Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Impact of Different Attitudes toward Face-to-Face and Online Classes on Learning Outcomes in Japan.

During the coronavirus disease 2019 (COVID-19) pandemic, online-based learning has become mainstream in many countries, and its learning outcomes have been evaluated. However, various studies have shown that online-based learning needs to be optimized in the future, and the number of reports for this purpose is currently not sufficient. The purpose in this study was to determine the relationship between academic performance and attitudes toward face-to-face and remote formats among Japanese pharmacy students enrolled in a course designed for knowledge acquisition. A combination of face-to-face and remote formats was used in a practice course for sixth-year pharmacy students, designed to improve academic performance through knowledge acquisition. To evaluate learning outcomes, we used a questionnaire that was administered to the course participants and the results of examinations conducted before and after the course. Online-oriented and face-to-face-oriented groups differed in their attitudes toward the ease of asking questions of faculty and communicating with the faculty members and classmates in each format. In a knowledge acquisition course for Japanese pharmacy students, the study revealed that the same academic outcomes were achieved, regardless of the students' own perceptions of their aptitude for face-to-face or remote learning style.

Liver-specific overexpression of lipoprotein lipase improves glucose metabolism in high-fat diet-fed mice.

The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7-2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease.

Porcine aldo-keto reductase 1C subfamily members AKR1C1 and AKR1C4: Substrate specificity, inhibitor sensitivity and activators.

Most members of the aldo-keto reductase (AKR) 1 C subfamily are hydroxysteroid dehydrogenases (HSDs). Similarly to humans, four genes for AKR1C proteins (AKR1C1-AKR1C4) have been identified in the pig, which is a suitable species for biomedical research model of human diseases and optimal organ donor for xenotransplantation. Previous study suggested that, among the porcine AKR1Cs, AKR1C1 and AKR1C4 play important roles in steroid hormone metabolism in the reproductive tissues; however, their biological functions are still unknown. Herein, we report the biochemical properties of the two recombinant enzymes. Kinetic and product analyses of steroid specificity indicated that AKR1C1 is a multi-specific reductase, which acts as 3α-HSD for 3-keto-5ß-dihydro-C19/C21-steroids, 3ß-HSD for 3-keto-5α-dihydro-C19-steroids including androstenone, 17ß-HSD for 17-keto-C19-steroids including estrone, and 20α-HSD for progesterone, showing Km values of 0.5-11 µM. By contrast, AKR1C4 exhibited only 3α-HSD activity for 3-keto groups of 5α/ß-dihydro-C19-steroids, 5ß-dihydro-C21-steroids and bile acids (Km: 1.0-1.9 µM). AKR1C1 and AKR1C4 also showed broad substrate specificity for nonsteroidal carbonyl compounds including endogenous 4-oxo-2-nonenal, 4-hydroxy-nonenal, acrolein, isocaproaldehyde, farnesal, isatin and methylglyoxal, of which 4-oxo-2-nonenal was reduced with the lowest Km value of 0.9 µM. Moreover, AKR1C1 had the characteristic of reducing aliphatic ketones and all-trans-retinal. The enzymes were inhibited by flavonoids, synthetic estrogens, nonsteroidal anti-inflammatory drugs, triterpenoids and phenolphthalein, whereas only AKR1C4 was activated by bromosulfophthalein. These results suggest that AKR1C1 and AKR1C4 function as 3α/3ß/17ß/20α-HSD and 3α-HSD, respectively, in metabolism of steroid hormones and a sex pheromone androstenone, both of which also play roles in metabolism of nonsteroidal carbonyl compounds.

ZFAND3 Overexpression in the Mouse Liver Improves Glucose Tolerance and Hepatic Insulin Resistance.

Genome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo. This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus.

