Tankyrase inhibition ameliorates lipid disorder via suppression of PGC-1α PARylation in db/db mice.

OBJECTIVE: Human TNKS, encoding tankyrase 1 (TNKS1), localizes to a susceptibility locus for obesity and type 2 diabetes mellitus (T2DM). Here, we addressed the therapeutic potential of G007-LK, a TNKS-specific inhibitor, for obesity and T2DM. METHODS: We administered G007-LK to diabetic db/db mice and measured the impact on body weight, abdominal adiposity, and serum metabolites. Muscle, liver, and white adipose tissues were analyzed by quantitative RT-PCR and western blotting to determine TNKS inhibition, lipolysis, beiging, adiponectin level, mitochondrial oxidative metabolism and mass, and gluconeogenesis. Protein interaction and PARylation analyses were carried out by immunoprecipitation, pull-down and in situ proximity ligation assays. RESULTS: TNKS inhibition reduced body weight gain, abdominal fat content, serum cholesterol levels, steatosis, and proteins associated with lipolysis in diabetic db/db mice. We discovered that TNKS associates with PGC-1α and that TNKS inhibition attenuates PARylation of PGC-1α, contributing to increased PGC-1α level in WAT and muscle in db/db mice. PGC-1α upregulation apparently modulated transcriptional reprogramming to increase mitochondrial mass and fatty acid oxidative metabolism in muscle, beiging of WAT, and raised circulating adiponectin level in db/db mice. This was in sharp contrast to the liver, where TNKS inhibition in db/db mice had no effect on PGC-1α expression, lipid metabolism, or gluconeogenesis. CONCLUSION: Our study unravels a novel molecular mechanism whereby pharmacological inhibition of TNKS in obesity and diabetes enhances oxidative metabolism and ameliorates lipid disorder. This happens via tissue-specific PGC-1α-driven transcriptional reprogramming in muscle and WAT, without affecting liver. This highlights inhibition of TNKS as a potential pharmacotherapy for obesity and T2DM.

USF1 deficiency activates brown adipose tissue and improves cardiometabolic health.

USF1 (upstream stimulatory factor 1) is a transcription factor associated with familial combined hyperlipidemia and coronary artery disease in humans. However, whether USF1 is beneficial or detrimental to cardiometabolic health has not been addressed. By inactivating USF1 in mice, we demonstrate protection against diet-induced dyslipidemia, obesity, insulin resistance, hepatic steatosis, and atherosclerosis. The favorable plasma lipid profile, including increased high-density lipoprotein cholesterol and decreased triglycerides, was coupled with increased energy expenditure due to activation of brown adipose tissue (BAT). Usf1 inactivation directs triglycerides from the circulation to BAT for combustion via a lipoprotein lipase-dependent mechanism, thus enhancing plasma triglyceride clearance. Mice lacking Usf1 displayed increased BAT-facilitated, diet-induced thermogenesis with up-regulation of mitochondrial respiratory chain complexes, as well as increased BAT activity even at thermoneutrality and after BAT sympathectomy. A direct effect of USF1 on BAT activation was demonstrated by an amplified adrenergic response in brown adipocytes after Usf1 silencing, and by augmented norepinephrine-induced thermogenesis in mice lacking Usf1. In humans, individuals carrying SNP (single-nucleotide polymorphism) alleles that reduced USF1 mRNA expression also displayed a beneficial cardiometabolic profile, featuring improved insulin sensitivity, a favorable lipid profile, and reduced atherosclerosis. Our findings identify a new molecular link between lipid metabolism and energy expenditure, and point to the potential of USF1 as a therapeutic target for cardiometabolic disease.

Size-dependent passage of liposome nanocarriers with preserved posttransport integrity across the middle-inner ear barriers in rats.

