Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

Two infants with mild, atypical clinical features of Kagami-Ogata syndrome caused by epimutation.

Kagami-Ogata syndrome (KOS) is an imprinting disorder characterized by polyhydramnios, bell-shaped thorax with coat-hanger appearance (curved ribs), respiratory distress, abdominal wall defects, and distinct facial features, together with intellectual developmental delay with special needs. Abnormal expression of the imprinted genes on chromosome 14q32.2 causes KOS. Epimutation with aberrant hypermethylation of the MEG3/DLK1: intergenic differentially methylated region (MEG3/DLK1:IG-DMR) and the MEG3:TSS-DMR is one of the etiologies of KOS. We report two infants with KOS caused by epimutation presenting with some characteristic clinical features, mild clinical course, and almost normal motor and intellectual development. Methylation analysis for ten DMRs related to major imprinting disorders using pyrosequencing with genomic DNA (gDNA) extracted from leukocytes showed abnormally increased methylation levels of the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR in both patients, but lower than those in patients with paternal uniparental disomy chromosome 14 (upd(14)pat). The methylation levels in the DMRs other than both DMRs were within normal range. We also conducted methylation analysis for the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR with gDNA extracted from nails and buccal cells of both patients. Methylation levels in the MEG3:TSS-DMR, particularly in buccal cells, were closer to normal range compared to those in leukocytes. Microsatellite analysis for chromosome 14 and array comparative hybridization analysis showed no upd(14)pat or microdeletion involving the 14q32.2 imprinted region in either patient. A differential mosaic ratio of cells with aberrant methylation of DMRs at the 14q32.2 imprinted region among tissues (connective tissue, lung, and brain) might have led to their atypical clinical features. Further studies of patients with epimutation should further expand the phenotypic spectrum of KOS.

Actual incidence of severe neonatal hypoglycemia in non-risk term neonates: A 10 year survey of all cases in Toyama Prefecture, Japan.

BACKGROUND: Severe neonatal hypoglycemia may cause irreversible neurological sequelae. Although blood glucose (BG) screening in term neonates without risk factors for hypoglycemia (non-risk neonates) is not recommended in the current guidelines, severe hypoglycemia can occur in such neonates. To evaluate the necessity of BG screening in non-risk neonates, it is important to determine the accurate incidence of severe hypoglycemia in those neonates. METHODS: We conducted a 10 year survey of all normal-weight term neonates diagnosed with severe neonatal hypoglycemia who were treated at secondary- and tertiary-level neonatal centers in Toyama Prefecture, Japan, between January 2011 and December 2020. RESULTS: During the study period, 11 cases of severe neonatal hypoglycemia (six of which occurred in non-risk neonates) were identified. The overall incidence of severe hypoglycemia was 1 in 5,827 normal-weight term births, and the incidence in non-risk neonates was 1 in 10 682 normal-weight term births. All of the cases in non-risk neonates were diagnosed as hyperinsulinemic hypoglycemia. CONCLUSIONS: This is the first population-based study to have identified the actual incidence of severe pathological neonatal hypoglycemia in non-risk neonates. The incidence was not low compared with those of the newborn screening disorders, justifying the necessity of BG screening even in non-risk neonates.

Efficacy and safety of perampanel monotherapy in patients with focal-onset seizures with newly diagnosed epilepsy or recurrence of epilepsy after a period of remission: The open-label Study 342 (FREEDOM Study).

