Akemi Takahashi

MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages

Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by macrophages.

Evaluation of the Potential Risk of Drugs to Induce Hepatotoxicity in Human-Relationships between Hepatic Steatosis Observed in Non-Clinical Toxicity Study and Hepatotoxicity in Humans-

In the development of drugs, we sometimes encounter fatty change of the hepatocytes (steatosis) which is not accompanied by degenerative change in the liver in non-clinical toxicity studies. In this study, we investigated the relationships between fatty change of the hepatocytes noted in non-clinical toxicity studies of compound X, a candidate compound in drug development, and mitochondrial dysfunction in order to estimate the potential risk of the compound to induce drug-induced liver injury (DILI) in humans. We conducted in vivo and in vitro exploratory studies for this purpose. In vivo lipidomics analysis was conducted to investigate the relationships between alteration of the hepatic lipids and mitochondrial dysfunction. In the liver of rats treated with compound X, triglycerides containing long-chain fatty acids, which are the main energy source of the mitochondria, accumulated. Accumulation of these triglycerides was considered to be related to the inhibition of mitochondrial respiration based on the results of in vitro mitochondria toxicity studies. In conclusion, fatty change of the hepatocytes (steatosis) in non-clinical toxicity studies of drug candidates can be regarded as a critical finding for the estimation of their potential risk to induce DILI in humans when the fatty change is induced by mitochondrial dysfunction.

Spontaneous Histiocytic Sarcoma of the Popliteal Lymph Node in a Young Sprague-Dawley Rat

The present report describes a rare case of spontaneous primary histiocytic sarcoma of the popliteal lymph node in a 19-week-old female Sprague-Dawley (SD) rat. At necropsy, a 10 mm-diameter whitish nodule was found at the site of the femoral muscle in the right hindlimb. Histopathologically, the nodule comprised large pleomorphic histiocyte-like cells with abundant eosinophilic or foamy cytoplasm. Multinucleated giant cells, necrotic foci surrounded by palisading arrays of epithelioid histiocyte-like cells and phagocytosis of cell debris or erythrocytes by the neoplastic cells were occasionally observed. Invasion of the tumor cells into the surrounding adipose tissue was found focally, but there were no distal metastases. Immunohistochemically, the neoplastic cells were positive for vimentin, CD68 (ED1) and lysozyme. We concluded that this tumor occurred in the popliteal lymph node, considering the anatomical location of the lesion and the presence of the remnants of lymphoid tissue involved in the tumor.

Pathophysiological analyses of skeletal muscle in obese type 2 diabetes SDT fatty rats

Sarcopenia is the age-related decrease of muscle mass and function. Diabetes and obesity are known to be risk factors that exacerbate sarcopenia, but the underlying mechanism of diabetes-related sarcopenia is still unknown. Obese type 2 diabetes SDT fatty rats show early onset of severe diabetes and there have been no reports on the characteristics of their skeletal muscle. Therefore, pathophysiological analyses were performed for the skeletal muscle in these rats. Diabetic male SDT fatty rats were sacrificed at 8, 16, 24, 32 and 40 weeks of age. Age-matched Sprague Dawley (SD) rats were used as the normal control. In addition to biological blood parameters, the soleus and the extensor digitorum longus muscles were examined for muscle weight, histopathology, and protein synthesis and degradation. Muscle grip strength was also examined. These results revealed that the muscle weights of the SDT fatty rats were significantly decreased from 16 weeks of age. The mean cross-sectional area of muscle fibers in the SDT fatty rats decreased from 24 weeks of age. Increased intramyocellular lipid accumulation, identified by immunohistochemistry for adipophilin and TEM, was observed in the SDT fatty rats from 8 weeks of age. Plasma insulin-like growth factor (IGF)-1 levels and muscle strength in the SDT fatty rats decreased at 24 weeks of age and thereafter. These pathophysiological findings have been reported both in sarcopenia in aged humans and in patients with diabetes. In conclusion, the SDT fatty rat was considered to be a useful model for analysis of diabetes-related sarcopenia.

Immunohistochemical Characterization of Spontaneous Sertoli Cell Clusters in the Seminiferous Tubules of C57BL/6J Mice

Cell clusters were observed in the seminiferous tubules of C57BL/6J mice as a spontaneous lesion in a 2-week toxicity study, and they were demonstrated to be basically composed of Sertoli cells by immunohistochemistry for claudin-11 and GATA-4 (GATA-binding protein 4), which are both Sertoli cell markers. The clusters were composed of about 5 to 50 cells, which had eosinophilic and occasionally vacuolated cytoplasm with an unclear cell boundary. The cell clusters involved some sperm. No mitotic figures were observed and no immunoreactivity for proliferating cell nuclear antigen (PCNA) was detected in the clusters. In most cases, the cell clusters were observed in seminiferous tubules that also showed degenerative changes. In rare instances, cell aggregates immunohistochemically positive for claudin-11 were observed in the lumen of the epididymis, suggesting that some of the Sertoli cell clusters were sloughed off from the seminiferous epithelium into the epididymal ducts. To our knowledge, this is the first report of Sertoli cell clusters in any animal species except for transgenic or surgically altered animals.

Hepatocellular adenoma with severe fatty change in a male Spontaneously Diabetic Torii rat

The Spontaneously Diabetic Torii (SDT) rat is a rat model of nonobese type 2 diabetes mellitus, and hepatocellular adenomas have not been reported in this model. We report a hepatocellular adenoma with severe fatty change in a male 42-week-old SDT rat fed a high-fat diet. At necropsy, the animal had a whitish nodular mass of approximately 2 cm in diameter in the right medial lobe. Histologically, the mass was well demarcated from the surrounding tissues, slightly compressing the adjacent hepatic parenchyma and widely compartmented by fibrous connective tissues. The mass consisted of vacuolated tumor cells resembling hepatocytes with a solid and occasionally trabecular growth pattern. Abundant neutral lipids, which were positive for fat with Oil Red O stain and which ultrastructurally had moderately dense material, were contained within the vacuoles of the tumor cells. Immunohistochemically, the tumor cells showed an increase in immunoreactivity or number for Cytokeratin 8/18 and proliferating cell nuclear antigen but were negative for mesenchymal markers. From these findings, the mass could be distinguished from hepatocellular hyperplasia and was diagnosed as hepatocellular adenoma. In rats, hepatocellular adenoma accompanied by severe fatty change is rare, and this is the first report of a hepatocellular tumor with severe fatty change in a SDT rat.