OBJECTIVE: The goal of this study was to evaluate the impact of liposome nanocarrier size on the efficacy of its transport across the middle-inner ear barriers. MATERIALS AND METHODS: The dynamic distribution of liposome nanocarriers encapsulating gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (LPS+Gd-DOTA) of sizes 95, 130, and 240 nm were observed with a 4.7 T magnetic resonance machine after transtympanic injection in Wistar rats. Histology was performed with confocal microscopy using TRITC conjugated LPS+Gd-DOTA. The integrity of the LPS+Gd-DOTA after transportation was evaluated using cryo-transmission electron microscopy (Cryo-TEM). RESULTS: Size-dependent transport of the LPS+Gd-DOTA across the middle-inner ear barriers was shown using magnetic resonance imaging, which indicated that the 95-nm nanocarrier showed the significantly highest transport percentage, that the 130-nm nanocarrier showed moderate transport, and that the 240 nm nanocarrier showed the lowest transport. Histologic examinations showed that the LPS+Gd-DOTA were distributed in the epithelial cells of the utricle, capillaries of the spiral ligament, and the spiral ganglion cells. LPS+Gd-DOTA remained intact in the perilymph after transportation. CONCLUSION: The nanocarrier delivery strategy used in this work could be effective in the development of novel inner ear treatments.

Oval window transport of Gd-dOTA from rat middle ear to vestibulum and scala vestibuli visualized by in vivo magnetic resonance imaging.

OBJECTIVES: We tested our hypothesis that the oval window (OW) potentially functions as a route to carry substances from the middle ear to the vestibulum and then the scala vestibuli through the annular ligament across the stapediovestibular joint. METHODS: Gd-DOTA was either injected into the lateral attic compartment of rats with a high-performance polyimide tube in a selective OW delivery group, or administered to the middle ear cavity of two groups of rats in which the OW was either sealed or not sealed. The dynamic uptake of Gd-DOTA in the inner ear was visualized with a 4.7-T magnetic resonance imaging machine. RESULTS: In the selective OW delivery group, Gd-DOTA appeared in the vestibulum and in the basal turn of the scala vestibuli but not in the scala tympani on T1-weighted images acquired at 10 minutes after Gd-DOTA administration (the earliest available time point of magnetic resonance imaging). In the sealed-OW group, immediate uptake of Gd-DOTA was absent in the vestibulum and scala vestibuli. Measurement of the signal ratio of the vestibulum to that of the scala tympani showed that selective OW delivery induced the greatest signal ratio and that sealing of the OW induced the lowest signal ratio. CONCLUSIONS: The OW is a genuine and efficient pathway to transport Gd-DOTA from the middle ear to the vestibulum.

MRI manifestation of novel superparamagnetic iron oxide nanoparticles in the rat inner ear.

AIM: Superparamagnetic iron oxide nanoparticles hierarchically coated with oleic acid and Pluronic F127 copolymers (POA@SPION) have shown exceptional T2 contrast enhancement. The aim of the present work was to investigate the MRI manifestation of POA@SPION in the inner ear. MATERIALS & METHODS: A total of 26 male Wister rats were selected for testing POA@SPION administered through intracochlear, intratympanic and intravenous routes. MRI was performed with a 4.7 T MR scanner. RESULTS & CONCLUSION: POA@SPION can be introduced into the perilymph space, after which it becomes widely distributed and can demonstrate the integrity of the perilymph-endolymph barrier. Positive highlighting of the endolymph compartment against the darkened perilymph was visualized for the first time. POA@SPION passed through the middle-inner ear barriers in only small amounts, but stayed in the perilymph for 3 days. They did not traverse the blood-perilymph barrier or blood-endolymph barrier. The inner ear distribution of POA@SPION was confirmed by histology. POA@SPION is a promising T2 negative contrast agent.

Gadolinium uptake in the rat inner ear perilymph evaluated with 4.7 T MRI: a comparison between transtympanic injection and gelatin sponge-based diffusion through the round window membrane.

