AST-120 Treatment Alters the Gut Microbiota Composition and Suppresses Hepatic Triglyceride Levels in Obese Mice.

Background: The prevalence of nonalcoholic fatty liver disease (NAFLD) has been increasing worldwide. The existence of a relationship between the microbiota and the pathology of hepatic steatosis is also becoming increasingly clear. AST-120, an oral spherical carbon adsorbent, has been shown to be useful for delaying dialysis initiation and improving uremic symptoms in patients with chronic kidney disease. However, little is known about the effect of AST-120 on fatty liver.Methods: AST-120 (5% w/w) was administrated to 6-week-old male db/db mice for 8 weeks. The body weight, blood glucose and food consumption were examined. Hepatic triglyceride (TG) levels, lipid droplets and epididymal fat cell size were measured. The gut microbiota compositions were investigated in feces and cecum.Results: Significant decreases of the hepatic weight and hepatic TG levels were observed in the AST-120-treated db/db mice. Furthermore, AST-120 treatment was also associated with a decrease of Bacteroidetes, increase of Firmicutes, and a reduced ratio of Bacteroidetes to Firmicutes (B/F ratio) in the feces in the db/db mice. The B/F ratio in the feces was correlated with the liver weight and area of the liver occupied by lipid droplets in the db/db mice.Conclusions: These data suggest that AST-120 treatment alters the composition of the fecal microbiota and suppresses hepatic TG levels in the db/db mice.

Current status of pathological image analysis technology in pharmaceutical companies: a questionnaire survey of the Japan Pharmaceutical Manufacturers Association.

The Japan Pharmaceutical Manufacturers Association (JPMA) has instituted a task force (TF) for the "development of image analysis technology for histopathological changes" as part of the collaboration for realizing cutting-edge drug development since 2016. In recent years, there has been progress in the digital pathology technology; however, few applications in nonclinical drug development studies have been observed. Therefore, TF performed a questionnaire survey to investigate the current status, needs, possibility, and development of image analysis. The subjects were 35 member companies of the JPMA. The questionnaire was set to assess the efficacy and/or safety of researchers engaged in pathological evaluations for each company. The questions focused on the experiences, implementation, and issues regarding histopathological examinations; the need for image analysis software; and future views. Valid responses were obtained from 26 companies. Most companies assumed that the beneficial aspect of image analysis is to gain objectivity and persuasiveness; however, challenges in the analysis conditions with regard to accuracy and without subjectivity persist. Additionally, there seems to be a need for image analysis software with advanced digital pathology technology, with most companies believing that, in the future, pathological evaluations will be partly performed by computers. In conclusion, in this questionnaire survey, TF extracted the current status of image analysis in nonclinical studies performed by pharmaceutical companies and collected opinions on future prospects regarding the development of image analysis software with advanced digital pathology technology.

Quantification of histopathological findings using a novel image analysis platform.

Digital pathology, including image analysis and automatic diagnosis of pathological tissue, has been developed remarkably. HALO is an image analysis platform specialized for the study of pathological tissues, which enables tissue segmentation by using artificial intelligence. In this study, we used HALO to quantify various histopathological changes and findings that were difficult to analyze using conventional image processing software. Using the tissue classifier module, the morphological features of degeneration/necrosis of the hepatocytes and muscle fibers, bile duct in the liver, basophilic tubules and hyaline casts in the kidney, cortex in the thymus, and red pulp, white pulp, and marginal zone in the spleen were learned and separated, and areas of interest were quantified. Furthermore, using the cytonuclear module and vacuole module in combination with the tissue classifier module, the number of erythroblasts in the red pulp of the spleen and each area of acinar cells in the parotid gland were quantified. The results of quantitative analysis were correlated with the histopathological grades evaluated by pathologists. By using artificial intelligence and other functions of HALO, we recognized morphological features, analyzed histopathological changes, and quantified the histopathological grades of various findings. The analysis of histopathological changes using HALO is expected to support pathology evaluations.

Quantitative analysis of histopathological findings using image processing software.

In evaluating pathological changes in drug efficacy and toxicity studies, morphometric analysis can be quite robust. In this experiment, we examined whether morphometric changes of major pathological findings in various tissue specimens stained with hematoxylin and eosin could be recognized and quantified using image processing software. Using Tissue Studio, hypertrophy of hepatocytes and adrenocortical cells could be quantified based on the method of a previous report, but the regions of red pulp, white pulp, and marginal zones in the spleen could not be recognized when using one setting condition. Using Image-Pro Plus, lipid-derived vacuoles in the liver and mucin-derived vacuoles in the intestinal mucosa could be quantified using two criteria (area and/or roundness). Vacuoles derived from phospholipid could not be quantified when small lipid deposition coexisted in the liver and adrenal cortex. Mononuclear inflammatory cell infiltration in the liver could be quantified to some extent, except for specimens with many clustered infiltrating cells. Adipocyte size and the mean linear intercept could be quantified easily and efficiently using morphological processing and the macro tool equipped in Image-Pro Plus. These methodologies are expected to form a base system that can recognize morphometric features and analyze quantitatively pathological findings through the use of information technology.