OBJECTIVE: Our study assessed perampanel monotherapy in patients (aged ≥12 years) with focal-onset seizures (FOS) with or without focal to bilateral tonic-clonic seizures (FBTCS) in Japan and South Korea. METHODS: Study 342 (NCT03201900; FREEDOM) is a single-arm, open-label, Phase III study. Patients initially received perampanel in a 32-week 4-mg/d Treatment Phase (6-week Titration; 26-week Maintenance Periods). If they experienced a seizure during the 4-mg/d Maintenance Period, they could be up-titrated to 8 mg/d across an additional 30-week Treatment Phase (4-week Titration; 26-week Maintenance Periods). Primary endpoint was the seizure-freedom rate during the Maintenance Period (4 mg/d and last evaluated dose [4 or 8 mg/d]). Secondary endpoints included time to first seizure onset and to withdrawal during Maintenance. Treatment-emergent adverse events (TEAEs) were monitored. RESULTS: At data cutoff (February 28, 2019), 89 patients with FOS (84 [94.4%] with newly diagnosed epilepsy and 5 [5.6%] with recurrence of epilepsy after a period of remission) had received ≥1 perampanel dose; 16 patients discontinued during the 4-mg/d Titration Period, meaning 73 patients entered the 4-mg/d Maintenance Period and were included in the primary analysis set for efficacy. Seizure-freedom rate in the 26-week Maintenance Period was 46/73 (63.0%; 95% confidence interval [CI]: 50.9-74.0) at 4 mg/d and 54/73 (74.0%; 95% CI: 62.4-83.5) at 4 or 8 mg/d. Cumulative probability of seizure-onset and withdrawal rates during Maintenance was 30.8% (95% CI: 21.5-43.0) and 23.7% (95% CI: 15.4-35.3) at 4 mg/d, and 18.2% (95% CI: 11.0-29.3) and 23.3% (95% CI: 15.2-34.8) at 4 or 8 mg/d. Perampanel was generally well tolerated, and the most common TEAE was dizziness. SIGNIFICANCE: Perampanel monotherapy (4 to 8 mg/d) was efficacious and consistent with the known safety profile up to 26 weeks in patients (≥12 years) with primarily newly diagnosed FOS with or without FBTCS.

Exclusive breast-feeding and postnatal changes in blood sodium, ketone, and glucose levels.

BACKGROUND: Blood sodium and ketone are parameters of dehydration and fasting, respectively. Little is known, however, about the postnatal changes in these parameters in healthy, term, exclusively breast-fed neonates. METHODS: Capillary blood sodium, ß-hydroxybutyrate (ß-OHB), and glucose levels in 628 samples obtained from 392 healthy, term, exclusively breast-fed neonates during the first 12-143 h of life were examined. RESULTS: Blood sodium and ß-OHB gradually increased and reached a peak at 48-59 h of life (mean blood sodium, 142.3 ± 2.8 mEq/L; mean blood sodium increase, 3.3 mEq/L; mean ß-OHB, 1.16 ± 0.46 mmol/L; mean ß-OHB increase, 0.65 mmol/L), and then gradually decreased and reached a nadir at 120-143 h of life. Blood glucose gradually decreased and reached a nadir at 48-59 h of life (mean, 62.4 ± 12.2 mg/dL; mean decrease, 4.7 mg/dL), and then gradually increased and peaked at 120-143 h of life. These changes were synchronized with changes in weight-loss percentage. CONCLUSIONS: The postnatal changes in blood sodium, ketone, and glucose levels during the first 12-143 h of life are described in healthy, term, exclusively breast-fed neonates. The parameters seemed to be associated with the sufficiency of the breast-milk supply. These results can serve as normal reference values for healthy, term, exclusively breast-fed neonates during the early postnatal period.

Capillary Blood Ketone Levels as an Indicator of Inadequate Breast Milk Intake in the Early Neonatal Period.

OBJECTIVE: To determine the utility of capillary blood ketone levels as an indicator of inadequate intake of breast milk in the early postnatal period. STUDY DESIGN: Levels of capillary blood beta-hydroxybutyrate (ßOHB), the main ketone body in the blood, were measured with a bedside ketone meter in 585 full-term neonates aged 48-95 hours who were breastfed exclusively. Relationships between weight-loss percentage, blood sodium, glucose, pH, partial pressure of carbon dioxide, base-deficit levels, and ßOHB levels were investigated. The diagnostic accuracy of ßOHB for predicting excessive weight loss (weight loss ≥10% of birth weight) and hypernatremic dehydration (blood sodium level ≥150 mEq/L) was determined. RESULTS: ßOHB levels were correlated positively with weight-loss percentage and blood sodium levels and were correlated negatively with blood glucose levels. The diagnostic accuracy of ßOHB was 0.846 (optimal cut off, 1.55 mmol/L; sensitivity, 80.9%, specificity, 74.0%) for predicting excessive weight loss and 0.868 (optimal cut off, 1.85 mmol/L; sensitivity, 94.3%; specificity, 69.9%) for predicting hypernatremic dehydration according to the area under the receiver operating characteristic curve. Multiple logistic analysis revealed that ßOHB and weight loss percentage were the only independent predictors of hypernatremic dehydration. Increases in ßOHB levels also were associated with worsening metabolic acidosis and hypocapnia. CONCLUSION: High ßOHB levels were associated with inadequate intake of breast milk in the early postnatal period. The use of bedside capillary blood ketone levels may be clinically useful as an indicator of dehydration, energy depletion, and acid-base imbalance in breastfeeding infants in the early postnatal period.

