Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway.

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α-FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK-PGC-1α-FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK-PGC-1α-FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

Intratympanic administration of alpha-lipoic acid-loaded pluronic F-127 nanoparticles ameliorates acute hearing loss.

We used antioxidant-containing nanoparticles (NPs) to treat acute hearing loss. Alpha-lipoic acid (ALA) served as the antioxidant; we employed Pluronic F127 to fabricate NPs. In vitro, ALA-NPs protected cells of the organ of Corti in HEI-OC1 mice, triggering nuclear translocation of NRF2 and increases in the levels of antioxidant proteins, including Nrf2, HO-1, SOD-1, and SOD-2. In vivo, the hearing of mice that received ALA-NP injections into the middle ear cavity was better preserved after induction of ototoxicity than in control animals. The cochlear Nrf2 level increased in test mice, indicating that the ALA-NPs protected hearing via the antioxidant mechanism observed in vitro. ALA-NPs effectively protected against acute hearing loss by activating the Nrf2/HO-1 pathway.

The relationship between miRNA-26b and connective tissue growth factor in rat models of aortic banding and debanding.

BACKGROUND/AIMS: Connective tissue growth factor (CTGF) is a profibrotic factor implicated in pressure overload-mediated myocardial fibrosis. In this study, we determined the role of predicted CTGF-targeting microRNAs (miRNAs) in rat models of aortic stenosis and reverse cardiac remodeling. METHODS: Minimally invasive ascending aortic banding was performed in 24 7-week-old male Sprague-Dawley rats, which were divided into three groups. The banding group consisted of eight rats that were sacrificed immediately after 6 weeks of aortic constriction. The debanding group underwent aortic constriction for 4 weeks and was sacrificed 2 weeks after band removal. The third group underwent sham surgery. We investigated the expression of CTGF, transforming growth factor-ß1 (TGFß1), and matrix metalloproteinase-2 using ELISA and examined miRNA-26b, miRNA-133a, and miRNA-19b as predicted CTGF-targeting miRNAs based on miRNA databases in 24-hour TGFß-stimulated and TGFß- washed fibroblasts and myocardial tissues from all subjects. RESULTS: CTGF was elevated in 24-hour TGFß-stimulated fibroblasts and decreased in 24-hour TGFß-washed fibroblasts. miRNA-26b was significantly increased in TGFß-washed fibroblasts compared with control and TGFß-stimulated fibroblasts (p < 0.05). CTGF expression was significantly higher in the banding group than that in the sham and debanding groups. The relative expression levels of miRNA-26b were higher in the debanding group than in the banding group. CONCLUSION: The results of our study using models of aortic banding and debanding suggested that miRNA-26b was significantly increased after aortic debanding. The in vitro model yielded the same results: miRNA-26b was upregulated after removal of TGFß from fibroblasts.

α-Lipoic acid prevents against cisplatin cytotoxicity via activation of the NRF2/HO-1 antioxidant pathway.

The production of reactive oxygen species (ROS) by cisplatin is one of the major mechanisms of cisplatin-induced cytotoxicity. We examined the preventive effect of α-lipoic acid (LA) on cisplatin-induced toxicity via its antioxidant effects on in vitro and ex vivo culture systems. To elucidate the mechanism of the antioxidant activity of LA, NRF2 was inhibited using NRF2 siRNA, and the change in antioxidant activity of LA was characterized. MTT assays showed that LA was safe at concentrations up to 0.5 mM in HEI-OC1 cells and had a protective effect against cisplatin-induced cytotoxicity. Intracellular ROS production in HEI-OC1 cells was rapidly increased by cisplatin for up to 48 h. However, treatment with LA significantly reduced the production of ROS and increased the expression of the antioxidant proteins HO-1 and SOD1. Ex vivo, the organs of Corti of the group pretreated with LA exhibited better preservation than the group that received cisplatin alone. We also confirmed the nuclear translocation of NRF2 after LA administration, and that NRF2 inhibition decreased the antioxidant activity of LA. Together, these results indicate that the antioxidant activity of LA was through the activation of the NRF2/HO-1 antioxidant pathway.