Pathological characterization and morphometric analysis of hepatic lesions in SHRSP5/Dmcr, an experimental non-alcoholic steatohepatitis model, induced by high-fat and high-cholesterol diet.

SHRSP5/Dmcr is a newly established substrain of stroke-prone spontaneously hypertensive rat (SHRSP). Recently, high-fat and high-cholesterol (HFC) diet-fed SHRSP5/Dmcr has been reported as a novel rat model of developing hepatic lesions similar to human non-alcoholic steatohepatitis (NASH). The aim of this study was to investigate the detailed pathological conditions induced by HFC diet in SHRSP5/Dmcr rats using molecular biological methods and morphometric analysis. SHRSP5/Dmcr rats at 6 weeks of age were fed on either HFC diet or stroke-prone (SP) diet for 2, 4, 6, 8 and 16 weeks and histopathological changes in the liver, blood chemistry and mRNA expression levels in the liver were investigated. As evidenced by the histopathological examination of the liver of the SHRSP5/Dmcr rats, hepatic steatosis and lobular inflammation were present, with gradual increasing severity from 2 weeks after the introduction of the HFC diet. Partial hepatic fibrosis was detected at 6 weeks and spread over the entire region of the liver with more severe bridging formation by 16 weeks. The degrees of NASH-like hepatic lesions such as steatosis (the size distribution of lipid droplets), inflammation and fibrosis were quantified by morphometric analysis. Eosinophilic inclusion bodies encountered in the hepatocytes had immunoreactivity with Cox-4 and double-membrane walls, identified as mega-mitochondria. Serum ALT and bilirubins, and the mRNA expression levels related to fibrosis were closely correlated with hepatic histopathological changes. The clear feeding time-dependent progression of NASH-like hepatic lesion in HFC diet-fed SHRSP5/Dmcr rats reinforced the conclusion that this strain might be a useful model of NASH and of inflammatory fibrotic liver disease.

Tysnd1 deficiency in mice interferes with the peroxisomal localization of PTS2 enzymes, causing lipid metabolic abnormalities and male infertility.

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids ß-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1(-/-) mice. Male Tysnd1(-/-) mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1(-/-) mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.

Inhibition of repulsive guidance molecule-a ameliorates compromised blood-spinal cord barrier integrity associated with neuromyelitis optica in rats.

The influx of pathogenic aquaporin-4 antibodies (AQP4-Abs) across the blood-spinal cord barrier (BSCB) is crucial for the development and exacerbation of neuromyelitis optica (NMO). We examined whether prophylactic intravenous administration of anti-repulsive guidance molecule-a antibodies (RGMa-Abs) has disease-modifying effects on BSCB dysfunction using an NMO model elicited by peripheral administration of AQP4-Abs to rats. RGMa-Ab treatment attenuated the acute exacerbation of perivascular astrocytopathy in the spinal cord and clinical symptoms, which were highly correlated with neurofilament light chain levels in both the cerebrospinal fluid (CSF) and serum. Additionally, RGMa-Ab treatment suppressed the expression of proinflammatory cytokines/chemokines and the infiltration of inflammatory cells into the spinal cord. CSF analysis of NMO rats revealed that RGMa-Ab treatment improved the CSF/serum albumin ratio and suppressed AQP4-Abs influx. RGMa inhibition using RGMa-Abs is suggested as a potential therapeutic option for BSCB dysfunction associated with NMO.

Lowering bile acid pool size with a synthetic farnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energy expenditure.

We evaluated the metabolic impact of farnesoid X receptor (FXR) activation by administering a synthetic FXR agonist (GW4064) to mice in which obesity was induced by a high fat diet. Administration of GW4064 accentuated body weight gain and glucose intolerance induced by the high fat diet and led to a pronounced worsening of the changes in liver and adipose tissue. Mechanistically, treatment with GW4064 decreased bile acid (BA) biosynthesis, BA pool size, and energy expenditure, whereas reconstitution of the BA pool in these GW4064-treated animals by BA administration dose-dependently reverted the metabolic abnormalities. Our data therefore suggest that activation of FXR with synthetic agonists is not useful for long term management of the metabolic syndrome, as it reduces the BA pool size and subsequently decreases energy expenditure, translating as weight gain and insulin resistance. In contrast, expansion of the BA pool size, which can be achieved by BA administration, could be an interesting strategy to manage the metabolic syndrome.

Automated recognition of glomerular lesions in the kidneys of mice by using deep learning.