Loxoprofen enhances intestinal barrier function via generation of its active metabolite by carbonyl reductase 1 in differentiated Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide as antipyretic analgesics and agents for rheumatoid arthritis and osteoarthritis, but known to cause damage to the gastrointestinal mucosae as their serious adverse effects. Few studies showed the impairment of intestinal epithelial barrier function (EBF) by high concentrations (0.5-1 mM) of NSAIDs, but the underlying mechanism is not fully understood. This study is aimed at clarifying effects at a low concentration (50 µM) of three NSAIDs, loxoprofen (Lox), ibuprofen and indomethacin, on intestinal EBF using human intestinal epithelial-like Caco-2 cells. Among those NSAIDs, Lox increased the transepithelial electric resistance (TER) value, decreased the paracellular Lucifer yellow CH (LYCH) permeability, and upregulated claudin (CLDN)-1, -3 and -5, indicating that low doses of Lox enhanced EBF through increasing expression of CLDNs. Lox is known to be metabolized to a pharmacologically active metabolite, (2S,1'R,2'S)-loxoprofen alcohol (Lox-RS), by carbonyl reductase 1 (CBR1), which is highly expressed in human intestine. CBR1 was expressed in the Caco-2 cells, and the pretreatment with a CBR1 inhibitor suppressed both the Lox-evoked CLDN upregulation and EBF enhancement. In addition, the treatment of the cells with Lox-RS resulted in higher TER value and lower LYCH permeability than those with Lox. Thus, Lox-RS synthesized by CBR1 may greatly contribute to the improving efficacy of Lox on the barrier function. Since EBF is decreased in inflammatory bowel disease, we finally examined the effect of Lox on EBF using the Caco-2/THP-1 co-culture system, which is used as an in vitro inflammatory bowel disease model. Lox significantly recovered EBF which was impaired by inflammatory cytokines secreted from THP-1 macrophages. These in vitro observations suggest that Lox enhances intestinal EBF, for which the metabolism of Lox to Lox-RS by CBR1 has an important role.

The Role of AKR1B10 in Physiology and Pathophysiology.

AKR1B10 is a human nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase belonging to the aldo-keto reductase (AKR) 1B subfamily. It catalyzes the reduction of aldehydes, some ketones and quinones, and interacts with acetyl-CoA carboxylase and heat shock protein 90α. The enzyme is highly expressed in epithelial cells of the stomach and intestine, but down-regulated in gastrointestinal cancers and inflammatory bowel diseases. In contrast, AKR1B10 expression is low in other tissues, where the enzyme is upregulated in cancers, as well as in non-alcoholic fatty liver disease and several skin diseases. In addition, the enzyme's expression is elevated in cancer cells resistant to clinical anti-cancer drugs. Thus, growing evidence supports AKR1B10 as a potential target for diagnosing and treating these diseases. Herein, we reviewed the literature on the roles of AKR1B10 in a healthy gastrointestinal tract, the development and progression of cancers and acquired chemoresistance, in addition to its gene regulation, functions, and inhibitors.

Dietary calamondin supplementation slows the progression of non-alcoholic fatty liver disease in C57BL/6 mice fed a high-fat diet.

Obesity is associated with an increased risk of metabolic abnormalities. The citrus fruit calamondin contains nobiletin and hesperidin, which are involved in lipid metabolism, and vitamin C, which is an antioxidant. We investigated the metabolic profiles of C57BL/6 mice fed a normal diet, high-fat diet (HFD), HFD + 1% (w/w) calamondin puree (HFD + CL1), or HFD + 5% (w/w) calamondin puree (HFD + CL5). Glucose tolerance was significantly higher in HFD + CL than in HFD-fed mice. Histological analysis revealed less lipid accumulation in the livers of HFD + CL-fed mice than in those of HFD-fed control mice. Hepatocyte ballooning and large lipid droplets - key non-alcoholic fatty liver disease characteristics - were observed in HFD-fed mice after 4 weeks; however, they were nearly absent in HFD + CL-fed mice. The serum expression level of inflammation-associated Ccl2 was lower in HFD + CL-fed mice than in HFD-fed mice. Thus, calamondin may ameliorate HFD-induced metabolic disturbances, including the progression of non-alcoholic fatty liver disease.

Adenovirus Vector-Induced IL-6 Promotes Leaky Adenoviral Gene Expression, Leading to Acute Hepatotoxicity.

Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.

Dimeric dihydrodiol dehydrogenase is an efficient primate 1,5-anhydro-D-fructose reductase.

1,5-Anhydro-D-fructose (AF), a metabolite of the anhydrofructose pathway of glycogen metabolism, has recently been shown to react with intracellular proteins and form advanced glycation end-products. The reactive AF is metabolized to non-reactive 1,5-anhydro-D-glucitol by AF reductase in animal tissues and human cells. Pig and mouse AF reductases were characterized, but primate AF reductase remains unknown. Here, we examined the AF-reducing activity of eleven primate NADPH-dependent reductases with broad substrate specificity for carbonyl compounds. AF was reduced by monkey dimeric dihydrodiol dehydrogenase (DHDH), human aldehyde reductase (AKR1A1) and human dicarbonyl/L-xylulose reductase (DCXR). DHDH showed the lowest KM (21 µM) for AF, and its kcat/KM value (1208 s-1mM-1) was much higher than those of AKR1A1 (1.3 s-1mM-1), DCXR (1.1 s-1mM-1) and the pig and mouse AF reductases. AF is a novel substrate with higher affinity and catalytic efficiency than known substrates of DHDH. Docking simulation study suggested that Lys156 in the substrate-binding site of DHDH contributes to the high affinity for AF. Gene database searches identified DHDH homologues (with >95% amino acid sequence identity) in humans and apes. Thus, DHDH acts as an efficient AF reductase in primates.