Inhibition of xanthine oxidoreductase protects against contrast-induced renal tubular injury by activating adenosine monophosphate-activated protein kinase.

Reactive oxygen species (ROS) play a pivotal role in the development of contrast-induced nephropathy (CIN). The inhibition of xanthine oxidoreductase is known to reduce levels of ROS. We investigated whether febuxostat could attenuate oxidative stress via the activation of adenosine monophosphate-activated protein kinase (AMPK) against CIN. In a mouse model of CIN, renal impairment and tubular injury substantially increased, whereas febuxostat attenuated renal injury. Plasma and kidney xanthine oxidoreductase levels were decreased by febuxostat. Febuxostat administration was accompanied by the upregulation of AMPK phosphorylation and the inhibition of nicotinamide-adenine dinucleotide phosphate oxidase (Nox)1 and Nox2, followed by the inhibition of hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 expressions and the suppression of transcription factor forkhead box O (FoxO)1 and FoxO3a phosphorylation. Cell survival was significantly reduced after iohexol administration and febuxostat ameliorated iohexol-induced cell death in proximal tubular (HK-2) cells. Furthermore, febuxostat enhanced AMPK phosphorylation and inhibited Nox1, Nox2, and HIF-1α expression in iohexol-exposed HK-2 cells. Finally, these processes decrease ROS in both in vivo and in vitro models of CIN. AMPK inhibition using small interfering RNA blunted the antioxidative effects of febuxostat in iohexol-treated HK-2 cells. Febuxostat attenuated CIN by modulating oxidative stress through AMPK-NADPH oxidase-HIF-1α signaling.

Expression profile of microRNAs following bone marrow-derived mesenchymal stem cell treatment in lipopolysaccharide-induced acute lung injury.

Immunomodulatory or immunosuppressive properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) facilitate the treatment of acute respiratory distress syndrome and acute lung injury (ALI). Dysregulated miRNA (miRNA or miR) expression associated with the effects of BM-MSCs was assessed in a rat model of lipopolysaccharide (LPS)-induced ALI. The present study performed biochemical tests to assess five analytes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, blood urea nitrogen (BUN), and creatinine (CREA). Total cell count was assessed and the percentage of bronchoalveolar lavage neutrophil content was also examined. The results Histopathological examination of rat upper lobe lung tissue was then used to estimate lung injury score (LIS). The levels of AST, lactate, BUN and creatinine (excluding ALT), released into the circulation upon injury, were significantly lower in ALI rats treated with BM-MSCs than in ALI rats alone (P<0.05). BM-MSC rats exhibited a significantly decreased bronchoalveolar lavage neutrophil percentage and LIS compared with that of LPS treated rats alone (P<0.05). In addition, the miRNA expression profile was determined following treatment with BM-MSCs via microarray analysis. A total of 95/690 miRNAs were differentially expressed following the treatment of BM-MSCs in rats with ALI. Among the 95 miRNAs, 66 were upregulated and 29 were downregulated; 9 miRNAs were significantly upregulated (miR-1843-3p, miR-323-3p, miR-183-5p, miR-182 and miR-196b-3p) or downregulated (miR-547-3p, miR-301b-5p, miR-503-3p and miR-142-3p). A total of 3 miRNAs were inversely expressed in ALI treated with BM-MSCs compared with untreated ALI. Of these 3 miRNAs, the expression of miR-142-3p and miR-503-3p was upregulated in the LPS groups and downregulated in the BM-MSC groups. miR-196b-3p was downregulated in the LPS group and upregulated in the BM-MSC groups. miRNAs have a role in cell proliferation, immune response, inflammation and apoptosis, which may be associated with the therapeutic effects of BM-MSCs in ALI. In summary, BM-MSCs improved multi-organ damage and attenuated lung injury. Different miRNA profiles were expressed following BM-MSC treatment of ALI. These dysregulated miRNAs participated in BM-MSC-mediated immunomodulation of ALI.