Background: In recent years, digital pathology has been rapidly developing and applied throughout the world. Especially in clinical settings, it has been utilized in a variety of situations, including automated cancer diagnosis. Conversely, in non-clinical research, it has not yet been utilized as much as in clinical settings. We have been performing automated recognition of various pathological animal tissues and quantitative analysis of pathological findings, including liver and lung. In this study, we attempted to construct an artificial intelligence (AI)-based trained model that can automatedly recognize glomerular lesions in mouse kidneys that are characterized by complex structures. Materials and methods: By using hematoxylin and eosin (HE)-stained whole slide images (WSI) from Col4a3 KO mice as variation data, normal glomeruli and glomerular lesions were annotated, and deep learning (DL) was performed with the use of the neural network classifier DenseNet system in HALO AI. The trained model was refined by correcting the annotation of misrecognized tissue area and reperforming DL. The accuracy of the trained model was confirmed by comparing the AI-obtained results with the pathological grades evaluated by pathologists. The generality of the trained model was also confirmed by analyzing the WSI of adriamycin (ADR)-induced nephropathy mice, which is a different disease model. Results: Glomerular lesions (including mesangial proliferation, crescent formation, and sclerosis) observed in Col4a3 KO mice and ADR mice were detected by our trained model. The number of glomerular lesions detected by our trained model were also highly correlated with that of counted by pathologists. Conclusion: In this study, we constructed a trained model allowing us to automatedly recognize glomerular lesions in the mouse kidney with the use of the HALO AI system. The findings and insights of this study will facilitate the development of digital pathology in non-clinical research and improve the probability of success in drug discovery research.

RNAi-mediated knockdown of mouse melanocortin-4 receptor in vitro and in vivo, using an siRNA expression construct based on the mir-187 precursor.

RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken ß-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.

Beneficial effects of canagliflozin in combination with pioglitazone on insulin sensitivity in rodent models of obese type 2 diabetes.

BACKGROUND: Despite its insulin sensitizing effects, pioglitazone may induce weight gain leading to an increased risk of development of insulin resistance. A novel sodium glucose co-transporter 2 (SGLT2) inhibitor, canagliflozin, provides not only glycemic control but also body weight reduction through an insulin-independent mechanism. The aim of this study was to investigate the combined effects of these agents on body weight control and insulin sensitivity. METHODS: Effects of combination therapy with canagliflozin and pioglitazone were evaluated in established diabetic KK-Ay mice and prediabetic Zucker diabetic fatty (ZDF) rats. RESULTS: In the KK-Ay mice, the combination therapy further improved glycemic control compared with canagliflozin or pioglitazone monotherapy. Furthermore, the combination significantly attenuated body weight and fat gain induced by pioglitazone and improved hyperinsulinemia. In the ZDF rats, early intervention with pioglitazone monotherapy almost completely prevented the progressive development of hyperglycemia, and no further improvement was observed by add-on treatment with canagliflozin. However, the combination significantly reduced pioglitazone-induced weight gain and adiposity and improved the Matsuda index, suggesting improved whole-body insulin sensitivity. CONCLUSIONS: Our study indicates that combination therapy with canagliflozin and pioglitazone improves insulin sensitivity partly by preventing glucotoxicity and, at least partly, by attenuating pioglitazone-induced body weight gain in two different obese diabetic animal models. This combination therapy may prove to be a valuable option for the treatment and prevention of obese type 2 diabetes.

The Niemann-Pick C1 like 1 (NPC1L1) inhibitor ezetimibe improves metabolic disease via decreased liver X receptor (LXR) activity in liver of obese male mice.

Dyslipidemic patients with diabetes mellitus, including metabolic syndrome, are at increased risk of coronary heart disease. It has been reported that ezetimibe, a cholesterol absorption inhibitor, improves metabolic diseases in mice and humans. However, the underlying mechanism has been unclear. Here we explored the effects of ezetimibe on lipid and glucose homeostasis. Male KK-A(y) mice were fed a high-fat diet, which is the mouse model of metabolic syndrome, with or without ezetimibe for 14 weeks. Ezetimibe improved dyslipidemia, steatosis, and insulin resistance. Ezetimibe decreased hepatic oxysterols, which are endogenous agonists of liver X receptor (LXR), to decrease hepatic lipogenic gene expressions, especially in stearoyl-CoA desaturase-1 (SCD1), leading to a remarkable reduction of hepatic oleate content that would contribute to the improvement of steatosis by reducing triglycerides and cholesterol esters. Simultaneously, hepatic ß-oxidation, NADPH oxidase and cytochrome P450 2E1 (CYP2E1) were reduced, and thus reactive oxygen species (ROS) and inflammatory cytokines were also decreased. Consistent with these changes, ezetimibe diminished c-Jun N-terminal kinase (JNK) phosphorylation and improved insulin signaling in the liver. In vitro study using primary hepatocytes obtained from male SD rats, treated with oleate and LXR agonist, showed excess lipid accumulation, increased oxidative stress and impaired insulin signaling. Therefore, in obese subjects, ezetimibe reduces hepatic LXR activity by reducing hepatic oxysterols to lower hepatic oleate content. This improves steatosis and reduces oxidative stress, and this reduction improves insulin signaling in the liver. These results provide insight into pathogenesis and strategies for treatment of the metabolic syndrome.