In Vitro and In Silico Characterization of Lemborexant (E2006), a Novel Dual Orexin Receptor Antagonist.

Orexin (hypocretin) neuropeptides have, among others, been implicated in arousal/sleep control, and antagonizing the orexin signaling pathway has been previously demonstrated to promote sleep in animals and humans. This mechanism opens up a new therapeutic approach to curb excessive wakefulness in insomnia disorder rather than to promote sleep-related signaling. Here we describe the preclinical pharmacological in vitro and in silico characterization of lemborexant ((1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide)), a dual orexin receptor antagonist (DORA), as a novel experimental therapeutic agent for the symptomatic treatment of insomnia disorder and compare its properties to two other DORAs, almorexant and suvorexant. Lemborexant binds to both orexin receptors and functionally inhibits them in a competitive manner with low nanomolar potency, without any species difference apparent among human, rat, and mouse receptors. Binding and dissociation kinetics on both orexin receptors are rapid. Lemborexant is selective for both orexin receptors over 88 other receptors, transporters, and ion channels of important physiologic function. In silico modeling of lemborexant into the orexin receptors showed that it assumes the same type of conformation within the receptor-binding pocket as suvorexant, the π-stacked horseshoe-like conformation.

Virus myocarditis in a 1-month-old boy presenting as two types of paroxysmal supraventricular tachycardia.

Herein we describe the case of a 1-month-old boy with acute viral myocarditis, who presented with two kinds of paroxysmal supraventricular tachycardia, and who was cured after medical treatment. He was brought to the emergency room with poor feeding due to fever. On the third day of hospitalization, a narrow QRS tachycardia (180-200 beats/min) was detected. Echocardiography showed a high echoic area at the atrial septum around the atrioventricular node. The patient was clinically diagnosed with acute myocarditis. The narrow QRS tachycardia was diagnosed as incessant junctional ectopic tachycardia. The patient was treated with propranolol and landiolol. The frequency of the tachycardia decreased, but a different narrow QRS tachycardia was detected on the 15th day of hospitalization on electrocardiogram (220 beats/min), which was ascribed to atrioventricular nodal re-entrant tachycardia. Atenolol was effective for the tachycardia. At 2 years follow up, cardiac function was normal and tachycardia had not recurred.

Left atrium thrombus in an extremely low-birthweight infant with late-onset circulatory dysfunction.

A left atrium thrombus, potentially a life-threatening complication, is an extremely rare in early infancy. Most cases are caused by mal-placement of central venous catheters or related to congenital heart diseases with left atrial blood congestion. Here we present an extremely low birth weight infant who developed a left atrial thrombus during the course of late onset circulatory dysfunction. The thrombus was successfully treated by recombinant tissue plasminogen activator. A hemodynamically unstable condition like late onset circulatory dysfunction should be taken into consideration as a potential risk condition of this rare disease.

Intracranial calcification in a uremic infant with Wilms' tumor in a solitary kidney.