Optimized phospholipid-based nanoparticles for inner ear drug delivery and therapy.

To develop efficient carriers for inner ear drug delivery, we prepared four kinds of phospholipid-based nanoparticles: neutral, anionic, cationic, and cationic-PEG (polyethyleneglycol) particles. PEG was used to maintain long-term particle circulation in the perilymph, avoiding non-specific binding of particles to proteins. All four nanoparticles were about 200 nm in diameter, and their zeta potentials were -4.32, -26.0, +25.8, and -0.28, respectively, for neutral, anionic, cationic, and cationic-PEG nanoparticles. To test particle efficacy in vitro, we used an artificial mucosa 100 µm in thickness to model the round window membrane (RWM) and HEI-OC1 cells, which were treated with particles containing Nile Red dye. Based on the levels of particle penetration and cellular uptake in this model system, we selected an optimal particle for further study. We also observed the movement of particles in ex vivo organotypic cultures of the organ of Corti. In mice, we analyzed the biodistribution of dexamethasone (Dex) in the inner ear after intratympanic injection of Dex-loaded nanoparticles. Then, we tested the therapeutic utility of the Dex-loaded nanoparticles in a mouse model of ototoxicity. In the auditory brainstem response (ABR) test, particle provided improved hearing loss recovery at all tested frequencies, more so than did the Dex sodium phosphate (Dex-SP) solution in current clinical use. Furthermore, quantitative PCR showed that nanoparticles reduced the levels of pro-inflammatory cytokines, exhibiting anti-inflammatory effects superior to those of Dex-SP. Thus, the surface properties of nanoparticles play pivotal roles in particle penetration and distribution after intratympanic injection. Our in vitro screening system using an artificial mucosa will also be valuable in the development of carriers for inner ear drug delivery.

Modulation of PI3K/PTEN Pathway Does Not Affect Catalytic Activity of PDK1 in Jurkat Cells.

Unopposed phosphoinositide 3-kinase (PI3K) activity and 3-phosphoinositide production in Jurkat cells, due to a mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor-suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB. In Jurkat cells, PKB is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3-Phosphoinositide-dependent protein kinase-1 (PDK1), an enzyme that also contains a PH domain, catalyses Thr308 phosphorylation of PKB in addition to other kinase families such as PKC isoforms. It is unknown, however, whether the loss of PTEN in Jurkat cells also results in unregulated PDK1 activity and whether such loss has an impact on activation-loop phosphorylation of other PDK1 substrates e.g. PKC. In this study, we addressed whether loss of PTEN in Jurkat cells affects PDK1 catalytic activity and intracellular localization. We demonstrated that reducing the level of 3-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3K or expression of PTEN does not affect PDK1 activity or its intracellular localization. We conclude, therefore, that although Jurkat cells lack PTEN expression, only a subset of pathways downstream of PDK1 are perturbed as a consequence of PTEN loss.

Regeneration of thyroid follicles from primordial cells in a murine thyroidectomized model.

The functional unit of the thyroid gland, the thyroid follicle, dynamically responds to various stimuli to maintain thyroid hormone homeostasis. However, thyroid follicles in the adult human thyroid gland have a very limited regenerative capacity following partial resection of the thyroid gland. To gain insight into follicle regeneration in the adult thyroid gland, we observed the regeneration processes of murine thyroid follicles after partial resection of the lower third of the thyroid gland in 10-week-old male C57BL/6 mice. Based on sequential observation of the partially resected thyroid lobe, we found primitive follicles forming in the area corresponding to the central zone of the intact lateral thyroid lobe. The primitive thyroid follicles were multiciliated and had coarsely vacuolated cytoplasm and large vesicular nuclei. Consistently, these primitive follicular cells did not express the differentiation markers paired box gene-8 and thyroid transcription factor-1 (clone SPT24), but were positive for forkhead box protein A2 and leucine-rich repeat-containing G-protein-coupled receptor 4/GPR48. Follicles newly generated from the primitive follicles had clear or vacuolar cytoplasm with dense, darkly stained nuclei. At day 21 after partial thyroidectomy, the tall cuboidal follicular epithelial cells had clear or vacuolar cytoplasm, and the intraluminal colloid displayed pale staining. Smaller activated follicles were found in the central zone of the lateral lobe, whereas larger mature follicles were located in the peripheral zone. Based on these observations, we propose that the follicle regeneration process in the partially resected adult murine thyroid gland associated with the appearance of primitive follicular cells may be a platform for the budding of differentiated follicles in mice.

