gene during postnatal development.

The expression of intestinal lactase-phlorizin hydrolase (LPH) gene normally decreases after completion of weaning in almost all mammals. To elucidate the mechanism whereby LPH gene expression is regulated during the suckling-weaning period, we studied the effects of the thyroid hormone (T(3)) on LPH gene expression in the small intestine during postnatal development in the rat. Firstly, we measured LPH mRNA level in rat jejunum at 5, 13, 20 and 27 days after birth. The amount of LPH mRNA at 27 days was significantly lower than that at 5 days. The transcript level of Cdx-2, which is a putative transcriptional factor for regulation of LPH gene expression, was also significantly decreased after 21 days. The binding of nuclear protein to the cis element CE-LPH1 on the promoter region of the LPH gene was reduced at the end of the weaning period. Daily intraperitoneal (i.p.) injection of T(3) for 6 days during days 22-27 significantly reduced LPH mRNA level by day 27 (50%, P<0.01), but injection of T(3) during days 8-13 did not. Moreover, i.p. T(3) injection during days 22-27 was accompanied by a reduction in the level of Cdx-2 mRNA. Our study suggests that the decrease in the LPH gene expression during the weaning period is associated with a reduction of Cdx-2 expression caused by thyroid hormone.

Dietary sucrose enhances intestinal lactase gene expression in euthyroid rats.

It is postulated that dietary carbohydrates and thyroid hormones are major regulators for expression of the lactase/phlorizin hydrolase (LPH) gene in rat jejunum. In this study, we investigated the effects of thyroid hormones and dietary sucrose on LPH gene expression and lactase activity in starved rats. Firstly, animals at 8 wk of age were fed a low-starch diet (5.5% energy as cornstarch) or high-starch diet (71% energy as cornstarch) for 7 d (experiment 1). The mRNA level of LPH as well as lactase activity significantly decreased in rats fed the low-starch diet as compared to those fed the high-starch diet. To investigate the effects of thyroid hormone status, the animals previously fed the low-starch diet were starved for 3 d, and half of the animals were given intraperitoneal (i.p.) injections of 20 microg/ 100 g body weight triiodothyronine (T3) twice daily (experiment 2). The LPH mRNA level and lactase activity were elevated by starvation for 3 d, but they were repressed by the injection of T3 during starvation. To investigate the effects of dietary sucrose in starved rats, they were force-fed a sucrose diet for 6 h (experiment 3). The LPH gene expression and lactase activity were up-regulated by force-feeding a sucrose diet, only when the animals were kept in euthyroid status by daily T3 administrations. In contrast, the sucrase-isomaltase mRNA levels and sucrase activity were unaffected by force-feeding the sucrose diet for both T3-treated and untreated starved rats. Our work suggests that dietary sucrose is capable of enhancing lactase gene expression in starved rats when they have a sustainable thyroid hormone level.