Hiroyuki Higashiyama

Adipose Mitochondrial Biogenesis Is Suppressed in db/db and High-Fat Diet–Fed Mice and Improved by Rosiglitazone

The objective of this study was to further establish and confirm the relationship of adipose mitochondrial biogenesis in diabetes/obesity and the effects of rosiglitazone (RSG), a peroxisome proliferator–activated receptor (PPAR) γ agonist, by systematically analyzing mitochondrial gene expression and function in two mouse models of obesity and type 2 diabetes. Using microarray technology, adipose mitochondrial gene transcription was studied in db/db, high-fat diet–fed C57BL/6 (HFD) and respective control mice with or without RSG treatment. The findings were extended using mitochondrial staining, DNA quantification, and measurements of citrate synthase activity. In db/db and HFD mice, gene transcripts associated with mitochondrial ATP production, energy uncoupling, mitochondrial ribosomal proteins, outer and inner membrane translocases, and mitochondrial heat-shock proteins were decreased in abundance, compared with db/+ and standard-fat diet–fed control mice, respectively. RSG dose-dependently increased these transcripts in both db/db and HFD mice and induced transcription of mitochondrial structural proteins and cellular antioxidant enzymes responsible for removal of reactive oxygen species generated by increased mitochondrial activity. Transcription factors, including PPAR coactivator (PGC)-1β, PGC-1α, estrogen-related receptor α, and PPARα, were suppressed in both models and induced by RSG. The effects of RSG on adipose mitochondrial genes were confirmed by quantitative RT-PCR and further supported by mitochondrial staining, mitochondrial DNA quantification, and citrate synthase activity. Adipose mitochondrial biogenesis was overwhelmingly suppressed in both mouse models of diabetes/obesity and globally induced by RSG. These findings suggest an important role of adipose mitochondria in diabetes/obesity and the potential for new treatment approaches targeting adipose mitochondria.

Expression profiling of Peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis.

Background: Recent advances in fibrosis biology have identified transforming growth factor (TGF)-β type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression. Aims: To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis. Methods: Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson’s trichrome stain, and scored. TGF-β1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry. Results: The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-β1 expression in infiltrating macrophages. With the progression in fibrosis (day 7–14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells. Conclusions: The time-course data of TGF-β1 and R-Smads indicate that progressive enhancement of TGF-β1 signalling via R-Smad is activated in the process of fibrosis progression.

A new method for producing urinary bladder hyperactivity using a non-invasive transient intravesical infusion of acetic acid in conscious rats

INTRODUCTION Animal models that closely resemble the pathophysiology of human overactive bladder are important for evaluating novel therapeutics to treat the disorder. We established a non-invasive hyperactive bladder model that is sensitive to anti-muscarinic drugs and without bladder inflammation. METHODS Acetic acid solution was infused into the bladder for 5 min via the urethral orifice without any surgical procedures under isoflurane anaesthesia. After washing the bladder with saline, voiding frequency (VF) and total urine volume were determined for 9 h under conscious conditions. RESULTS Infusion of a 0.5% acetic acid solution caused a significant increase in VF, without influencing total urine volume or inducing significant histopathological inflammatory alterations in the bladder urothelium. Oral administration of oxybutynin (3 and 10 mg/kg) significantly ameliorated increases in VF induced by 0.5% acetic acid. Infusion of 0.75% acetic acid induced intensive urinary inflammation and a decrease in total urine volume as well as an increase in VF. Oral treatment with oxybutynin (10 mg/kg) did not significantly improve the increased VF due to 0.75% acetic acid. Acetic acid (0.5%) infusion evoked bladder hyper-responsiveness whether applied at night or during the day. However, VF was increased more by the nighttime application of acetic acid, while there were no significant differences in basal levels of VF between daytime and nighttime. DISCUSSION In this study, the non-invasive rat urinary hyperactive bladder model indicated minimizes the secondary effects of experimental procedures such as surgical operations and anesthesia on bladder function and is sensitive to oxybutynin. Thus, the model may be useful for investigating novel therapeutics for OAB treatment.

