Active-phase Plasma Alkaline Phosphatase Isozyme Activity Is a Sensitive Biomarker for Excessive Fructose Intake.

BACKGROUND/AIM: Excessive fructose intake reportedly leads to the development of nonalcoholic fatty liver disease (NAFLD). In our previous study, we reported that plasma activities of alkaline phosphatase (ALP) isozymes were markedly changed in rats with excessive fructose intake-induced hepatomegaly. In this study, we examined ALP isozyme activity prior to the occurrence of hepatomegaly, and investigated the effect of the timing of sample collection, to explore its potential as a biomarker. MATERIALS AND METHODS: After 1-week intake of a 63% high-fructose diet (HFrD), blood samples were collected from male rats during sleep or active phases to analyze biochemical parameters. RESULTS: Body and liver weights were similar between the HFrD and control diet groups, indicating that hepatomegaly due to excessive fructose intake had not occurred. The triglyceride levels and glutamate dehydrogenase (GLDH) activity were significantly elevated to similar degrees at both time points. HFrD intake significantly increased liver-type ALP (L-ALP) activity, stimulating it by 12.7% at the sleep phase and by 124.3% at the active phase. HFrD consumption also significantly decreased intestinal-type ALP (I-ALP) at the active phase, but only showed a decreasing trend during the sleep phase. CONCLUSION: Measurements of plasma ALP isozyme and GLDH activity, and triglyceride levels are effective early biomarkers of impending NAFLD caused by excessive fructose intake. L-ALP and I-ALP activities during the active phase are particularly sensitive for detection of excessive fructose intake before the occurrence of NAFLD.

Novel Biomarker Establishment for Evaluation of Excessive Fructose Consumption Using a Rat Model.

BACKGROUND/AIM: The habitual consumption of excessive fructose is associated with the onset and progression of lifestyle-related diseases, such as nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the physiological changes observed when consuming a diet containing excessive fructose on the viewpoints of hepatotoxicity biological markers using a rat model and explored the biomarker candidates that could detect the effects of excessive fructose intake at an early stage. MATERIALS AND METHODS: Male rats were fed 63% high fructose diet (HFrD) ad libitum and their blood samples were collected before and at 1, 2, 3, and 4 weeks after allocation. The plasma biochemical parameters, hepatotoxic enzyme activities including alkaline phosphatase (ALP) isozymes were analyzed. RESULTS: HFrD consumption for 4-weeks created NAFLD-like symptoms, including elevated plasma lipid parameters and hepatotoxicity markers, as well as fat accumulation in the liver compared with rats consuming a control diet. Alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) were increased from the 3rd and 2nd weeks, respectively, but no changes were observed on ALP activity. However, the daily consumption of the HFrD increased the plasma activities of liver-type ALP isozyme, and decreased plasma small intestinal-type ALP isozyme soon after the start of feeding. CONCLUSION: ALP isozyme analysis in combination with GLDH and ALT activities in the plasma samples could be a useful tool to detect the physiological changes induced by excessive fructose intake at an early stage of the development of NAFLD.

Acyl-CoA:diacylglycerol acyltransferase 1 inhibition in the small intestine increases plasma transaminase activity via the activation of protein kinase C pathway.

Acyl-CoAdiacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the fat absorption step in enterocytes. We previously reported that the pharmacological inhibition of DGAT1 increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in corn oil-loaded rats without any sign of hepatotoxicity. In this study, we investigated this mechanism. We found that this elevation occurred only during the pharmacologically active period of a DGAT1 inhibitor and the magnitude did not depend on the volume of corn oil. In addition, this elevation was not accompanied by increases in ALT or AST mRNA levels in the small intestine and liver. To clarify a lipid component responsible for this elevation, rats were treated with free fatty acids instead of corn oil and no plasma ALT elevation was observed. Next, rats were pretreated with inhibitors of monoacylglycerol acyltransferase 2 and intestinal microsomal triglyceride transfer protein instead of the DGAT1 inhibitor, but no plasma ALT elevation was observed after corn oil loading. Since the results suggested a possible role of diacylglycerol (DAG), which activates protein kinase C (PKC), we measured PKC activity in the small intestine and found that the activity was increased by treatment with the DGAT1 inhibitor and corn oil. Moreover, rats pretreated with a PKC inhibitor in combination with the DGAT1 inhibitor showed suppression of plasma ALT elevation. Taken together, the present results suggest that DAG accumulation induced by pharmacological DGAT1 inhibition and resultant PKC activation in enterocytes are involved in the increase in plasma ALT and AST activity in rats.

Sensor-Based Human Activity Recognition Using Adaptive Class Hierarchy.

In sensor-based human activity recognition, many methods based on convolutional neural networks (CNNs) have been proposed. In the typical CNN-based activity recognition model, each class is treated independently of others. However, actual activity classes often have hierarchical relationships. It is important to consider an activity recognition model that uses the hierarchical relationship among classes to improve recognition performance. In image recognition, branch CNNs (B-CNNs) have been proposed for classification using class hierarchies. B-CNNs can easily perform classification using hand-crafted class hierarchies, but it is difficult to manually design an appropriate class hierarchy when the number of classes is large or there is little prior knowledge. Therefore, in our study, we propose a class hierarchy-adaptive B-CNN, which adds a method to the B-CNN for automatically constructing class hierarchies. Our method constructs the class hierarchy from training data automatically to effectively train the B-CNN without prior knowledge. We evaluated our method on several benchmark datasets for activity recognition. As a result, our method outperformed standard CNN models without considering the hierarchical relationship among classes. In addition, we confirmed that our method has performance comparable to a B-CNN model with a class hierarchy based on human prior knowledge.