The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy.

Dexamethasone (Dex)-loaded PHEA-g-C18-Arg8 (PCA) nanoparticles (PCA/Dex) were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18) core with Dex and an arginine 8 (Arg8) peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA). The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120-140 nm and a zeta potential of 20-25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene) for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr) than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30 h, and its signaling pathway resulted in an increase in phosphorylation of Akt, observed by Western blotting. Thus, Dex within PCA/Dex/gene nanoparticles created an anti-inflammatory effect and enhanced gene expression.

Effects of Complementary Combination Therapy of Korean Red Ginseng and Antiviral Agents in Chronic Hepatitis B.

OBJECTIVES: Chronic hepatitis B management is commonly targeted at reducing viral replication. However, the currently available antiviral therapies are associated with some problems, including resistance and numerous adverse effects. Ginseng has been reported to be effective for treating viral infections such as influenza and human immunodeficiency virus. However, there are currently few studies on the effects of ginseng in chronic hepatitis B. Thus, this study investigated the effects of ginseng together with antiviral agents in chronic hepatitis B. SUBJECTS AND METHODS: This was a prospective, single-blinded, randomized controlled trial, and single-center study. Thirty-eight patients were enrolled. The control group (n = 19) was administered antiviral agents alone. The experimental group (n = 19) was administered antiviral agents along with Korean Red Ginseng powder capsules (each dose is 1 gram (two capsules), a one-day dose is 3 grams). The baseline characteristics did not differ between the two groups. Differences in several non-invasive fibrosis serologic markers (type IV collagen, hyaluronic acid, transforming growth factor-ß) and in the hepatitis B virus DNA levels were compared between the groups. RESULTS: The non-invasive fibrosis serologic markers were further decreased in the experimental group, with significant differences after treatment observed for hyaluronic acid (p = 0.032) and transforming growth factor-ß (p = 0.008), but not for type IV collagen (p = 0.174). CONCLUSIONS: This study suggests the possibility of Korean Red Ginseng as a complementary therapy for chronic hepatitis B.

Exenatide reverses dysregulated microRNAs in high-fat diet-induced obese mice.

Exenatide has beneficial effects on insulin sensitivity in several animal models; however, its mechanism of action remains unclear. Furthermore, the relationship between the effect of exenatide on the changes in the relative abundance of microRNAs (miRNAs), which play a role in regulating glucose and lipid metabolism, is not fully understood. Therefore, we assessed the effect of exenatide on miRNA expression in a high-fat diet (HFD)-induced mouse model of obesity. Both HFD control and exenatide-treated HFD mice showed similar body weight gain and increase in ß-cell mass. Insulin levels were significantly lower in exenatide-treated mice than in HFD control mice. The levels of miRNA-15a, 29c, 124a, and 375 in the pancreas were significantly increased in HFD control mice. Furthermore, the levels of miRNA-29c, 124a, and 146a in the liver and miRNA-15a, 29c, 124a, and 146a in the muscle were significantly increased. In contrast, the levels of miRNA-15a, 29c, 124a, and 375 in the serum were significantly decreased. These effects were reversed by treatment with exenatide. Our results provide experimental evidence that exenatide-mediated amelioration of insulin sensitivity is associated with antagonistic changes in the relative abundance of miRNA-15a, 29c, 124a, and 375 in tissues and serum, thus highlighting their usefulness as biomarkers for monitoring insulin sensitivity and response to exenatide treatment in experimental diabetes.