Bile acid binding resin improves metabolic control through the induction of energy expenditure.

BACKGROUND: Besides well-established roles of bile acids (BA) in dietary lipid absorption and cholesterol homeostasis, it has recently become clear that BA is also a biological signaling molecule. We have shown that strategies aimed at activating TGR5 by increasing the BA pool size with BA administration may constitute a significant therapeutic advance to combat the metabolic syndrome and suggest that such strategies are worth testing in a clinical setting. Bile acid binding resin (BABR) is known not only to reduce serum cholesterol levels but also to improve glucose tolerance and insulin resistance in animal models and humans. However, the mechanisms by which BABR affects glucose homeostasis have not been established. We investigated how BABR affects glycemic control in diet-induced obesity models. METHODS AND FINDINGS: We evaluated the metabolic effect of BABR by administrating colestimide to animal models for the metabolic syndrome. Administration of BABR increased energy expenditure, translating into significant weight reduction and insulin sensitization. The metabolic effects of BABR coincide with activation of cholesterol and BA synthesis in liver and thermogenesis in brown adipose tissue. Interestingly, these effects of BABR occur despite normal food intake and triglyceride absorption. Administration of BABR and BA had similar effects on BA composition and thermogenesis, suggesting that they both are mediated via TGR5 activation. CONCLUSION: Our data hence suggest that BABR could be useful for the management of the impaired glucose tolerance of the metabolic syndrome, since they not only lower cholesterol levels, but also reduce obesity and improve insulin resistance.

Dietary cholesterol reduces plasma triacylglycerol in apolipoprotein E-null mice: suppression of lipin-1 and -2 in the glycerol-3-phosphate pathway.

BACKGROUND: Cholesterol metabolism is tightly regulated by both cholesterol and its metabolites in the mammalian liver, but the regulatory mechanism of triacylglycerol (TG) synthesis remains to be elucidated. Lipin, which catalyzes the conversion of phosphatidate to diacylglycerol, is a key enzyme involved in de novo TG synthesis in the liver via the glycerol-3-phosphate (G3P) pathway. However, the regulatory mechanisms for the expression of lipin in the liver are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Apolipoprotein E-knock out (apoE-KO) mice were fed a chow supplemented with 1.25% cholesterol (high-Chol diet). Cholesterol and bile acids were highly increased in the liver within a week. However, the amount of TG in very low-density lipoprotein (VLDL), but not in the liver, was reduced by 78%. The epididymal adipose tissue was almost eradicated in the long term. DNA microarray and real-time RT-PCR analyses revealed that the mRNA expression of all the genes in the G3P pathway in the liver was suppressed in the high-Chol diet apoE-KO mice. In particular, the mRNA and protein expression of lipin-1 and lipin-2 was markedly decreased, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), which up-regulates the transcription of lipin-1, was also suppressed. In vitro analysis using HepG2 cells revealed that the protein expression of lipin-2 was suppressed by treatment with taurocholic acid. CONCLUSIONS/SIGNIFICANCE: These data using apoE-KO mice indicate that cholesterol and its metabolites are involved in regulating TG metabolism through a suppression of lipin-1 and lipin-2 in the liver. This research provides evidence for the mechanism of lipin expression in the liver.

Changes in pH increase perfusion pressure of coronary arteries in the rat.

Stricture of coronary arteries is closely related to ischemic heart disease. The purpose of this study was to examine whether changes in pH caused contraction of rat coronary arteries, as determined using Langendorff perfused hearts. Changing the pH of the perfusate increased perfusion pressure as an indication of the contractile state of coronary arteries. Alkaline pH-induced increase of perfusion pressure in Wistar Kyoto rats (WKY) was almost identical to that of spontaneously hypertensive rats (SHR), whereas acidic pH-induced increase in SHR was much greater than that in WKY. Acidic pH-induced increase in perfusion pressure was inhibited by verapamil, cromakalim, and adenosine. Feeding WKY with N(G)-nitro-L-arginine resulted in hypertension followed by enhanced acidic pH-induced increase in perfusion pressure. These results suggest that acidic-pH induced contraction of rat coronary arteries is caused by Ca(2+) influx through voltage-dependent Ca(2+) channels and the contraction is enhanced by hypertension.