Wilms' tumor (WT), also called nephroblastoma, is an embryonic neoplasm of the developing kidney. A previously healthy Japanese female infant had WT in a single kidney without associated congenital malformations. Preoperative chemotherapy was started for the preservation of renal tissue and function. Tumor lysis syndrome, disseminated intravascular coagulopathy, and acute renal failure were accompanying. The infant needed surgical intervention and permanent replacement therapy. At the start of emergency hemodialysis, the infant had posterior reversible leukoencephalopathy syndrome because of severe hypertension. During ongoing peritoneal dialysis, the infant suffered from anemia, dietary and fluid restriction, and restriction of time and mobility. Despite alfacalcidol and calcium supplementation, the infant had secondary hyperparathyroidism and remarkably short stature. After waiting for the completion of chemotherapy, renal transplantation from the mother was completed. Successful kidney transplantation promptly corrected preexisting metabolic abnormalities causing secondary hyperparathyroidism. Subsequently, the infant often complained of headache. Computed tomographic scanning revealed calcification in the cerebellum. Refractory secondary hyperparathyroidism was inferred as the cause. A well-functioning graft provided the infant with a greater sense of well-being and enabled her to enjoy a lifestyle free of dialysis, although the infant must continue taking transplant medications and has retained unresolved issues of short stature and ectopic intracranial calcification.

Commotio Cordis presenting as a temporary complete atrioventricular block in a 2-year-old girl with congenitally corrected transposition of the great arteries.

This report describes a 2-year-old girl with congenitally corrected transposition of the great arteries (ccTGA) who presented with transient complete atrioventricular (AV) block after a mild chest blow. Running around the house with her older sister, she fell to the floor. Her sister also fell and landed on her. The girl became cyanotic and pale and experienced a transient loss of consciousness. At arrival to the emergency department, she had regained consciousness, but she remained pale. An electrocardiogram (ECG) demonstrated complete AV block with a heart rate of 78 beats per minute (bpm). The ECG after admission showed a Wenckebach-type second-degree AV block. Day 2 after admission, a 12-lead ECG showed significant ST and T-wave abnormalities in the precordial leads, but the girl had no chest pain and a normal physical examination. Echocardiography demonstrated normal contractility of the systemic right ventricle. The first-degree AV block and the ST and T-wave abnormalities on the 12-lead ECG improved gradually without abnormal Q-waves. This is the first report of ccTGA in which a transient complete AV block naturally recovered after a presentation with commotio cordis.

Expansion of FOXP3-positive CD4+CD25+ T cells associated with disease activity in atopic dermatitis.

BACKGROUND: FOXP3-positive CD4+CD25+ T cells are known to have an immunoregulatory function by means of preventing T-cell reactivity to both self- and non-self-antigens. However, the role of these cells in the pathogenesis of allergic diseases is not clear. OBJECTIVE: To evaluate the quantity and quality of circulating FOXP3-positive T cells in patients with atopic dermatitis (AD). METHODS: Peripheral blood mononuclear cells were isolated from 35 AD patients (mean [SD] age, 27.1 [7.5] years) and 36 controls (mean [SD] age, 27.5 [10.0] years). Cellular FOXP3 expression was analyzed using flow cytometry. Characteristics of FOXP3-positive T cells were evaluated with respect to cytokine production capability and suppressive function. RESULTS: Frequencies of circulating FOXP3+CD25+ cells in the CD4+ T-cell population of AD patients were significantly higher than those in controls (mean [SD], 7.4% [4.6%] vs 4.5% [1.3%]; P = .002) and correlated with their Scoring Atopic Dermatitis (SCORAD) scores (r = 0.74, P = .008) and peripheral blood eosinophil counts (r = 0.72, P < .001). In the patients whose samples were analyzed at intervals of 1 to 2 months, frequencies of FOXP3-positive T cells were decreased as their skin lesions improved, regardless of medicines used. FOXP3-positive CD4+ T cells from patients, as well as those from controls, showed little capability to synthesize interferon gamma and interleukin 4. No differences were found in suppression abilities of CD4+CD25+ T cells between AD patients and controls. CONCLUSIONS: Our data suggest that dynamic fluctuation in numbers of circulating FOXP3-positive regulatory T cells might contribute to the pathogenesis of AD.

The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

BACKGROUND: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

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.

Immunolocalization of farnesoid X receptor (FXR) in mouse tissues using tissue microarray.