Histopathological study of time course changes in inter-renal aortic banding-induced left ventricular hypertrophy of mice

The left ventricular hypertrophy (LVH) in response to pressure overload is an important risk factor in cardiac morbidity and mortality. To investigate the time course of histopathological alterations in the LVH in response to pressure overload, histopathological and immunohistochemical examination was performed using the aortic banding‐induced mouse LVH model. Five‐week‐old male CD‐1 mice were subjected to the inter‐renal aortic banding. Major organs were sampled on 3, 10, 14, 21, 28 or 42 days after banding. Haematoxylin and eosin (H&E) staining, Massons trichrome staining and immunohistochemistry for proliferating cell nuclear antigen (PCNA), alpha‐smooth muscle actin (aSMA), ICAM‐1, type I collagen and CD31 was performed and microscopically examined. Three days after aortic banding, acute inflammatory changes, such as macrophages/neutrophil infiltration and vascular wall injury were observed on/around the coronary arteries/arterioles of both ventricles. Intense ICAM‐1 immunostaining was observed on the endothelium of the coronary arteries/arterioles. After day 10, vascular wall thickening and perivascular fibrosis was induced on the coronary arteries/arterioles. Immunohistochemistry for aSMA and PCNA demonstrated the proliferation of vascular smooth muscle cells in the media. After day 28, minimal cardiomyocyte hypertrophy was observed at the light microscope level. In the inter‐renal aortic banding LVH model, histopathological alterations in early phase were mainly observed on coronary arteries/arterioles. These early phase alterations were thought to be hypertension‐related changes in the coronary vasculatures. The cardiomyocyte hypertrophy observed in later phase was minimal at the light microscope level. These evidences would facilitate the understanding of pathophysiology of pressure overload LVH.

Quantitative image analysis in adipose tissue using an automated image analysis system: Differential effects of peroxisome proliferator‐activated receptor‐α and ‐γ agonist on white and brown adipose tissue morphology in AKR obese and db/db diabetic mice

Morphometric analysis of adipocytes is widely used to demonstrate the effects of antiobesity drugs or anti‐diabetic drugs on adipose tissues. However, adipocyte morphometry has been quantitatively performed by manual object extraction using conventional image analysis systems. The authors have developed an automated quantitative image analysis method for adipose tissues using an innovative object‐based quantitative image analysis system (eCognition). Using this system, it has been shown quantitatively that morphological features of adipose tissues of mice treated with peroxisome proliferator‐activated receptor (PPAR) agonists differ dramatically depending on the type of PPAR agonist. Marked alteration of morphological characteristics of brown adipose tissue (BAT) treated with GI259578A, a PPAR‐α agonist, was observed in AKR/J (AKR) obese mice. Furthermore, there was a 22.8% decrease in the mean size of adipocytes in white adipose tissue (WAT) compared with vehicle. In diabetic db/db mice, the PPAR‐γ agonist GW347845X decreased the mean size of adipocytes in WAT by 15.4% compared with vehicle. In contrast to changes in WAT, GW347845X increased the mean size of adipocytes in BAT greatly by 96.1% compared with vehicle. These findings suggest that GI259578A may activate fatty acid oxidation in BAT and that GW347845X may cause adipocyte differentiation in WAT and enhancement of lipid storage in BAT.

Expression profiling of liver receptor homologue 1 (LRH-1) in mouse tissues using tissue microarray

Liver receptor homologue 1 (LRH-1) is a nuclear receptor that plays important roles in lipid homeostasis and embryogenesis. To elucidate systemic physiological functions of LRH-1, we used tissue microarray-based immunohistochemistry to examine the tissue distribution and localization of LRH-1 in adult mouse tissues. LRH-1 immunoreactivity was observed in the nucleus of multiple epithelial lineage cells in the digestive system (including absorptive epithelial cells in the small and large intestines, goblet cells, acinar cells of the exocrine glands, chief cells and mucus neck cells in the stomach, granular and prickle layer cells in the tongue and forestomach, and gall bladder epithelium); respiratory system (alveolar type II cells); and urinary system (transitional epithelium). Nuclear LRH-1 immunoreactivity was also localized in cells involved in fatty acid/glucose metabolism, including hepatocytes, brown adipocytes, and cardiomyocytes, and neurons involved in the regulation of food intake, including the arcuate nucleus in the hypothalamus and paraventricular nucleus of thalamus. Additionally, LRH-1 immunoreactivity was observed in testicular Leydig cells and ovarian follicular cells. These data suggest that LRH-1 functions in multiple organ systems to regulate epithelial cell physiology and differentiation, energy metabolism, and reproduction.