OBJECTIVE: To investigate the inner ear uptake of the contrast agent gadolinium administered intratympanically through transtympanic injection or using a gelatin sponge placed on the round window. MATERIALS AND METHODS: The T1 contrast agent Gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) was administrated to the middle ear cavities of 12 male Wistar rats by a transtympanic injection technique (TTI group; 40 microl), gelatin sponge diffusion (GelD40 group; 40 microl), or gelatin sponge diffusion (GelD8 group; 8 microl). Magnetic resonance imaging was performed with a 4.7-T scanner using a T1-weighted 2-dimensional rapid acquisition with relaxation enhancement sequence and a high-resolution T1-weighted 3-dimensional rapid acquisition with relaxation enhancement sequence. RESULTS: The uptake of Gd-DOTA into the perilymph was more pronounced at 3 than at 1 hour after intratympanic administration for all methods studied. Transtympanic injection and GelD40 induced similar uptake of Gd-DOTA in the lower turns of the rat cochlea. Transtympanic injection induced less efficient Gd-DOTA uptake in the apex than GelD40. GelD8 was less efficient at Gd-DOTA uptake than either TTI or GelD40. CONCLUSION: Both TTI and GelD40 are able to efficiently deliver substances to inner ear destinations. Considering its simplicity, TTI is more practical for use in the clinic for the administration of substances to the inner ear, although it provided less efficient uptake in the apex than GelD40.

Differential passage of gadolinium through the mouse inner ear barriers evaluated with 4.7T MRI.

Magnetic resonance imaging (MRI), supplemented by contrast agents, is a powerful tool that can be used to visualise the structures of the inner ear in vivo and assess some aspects of physiology, such as the permeability of agents through membranes. The mouse is an excellent animal species for investigating human diseases, including hearing loss but detailed MRI studies with contrast have not been reported. In this work, we aimed to demonstrate the limits of MR imaging resolution of the fine inner ear structures in the mouse and to explore the permeability of the intracochlear barriers to gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) administered by intravenous injection (IV) or intratympanic (IT) routes. Twenty-three female FVB mice were imaged with a 4.7-T MR scanner using both 2D and high resolution 3D sequences. Inner ear region of interest (ROI) signal intensities and perilymph volumes were evaluated. Finer structures were studied using 3D acquisition and reconstruction techniques and comparisons were made to similarly oriented histological sections that were examined by light microscopy. Gd-DOTA enhancement occurred in the perilymphatic compartment and highlighted the contiguous inner ear structures, but enhancement did not appear within the endolymph. The dynamic uptake of Gd-DOTA in the perilymphatic compartments reached an initial plateau 80min after IV administration and continued to slightly increase to a maximum level by 100min. The perilymph volume demonstrated by Gd-DOTA uptake was statistically significantly larger in the IV group (1.72mm(3)) than in the IT group (1.28mm(3)) (p<0.05).

In vivo MRI reveals the dynamics of pathological changes in the brains of cathepsin D-deficient mice and correlates changes in manganese-enhanced MRI with microglial activation.

Cathepsin D (CTSD; EC 3.4.23.5) is essential for normal development and/or maintenance of neurons in the central nervous system: its deficiency causes a devastating neurological disorder with severely shortened life span in man, sheep and mouse. Neuropathologically, the CTSD deficiencies are characterized by selective neuronal degeneration, gliosis and accumulation of autofluorescent proteinaceous storage material in neurons. Our aim was to study the dynamics behind the pathological alterations occurring in the brains of CTSD-deficient (CTSD-/-) mice by using in vivo magnetic resonance imaging (MRI) and histology. In order to do this, we measured T(2) signal intensity (SI), apparent diffusion coefficient, area and volume of multiple brain structures from MR images acquired using T(2)-, T(1)- and diffusion-weighted sequences at three time points during disease progression. MRI revealed no differences in the brains between CTSD-/- and control mice at postnatal day 15+/-1 (P15+/-1), representing an initial stage of the disease. In the intermediate stage of the disease, P19(+/-1), SI alterations in the thalami of the affected mice became evident in both T(1)- and T(2)-weighted images. The terminal stage of the disease, P25, was characterized by marked alterations in the T(2) SI, apparent diffusion coefficient and volume of multiple brain structures in CTSD-/- mice. In addition, manganese enhanced high-resolution T(1)-weighted 3D sequences (MEMRI) and histological stainings revealed that the hyperintense signal areas in MEMRI matched perfectly with areas of microglial activation in the brains of CTSD-/- mice at the terminal disease stage. In conclusion, the SI alterations in the thalami of CTSD-/- mice preceded other changes, and the degenerative process was greatly enhanced at the age P19(+/-1), leading to severely reduced brain volume in just 6 days.