Cranberry Attenuates Progression of Non-alcoholic Fatty Liver Disease Induced by High-Fat Diet in Mice.

Obesity is characterized by abnormal or excessive fat accumulation, which leads to the development of metabolic syndrome. Because oxidative stress is increased in obesity, antioxidants are regarded as suitable agents for preventing metabolic syndrome. Here, we examined the impact of cranberry, which contains various antioxidants, on metabolic profiles, including that during the progression of non-alcoholic fatty liver disease (NAFLD), in high-fat diet (HFD)-fed C57BL/6 mice. We observed that oxidative stress was diminished in mice that were fed HFD diets supplemented with 1 and 5% cranberry powder as compared with that in HFD-fed control mice. Notably, from 1 week after beginning the diets to the end of the study, the body weight of mice in the cranberry-treatment groups was significantly lower than that of mice in the HFD-fed control group; during the early treatment phase, cranberry suppressed the elevation of serum triglycerides; and adipocytes in the adipose tissues of cranberry-supplemented-HFD-fed mice were smaller than these cells in HFD-fed control mice. Lastly, we examined the effect of cranberry on NAFLD, which is one of the manifestations of metabolic syndrome in the liver. Histological analysis of the liver revealed that lipid-droplet formation and hepatocyte ballooning, which are key NAFLD characteristics, were both drastically decreased in cranberry-supplemented-HFD-fed mice relative to the levels in HFD-fed control mice. Our results suggest that cranberry ameliorates HFD-induced metabolic disturbances, particularly during the early treatment stage, and exhibits considerable potential for preventing the progression of NAFLD.

Cooperative regulation of mouse aldose reductase (AKR1B3) gene transcription by Nrf2, TonEBP, and c-jun.

Aldose reductase (AR), a member of aldo-keto reductase family, is the rate-limiting enzyme in the polyol pathway, and is known to play a key role in the pathogenesis of diabetic complications. AR also catalyzes the reduction of reactive aldehydes, thereby detoxifying endogenous as well as xenobiotic aldehydes in various tissues. The transcription of the AR gene was previously shown to be augmented by various stimuli including osmotic and oxidative stresses. A highly conserved region composed of an antioxidant response element (ARE), AP-1 site, and tonicity responsive enhancer (TonE) has been identified within the 5'-flanking region of the AR genes of humans, rats, and mice, which we designated as the multiple stress response region (MSRR). We previously showed that the transcription factor Nrf2 activated AR transcription via ARE within MSRR. In the present study, we examined the interactions among Nrf2, c-Jun, and the TonE-binding protein (TonEBP) in the regulation of AR gene transcription. In gene reporter assays using luciferase reporter constructs containing the MSRR of the mouse AR (AKR1B3) gene with HepG2 cells, the forced expression of Nrf2 or TonEBP significantly increased promoter activity. The synergistic augmentation of promoter activity was observed when Nrf2 and TonEBP were co-introduced. On the other hand, the co-expression of c-Jun repressed promoter activation by Nrf2 and TonEBP. The mutation of the AP-1 site within MSRR did not affect the repressive effects of c-Jun, while the introduction of truncated c-Jun proteins lacking the leucine zipper domain no longer suppressed Nrf2-or TonEBP-mediated transactivation, suggesting that c-Jun repressed promoter activity independently of the AP-1 site and that interactions with protein factors via the leucine zipper domain were necessary for its negative effects on Nrf2 and TonEBP. These results indicate that AR promoter activity is cooperatively regulated by multiple transcription factors via MSRR.

Post-treatment with JP-1302 protects against renal ischemia/reperfusion-induced acute kidney injury in rats.

Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors.

[The Investigation of Genes, Using an Improved Adenovirus Vector, and Food for the Treatment and Prevention of Type 2 Diabetes Mellitus].