Farnesoid X receptor (FXR) is a member of the nuclear receptor family and is known to play important roles in bile acid homeostasis, and lipid and glucose metabolism. In this study, to elucidate the systemic physiological functions of FXR, comprehensive immunohistochemical analysis of cell/subcellular localization of FXR and its heterodimer partner, retinoid X receptor (RXR)-alpha, in adult mice tissues was performed using tissue microarray (TMA)-based immunohistochemistry. FXR immunolabeling was observed in the enterohepatic system--including absorptive epithelium in the intestines, hepatocytes and gall bladder epithelium, several epithelial lineage cells including the basal cells of stratified epithelium in the tongue, esophagus, forestomach--skin, corneal epithelium and ciliary body epithelium in the eye and adrenocortical cells--including glandular cells in the zona reticularis/fasciculata. In these FXP-positive cells, FXR was preferentially localized to the nucleus. RXR-alpha was ubiquitously distributed in the nucleus of most cell types, including FXR-positive cell types in the examined tissues. These data suggest that FXR might have various physiological roles, not only in bile acid homeostasis, and lipid and glucose metabolism, but also in the epithelial cell barrier, visual and urinary function through multiple organ systems.

Developmental changes of FOXP3-expressing CD4+CD25+ regulatory T cells and their impairment in patients with FOXP3 gene mutations.

FOXP3 is required for the generation and function of CD4(+)CD25(+) regulatory T (Treg) cells. To elucidate the biological role of Treg cells, we used a monoclonal anti-FOXP3 antibody to examine the frequencies of Treg cells during child development. The percentages of CD4(+)CD25(+)FOXP3(+) T cells were constant shortly from after birth through adulthood. CD4(+)CD25(+)FOXP3(+) T cells in cord blood showed the naive CD45RA(+)CD45RO(-) phenotype, whereas adult CD4(+)CD25(+)FOXP3(+) T cells expressed mostly the memory CD45RA(-)CD45RO(+) phenotype. The age-dependent dominance of memory CD4(+)CD25(+)FOXP3(+) T cells implies functional differences between naive and memory Treg cells. Notably, four patients with FOXP3 gene mutations revealed a paucity of CD4(+)CD25(+)FOXP3(+) T cells. Importantly, one patient with a frame shift mutation, who showed typical symptoms of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), exhibited marked T cell activation, whereas others with missense mutations, who were clinically milder, did not. This observation suggests a possible genotype-phenotype correlation in IPEX.

Quantitative image analysis in adipose tissue using an automated image analysis system: differential effects of peroxisome proliferator-activated receptor-alpha and -gamma 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-alpha 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-gamma 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.

Comparison of mitochondrial and macrophage content between subcutaneous and visceral fat in db/db mice.

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than peripheral (subcutaneous) obesity, however the underlying differences in morphology and pathophysiology between subcutaneous and visceral adipose are largely unknown. To evaluate the effects of diabetes and rosiglitazone (RSG) treatment, the expression of mitochondrial Hsp60, UCP-1 and F4/80 in inguinal subcutaneous (SC) fat, composed of white and brown adipose tissues, and epididymal (EP) fat, mainly white adipose tissue, were evaluated. In diabetic db/db mice, there was significant increased number of aggregated macrophage foci compared to db/+ mice, especially in EP fat. On the other hand, the expression of mitochondrial Hsp60 protein was suppressed in both SC and EP fat of db/db mice compared to db/+ mice, and the expression level of mitochondrial Hsp60 in db/+ mice was lower in EP fat compared with SC. In db/db mice, RSG suppressed the number of aggregated macrophage foci in EP fat, but not in SC fat. RSG ameliorated the mitochondrial Hsp60 expression and induced the expression of UCP-1 in both SC and EP fat. Taken together, these data suggest that differences exist in mitochondrial and macrophage content, and in the response to RSG between visceral and subcutaneous adipose tissue, and adipose type and distribution may be important for obesity-linked insulin resistance.

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) gamma 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)-1beta, PGC-1alpha, estrogen-related receptor alpha, and PPARalpha, 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.