Expression profiling of microRNAs in lipopolysaccharide-induced acute lung injury after hypothermia treatment.

We investigated the expression profiles of miRNAs in acute lung injury (ALI) rats after hypothermia treatment. ALI rats were induced with lipopolysaccharide (LPS) and maintained with hypothermia (HT) or normothermia (NT) for 6 hours. HT attenuated inflammatory cell infiltration in the lung and improved biochemical indicators of multi-organ dysfunction. Nineteen miRNAs were significantly differentially expressed in the HT group compared with the NT group. miR-142, miR-98, miR-541, miR-503, miR-653, miR- 223, miR-323 and miR-196b exhibited opposite patterns of expression between the two groups. These dysregulated miRNAs were mainly involved in the immune and inflammatory response on functional annotation analyses. This study shows that HT has lung protective effects and influences expression profiles of miRNAs in ALI. And dysregulated miRNAs after HT modulate the immune and inflammation in ALI. These results suggest that dysregulated miRNAs play a role in the mechanism of the lung protective effects of HT in ALI.

Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear.

A drug delivery system to the inner ear using nanoparticles consisting of oligoarginine peptide (Arg8) conjugated to poly(amino acid) (poly(2-hydroxyethyl L-aspartamide; PHEA) was investigated to determine whether the limitations of low drug transport levels across the round window membrane (RWM) and poor transport into inner ear target cells, including hair cells and spiral ganglion, could be overcome. Three types of carrier materials, PHEA-g-C18, PHEA-g-Arg8, and PHEA-g-C18-Arg8, were synthesized to examine the effects of oligoarginine and morphology of the synthesized carriers. Nile red (NR) was used as a fluorescent indicator as well as to model a hydrophobic drug. Compared with PHEA-g-C18-NR nanoparticles, the oligoarginine-conjugated nanoparticles of PHEA-g-C18-Arg8-NR and PHEA-g-Arg8-NR entered into HEI-OC1 cells at significant levels. Furthermore, the strongest fluorescence intensity was observed in nuclei when PHEA-g-C18-Arg8 nanoparticles were used. The high uptake rates of PHEA-g-C18 and PHEA-g-C18-Arg8 nanoparticles were observed in ex vivo experiments using hair cells. After the delivery of PHEA-g-C18-Arg8 nanoparticles with reporter gene transfer, EGFP (enhanced green fluorescent protein) expression was monitored as an indicator of gene delivery. In the inner ear cells, PHEA-g-C18-Arg8 nanoparticles showed comparable or better transfection capabilities than the commercially available Lipofectamine reagent. PHEA-g-C18-Arg8 penetrated in vivo across the RWM of C57/BL6 mice with Nile red staining and GFP expression in various inner ear tissues. In conclusion, PHEA-g-C18-Arg8 nanoparticles were successfully transported into the inner ear through the intratympanic route and are proposed as promising candidates as delivery carriers to address inner ear diseases.

Antiangiogenic effects of topically administered multiple kinase inhibitor, motesanib (AMG 706), on experimental choroidal neovascularization in mice.

PURPOSE: To investigate the effect of topical motesanib, an inhibitor of receptor tyrosine kinase, on experimental choroidal neovascularization (CNV). METHODS: CNV was induced in 46 nine-week-old male C57BL/6 mice using fundus laser photocoagulation. The right eye of each mouse was treated with motesanib eye drop (4 times daily) and the left eye with vehicle eye drop (4 times daily) for 14 days. To evaluate changes in the CNV lesions, fluorescein angiography, immunofluorescence staining with CD34, and histological examinations were performed 14 days after CNV induction. The expression of phosphorylated extracellular signal-regulated kinase (ERK1/2) in choroidal tissues was determined using western blot analysis to demonstrate the inhibitory effect of topically administered motesanib on intracellular signaling pathways involved in CNV development. RESULTS: Fluorescein angiography showed that fluorescence leakage in eyes treated with topical motesanib was significantly less than in mice treated with vehicle (P=0.01). On immunofluorescence staining, the CD34-labeled area was smaller in topical motesanib-treated eyes (P<0.001). The expression level of phosphorylated ERK1/2 relative to that of total ERK1/2 decreased in eyes treated with topical motesanib compared with eyes treated with vehicle. CONCLUSION: Topical motesanib significantly reduced laser-induced CNV in the experimental mouse model.

Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles.

CONTEXT: Local delivery systems for treatment of intractable inner ear disorders have been attempted by many investigators. OBJECTIVE: To evaluate the permeability and safety of a drug delivery system for the inner ear using a poly(2-hydroxyethyl aspartamide) (PHEA) polymersome. MATERIALS AND METHODS: One-month-old male C57/BL6 mice were used. We administered the same amount of the fluorescent dye, Nile red, into the middle ear in two forms: loaded in PHEA polymersomes (NP group) or diluted in ethanol (NR group). At 1 day after administration, we harvested the cochlea and counted visible red particles in the tissues of cochlea under confocal microscopy and compared the groups. In a safety evaluation, 1 week after the same surgery, we conducted hearing tests and histological evaluations of the bulla and cochlea, and compared the results with those of the sham operation and negative control groups. RESULTS: In terms of permeability, the number of red particles in the organ of Corti was increased significantly in the NP group, and three subjects in the NP group showed uptake of red particles in inner hair cells. However, there was no statistically significant difference in the observations in the lateral wall or modiolus. In safety tests, the NP and sham-operation groups showed decreased DPOAE responses and mildly swollen middle ear mucosa, compared with the negative control group, which was thought to be the result of postoperative changes. CONCLUSIONS: PHEA nanoparticles may have utility as a drug carrier into the inner ear in terms of both permeability and safety.

PKB/Akt phosphorylation of ERRγ contributes to insulin-mediated inhibition of hepatic gluconeogenesis.

AIMS/HYPOTHESIS: Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. METHODS: We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) ß-deficient (Pkbß (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. RESULTS: We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbß (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBß-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. CONCLUSIONS/INTERPRETATION: These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.

New players in high fat diet-induced obesity: LETM1 and CTMP.

OBJECTIVE: Obesity contributes to insulin resistance and is a risk factor for diabetes. C-terminal modulator protein (CTMP) and leucine zipper/EF-hand-containing transmembrane protein 1 (LETM1) have been reported to influence the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) signaling pathway via the modulation of PKB activity, a key player for insulin signaling. However, it remains unclear whether CTMP and LETM1 are associated with PI3K/PKB signaling in mouse models of obesity. MATERIALS/METHODS: To address this question, we used two different mouse models of obesity, including high-fat diet (HFD)-induced diabetic mice and genetically modified obese mice (ob/ob mice). The levels of insulin-signaling molecules in these mice were determined by immunohistochemical and Western blot analyses. The involvement of CTMP and LETM1 in PI3K/PKB signaling was investigated in HEK293 cells by transient transfection and adenovirus-mediated infection. RESULTS: We found that the levels of insulin receptor, phosphorylated PKB, and LETM1 were lower and the level of CTMP was higher in the adipose tissue of obese mice on an HFD compared to lean mice on a chow diet. Similar results were obtained in ob/ob mice. In HEK293 cells, the activation of PKB increased the LETM1 level, and inhibition of PKB increased the CTMP level. The overexpression of CTMP suppressed the insulin-induced increase in PKB phosphorylation, which was abrogated by co-overexpression with LETM1. CONCLUSION: These results suggest that CTMP and LETM1 may participate in impaired insulin signaling in the adipose tissue of obese mice, raising the possibility that these parameters may serve as new candidate biomarkers or targets in the development of new therapeutic approaches for diabetes.