Mast cell blocking reduces brain edema and hematoma volume and improves outcome after experimental intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is associated with high mortality and disability, and there is no widely approved clinical therapy. Poor outcome after ICH results mostly from a mass effect owing to enlargement of the hematoma and brain swelling, leading to displacement and disruption of brain structures. Cerebral mast cells (MC) are resident inflammatory cells that are located perivascularly and contain potent vasoactive, proteolytic, and fibrinolytic substances. We previously found pharmacological MC stabilization and genetic MC deficiency to be associated with up to 50% reduction of postischemic brain swelling in rats. Here, we studied the role of MC and MC stabilization in ICH using in vivo magnetic resonance imaging and ex vivo digital imaging for calculating brain edema and hematoma volume. In a rat ICH model of autologous blood injection into the basal ganglia, four groups of Wistar rats received either saline or sodium cromoglycate (MC stabilizer, two groups) or compound 48/80 (MC degranulator). Evaluated 24 h later, MC stabilization had resulted in highly significantly better neurologic scores (P<0.001), decrease mortality (P=0.002), less brain swelling (P<0.001), and smaller hematoma volume growth (P<0.001) compared with saline and compound 48/80. Moreover, to support our hypothesis, we induced ICH in MC-deficient rats and their wild-type littermates (WT). MC-deficient rats responded with significantly better neurologic scores (P<0.001), decrease mortality (0% versus 25%), less brain swelling (P<0.05), and smaller hematoma growth (P<0.05) than WT. The role of MC deserves a close evaluation as a potential target in the development of novel forms of ICH therapy.

Dose planning with comparison to in vivo dosimetry for epithermal neutron irradiation of the dog brain.

Boron neutron capture therapy (BNCT) is an experimental type of radiotherapy, presently being used to treat glioblastoma and melanoma. To improve patient safety and to determine the radiobiological characteristics of the epithermal neutron beam of Finnish BNCT facility (FiR 1) dose-response studies were carried on the brain of dogs before starting the clinical trials. A dose planning procedure was developed and uncertainties of the epithermal neutron-induced doses were estimated. The accuracy of the method of computing physical doses was assessed by comparing with in vivo dosimetry. Individual radiation dose plans were computed using magnetic resonance images of the heads of 15 Beagle dogs and the computational model of the FiR 1 epithermal neutron beam. For in vivo dosimetry, the thermal neutron fluences were measured using Mn activation foils and the gamma-ray doses with MCP-7s type thermoluminescent detectors placed both on the skin surface of the head and in the oral cavity. The degree of uncertainty of the reference doses at the thermal neutron maximum was estimated using a dose-planning program. The estimated uncertainty (+/-1 standard deviation) in the total physical reference dose was +/-8.9%. The calculated and the measured dose values agreed within the uncertainties at the point of beam entry. The conclusion is that the dose delivery to the tissue can be verified in a practical and reliable fashion by placing an activation dosimeter and a TL detector at the beam entry point on the skin surface with homogeneous tissues below. However, the point doses cannot be calculated correctly in the inhomogeneous area near air cavities of the head model with this type of dose-planning program. This calls for attention in dose planning in human clinical trials in the corresponding areas.