Although many treatments for type 2 diabetes mellitus (T2DM) have been developed, the quality of life for people with T2DM still tends to be lower than in those without the disease. Thus, the development of new T2DM treatments and prevention methods is required. Genetic predisposition and environmental factors are understood to be involved in the onset and pathology of T2DM. Therefore, we have attempted to explore genes and foods with potential for use in the treatment and prevention of T2DM. LipoQuality, which describes the functional features of diverse lipid species, has recently been a focus of study in the pathology of metabolic diseases. Phospholipids, the major components of biological membranes, are known to change in composition during the development of obesity and diabetes. Therefore, for our research, we focused on genes that regulate the composition of phospholipids. We examined the effects of such genes on T2DM using an improved adenovirus vector that demonstrates safer, higher, and longer-term transgene expression than that of the conventional adenovirus vector. We also found that certain foods inhibit the progression of non-alcoholic fatty liver disease, which is related to T2DM. In this review, we introduce our research results, demonstrating how genes and food independently contribute to the mechanisms of T2DM pathology.

Inhibition of α2C-adrenoceptors ameliorates cisplatin-induced acute renal failure in rats.

Nephrotoxicity is a major adverse reaction of the anticancer drug, cisplatin. We investigated the renoprotective effects of the α2-adrenoceptor antagonist, yohimbine and selective α2C-adrenoceptor antagonist, JP-1302, in cisplatin-treated Sprague Dawley rats. Rats were given a single intravenous dose of 7.5 mg/kg cisplatin and then yohimbine or JP-1302 was administered intraperitoneally at 0.1 or 3 mg/kg/day, respectively, for four days. Renal functional parameters, such as blood urea nitrogen, plasma creatinine, creatinine clearance and renal venous norepinephrine concentrations were measured. Kidney tissue damage and tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were assessed after the animals were euthanized. Cisplatin treatment aggravated the kidney functional parameters of blood urea nitrogen, plasma creatinine and creatinine clearance. Renal venous norepinephrine concentrations were also elevated after cisplatin administration. Treatment with yohimbine or JP-1302 clearly ameliorated kidney function and cell apoptosis. These treatments suppressed elevated renal plasma norepinephrine, TNF-α, MCP-1 and cleaved caspase 3 expressions which occurred after administration of cisplatin. These results suggest that yohimbine can prevent cisplatin-induced renal toxicity associated with acute kidney injury by suppressing cytokine expression through α2C-adrenoceptors.

Effect of monoamine oxidase inhibitors on ischaemia/reperfusion-induced acute kidney injury in rats.

Increases in renal sympathetic nerve activity during ischaemia and renal venous norepinephrine levels after reperfusion play important roles in the development of ischaemia/reperfusion-induced acute kidney injury. In the present study, we examined the effect of isatin, an endogenous monoamine oxidase inhibitor, on renal venous norepinephrine levels, superoxide production after reperfusion, and ischaemia/reperfusion-induced acute kidney injury. Ischaemia/reperfusion-induced acute kidney injury was accomplished by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal superoxide production and norepinephrine overflow were elevated and significant renal tissue damage was observed following ischaemia/reperfusion injury. Intravenous injection of isatin (10mg/kg) at 5min before ischaemia increased the renal venous plasma norepinephrine level after reperfusion and aggravated ischaemia/reperfusion-induced renal dysfunction and histological damage. The excessive superoxide production after reperfusion was significantly suppressed by isatin administration, indicating that the inhibition of oxidative deamination effectively suppressed superoxide production. These data suggest that the exacerbation effect of isatin is associated, at least in part, with increased norepinephrine levels but not with superoxide production. To the best of our knowledge, this is the first report of isatin involvement in the pathogenesis and/or development of acute kidney injury.

Adenovirus vector-mediated macrophage erythroblast attacher (MAEA) overexpression in primary mouse hepatocytes attenuates hepatic gluconeogenesis.

Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.

Human carbonyl reductase 1 participating in intestinal first-pass drug metabolism is inhibited by fatty acids and acyl-CoAs.