Enhancement of acetaminophen-induced chronic hepatotoxicity in spontaneously diabetic torii (SDT) rats.

Some patients encounter hepatotoxicity after repeated acetaminophen (APAP) dosing even at therapeutic doses. In the present study, we focused on the diabetic state as one of the suggested risk factors of drug-induced liver injury in humans and investigated the contribution of accelerated gluconeogenesis to the susceptibility to APAP-induced hepatotoxicity using an animal model of type 2 diabetes patients. Sprague-Dawley (SD) rats and spontaneously diabetic torii (SDT) rats were each given APAP at 0 mg/kg, 300 and 500 mg/kg for 35 days by oral gavage. Plasma and urinary glutathione-related metabolites, liver function parameters, and hepatic glutathione levels were compared between the non-APAP-treated SDT and SD rats and between the APAP-treated SDT and SD rats. Hepatic function parameters were not increased at either dose level in the APAP-treated SD rats, but were increased at both dose levels in the APAP-treated SDT rats. Increases in hepatic glutathione levels attributable to the treatment of APAP were noted only in the APAP-treated SD rats. There were differences in the profiles of plasma and urinary glutathione-related metabolites between the non-APAP-treated SD and SDT rats and the plasma/urinary endogenous metabolite profile after treatment with APAP in the SDT rats indicated that hepatic glutathione synthesis was decreased due to accelerated gluconeogenesis. In conclusion, SDT rats were more sensitive to APAP-induced chronic hepatotoxicity than SD rats and the high susceptibility of SDT rats was considered to be attributable to lowered hepatic glutathione levels induced by accelerated gluconeogenesis.

A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor.

Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.

[Investigation of Predisposition Biomarkers to Identify Risk Factors for Drug-induced Liver Injury in Humans: Analyses of Endogenous Metabolites in an Animal Model Mimicking Human Responders to APAP-induced Hepatotoxicity].

Drug-induced liver injury is a main reason of regulatory action pertaining to drugs, including restrictions to clinical indications and withdrawal from the marketplace. Acetaminophen (APAP) is a commonly used and effective analgesic/antipyretic agent and relatively safe drug even in long-term treatment. However, it is known that APAP at therapeutic doses may cause hepatotoxicity in some individuals. Hence great efforts have been made to identify risk factors for APAP-induced chronic hepatotoxicity. We investigated the contribution of undernourishment to susceptibility to APAP-induced chronic hepatotoxicity using an animal model. We employed daytime restricted fed (RF) rats as a modified-nutritional state model for human APAP-induced hepatotoxicity. RF and ad libitum fed (ALF) rats were given APAP at 0, 300, and 500 mg/kg for 3 months. Plasma and urinary glutathione-related metabolomes and liver function parameters were measured during the dosing period. Endogenous metabolites forming at different levels between the RF and ALF rats could be potential predisposition biomarkers for APAP-induced hepatotoxicity. In addition, RF rats were considered a useful model to estimate the contribution of nutritional state of patients to APAP-induced chronic hepatotoxicity. In this article we report our current research focusing on nutritional state as risk factor for APAP-induced chronic hepatotoxicity and our findings of hepatotoxicity biomarkers.

Enhancement of acetaminophen-induced chronic hepatotoxicity in restricted fed rats: a nonclinical approach to acetaminophen-induced chronic hepatotoxicity in susceptible patients.

Acetaminophen (APAP) is a commonly used and effective analgesic and antipyretic agent. However, some patients encounter hepatotoxicity after repeated APAP dosing at therapeutic doses. In the present study, we focused on the nutritional state as one of the risk factors of APAP-induced chronic hepatotoxicity in humans and investigated the contribution of undernourishment to susceptibility to APAP-induced chronic hepatotoxicity using an animal model mimicking undernourished patients. Rats were divided into 2 groups: the ad libitum fed (ALF) and the restricted fed (RF) rats and were assigned to 3 groups (n = 8/group) for each feeding condition. The animals were given APAP at 0, 300 and 500mg/kg for 99 days under each feeding condition. Plasma and urinary glutathione-related metabolites and liver function parameters were measured during the dosing period and hepatic glutathione levels were measured at the end of the dosing period. In the APAP-treated ALF rats hepatic glutathione levels were increased and hepatic function parameters were not changed, but in the APAP-treated RF rats hepatic glutathione levels were decreased at 500mg/kg and hepatic function parameters were increased at 300 and 500mg/kg. Moreover the urinary endogenous metabolite profile after long-term treatment with APAP in the ALF and RF rats was similar to that in human non-responders and responders to APAP-induced chronic hepatotoxicity, respectively. In conclusion, the RF rats were more sensitive to APAP-induced chronic hepatotoxicity than the ALF rats and were considered to be a useful model to estimate the contribution of the nutritional state of patients to APAP-induced chronic hepatotoxicity.

Difructose anhydrides III and IV equally promote calcium absorption from the luminally perfused rat small intestine.

We examined the effects of di-D-fructose anhydride (DFA) III and IV on Ca absorption in luminally perfused segments of the small intestine in anesthetized rats. The calcium absorption rate with perfusion of 10 mmol/l CaCl2 was similarly increased by addition of 100 mmol/l DFAIII or IV, and these promotive effects of both DFAs were pronounced at perfusion rate of 0.15 ml/min than at 0.3 ml/min. The promotive effects were higher in the duodenojejunum than in the ileum.