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, reduces a variety of carbonyl compounds including endogenous isatin, prostaglandin E2 and 4-oxo-2-nonenal. It is also a major non-cytochrome P450 enzyme in the phase I metabolism of carbonyl-containing drugs, and is highly expressed in the intestine. In this study, we found that long-chain fatty acids and their CoA ester derivatives inhibit CBR1. Among saturated fatty acids, myristic, palmitic and stearic acids were inhibitory, and stearic acid was the most potent (IC50 9µM). Unsaturated fatty acids (oleic, elaidic, γ-linolenic and docosahexaenoic acids) and acyl-CoAs (palmitoyl-, stearoyl- and oleoyl-CoAs) were more potent inhibitors (IC50 1.0-2.5µM), and showed high inhibitory selectivity to CBR1 over its isozyme CBR3 and other SDR superfamily enzymes (DCXR and DHRS4) with CBR activity. The inhibition by these fatty acids and acyl-CoAs was competitive with respect to the substrate, showing the Ki values of 0.49-1.2µM. Site-directed mutagenesis of the substrate-binding residues of CBR1 suggested that the interactions between the fatty acyl chain and the enzyme's Met141 and Trp229 are important for the inhibitory selectivity. We also examined CBR1 inhibition by oleic acid in cellular levels: The fatty acid effectively inhibited CBR1-mediated 4-oxo-2-nonenal metabolism in colon cancer DLD1 cells and increased sensitivity to doxorubicin in the drug-resistant gastric cancer MKN45 cells that highly express CBR1. The results suggest a possible new food-drug interaction through inhibition of CBR1-mediated intestinal first-pass drug metabolism by dietary fatty acids.

Vasodilatory effect of nitroglycerin in Japanese subjects with different aldehyde dehydrogenase 2 (ALDH2) genotypes.

The functional genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) influences the enzymatic activities of its wild type (Glu504 encoded by ALDH2*1) and mutant type (Lys504 encoded by ALDH2*2) proteins. The enzymatic activities of mutant-type ALDH2 are limited compared with those of the wild type. ALDH2 has been suggested as a critical factor for nitroglycerin-mediated vasodilation by some human studies and in vitro studies. Currently, there is no research on direct observations of the vasodilatory effect of nitroglycerin sublingual tablets, which is the generally used dosage form. In the present study, the contribution of ALDH2 to the vasodilatory effect of nitroglycerin sublingual tablets was investigated among three genotype groups (ALDH2*1/*1, ALDH2*1/*2, and ALDH2*2/*2) in Japanese. The results by direct assessments of in vivo nitroglycerin-mediated dilation showed no apparent difference in vasodilation among all genotypes of ALDH2. Furthermore, to analyze the effect of other factors (age and flow-mediated dilation), multiple regression analysis and Pearson's correlation coefficient analysis were carried out. These analyses also indicated that the genotypes of ALDH2 were not related to the degree of vasodilation. These results suggest the existence of other predominant pathway(s) for nitroglycerin biotransformation, at least with regard to clinical nitroglycerin (e.g., a sublingual tablet) in Japanese subjects.

Inhibitory Effect of Fruit Juices on the Doxorubicin Metabolizing Activity of Carbonyl Reductase 1.

BACKGROUND AND OBJECTIVE: Doxorubicin, an anthracycline anti-cancer drug, is effective for breast cancer and childhood lymphoma. Chronic cardiotoxicity has been known as a critical adverse effect of doxorubicin and is attributed to its metabolite doxorubicinol produced by carbonyl reductase 1 (CBR1, SDR21C1). Some flavonoids have been reported to act as inhibitors for CBR1, therefore, commercially available juices containing flavonoids are likely to be applicable as a prophylactic treatment against doxorubicin-induced cardiotoxicity by suppression of doxorubicinol production. In the study, fruit juices containing flavonoids were investigated for inhibitory effects on CBR1. METHOD: Recombinant CBR1 protein was subjected to in vitro enzymatic assays with/without juices. An apple juice and a grapefruit juice were selected in the present study as candidates capable of inhib-iting CBR1. RESULTS: The enzymatic assays revealed that both juices potently inhibit the CBR1-mediated metabolic conversion of doxorubicin to doxorubicinol in concertation-dependent manner. The concentrations required for obtaining 50% inhibition (IC50) were 0.0012% (v/v) and 0.0014% (v/v) for apple and grapefruit juices, respectively. Additionally, it is worth noting that these juices showed inhibitory effects on doxorubicin metabolism by CBR1 even at very low concentrations (0.0001% (v/v)). CONCLUSION: An apple juice and a grape fruit juice showed strong inhibitory effects on doxorubicin metabolism by CBR1 in vitro. These results suggest that the intake of flavonoid-containing juices can be a promising measure for protection against doxorubicin-induced cardiac toxicity, enabling patients to keep higher adherence with routine use in light of safety, economic performance and stable supply to market.