Attenuation of diet-induced obesity and induction of white fat browning with a chemical inhibitor of histone deacetylases.

BACKGROUND/OBJECTIVES: In the last decade, a strict link between epigenetics and metabolism has been demonstrated. Histone deacetylases (HDACs) have emerged as key epigenetic regulators involved in metabolic homeostasis in normal and pathologic conditions. Here we investigated the effect of the class I HDAC inhibitor MS-275 in a model of obesity induced by a high-fat diet (HFD). METHODS: C57BL6/J male mice were fed HFD for 17 weeks and then randomized in two groups, treated intraperitoneally with vehicle dimethylsulfoxide (DMSO) or with the class I selective HDAC inhibitor MS-275 every other day for 22 days. Glucose tolerance test and measurement of body temperature during cold exposure were performed. Adipose tissues and liver were phenotypically characterized through histological analysis. Gene and protein expression analysis of brown and white adipose tissues (WATs) were performed. RESULTS: MS-275 treated mice showed 10% reduction of body weight, lower adipocyte size and improved glucose tolerance. Inhibition of class I HDAC determined reduction of adipocyte size and of fat mass, paralleled by higher expression of adipose functionality markers and by increased rate of lipolysis and fatty acid ß-oxidation. MS-275 also promoted thermogenic capacity, related to 'browning' of visceral and subcutaneous WAT, showing increased expression of uncoupling protein 1. In brown adipose tissue, we observed limited effects on gene expression and only reduction of brown adipocyte size. CONCLUSIONS: This study provides evidence that class I HDAC inhibition stimulated functionality and oxidative potential of adipose tissue, improving glucose tolerance and ameliorating the metabolic profile in diet-induced obese mice.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Neuroprotective effect of simvastatin in stroke: a comparison between adult and neonatal rat models of cerebral ischemia.

Statins, the most widely used lipid lowering drugs, have been demonstrated to play a protective role in stroke. Animal studies confirmed the observations obtained in clinical trials and provided additional data on the putative mechanism/s of action underlying this beneficial effect. We have shown that simvastatin reduced the size of the infarct to a different extend, according to the animal model used. Indeed, in the rat neonatal model of hypoxia/ischemia simvastatin affords protection only when is administered before the ischemic insult. In contrast, in adult rats bearing middle cerebral artery occlusion, simvastatin exerted its beneficial effect on brain injury when injected for 3 days either before or after induction of ischemia. Studies carried out to determine the therapeutic window of simvastatin demonstrated that the protective effect is observed after a single dose and when the drug is administered within 3-6 hours after ischemia. Simvastatin-dependent activation of eNOS has been claimed to be one of the main mechanisms responsible for neuroprotection. This hypothesis is confirmed in the adult animal model where eNOS is activated by either pre- or post- simvastatin treatment but is not supported by the data obtained in the neonate where eNOS activity is not affected by drug treatment. These observations suggest that the protective effect of simvastatin on stroke may be mediated by multiple mechanisms as can be expected by its pleiotropic effects.

Myelin deterioration in Twitcher mice: motor evoked potentials and magnetic resonance imaging as in vivo monitoring tools.

We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.

Multiexponential T2-relaxation analysis in cerebrally damaged rats in the absence and presence of a gadolinium contrast agent.

An analysis of the multiexponential relaxation of transverse nuclear magnetization with and without a gadolinium-based paramagnetic contrast agent in spontaneously hypertensive stroke-prone rats (SHR-SP) and in the rat model of ischemia induced by middle cerebral artery occlusion is described. From the multiexponential relaxation, the presence of two T(2) relaxation times in the range of 0.03-0.5 s, T(2A) (shortest) and T(2B) (longest), with very different relative weights (respectively, A and B), is evidenced. In our models of cerebral damage, the changes in A and B were more evident than those in T(2A) and T(2B). The two T(2) values were interpreted as belonging to water molecules in two different compartments; therefore, the difference between the damaged and normal regions revealed by means of standard T(2)-weighted images is suggested to be due to a different water distribution in the two compartments, rather than different T(2)'s. The T(2) relaxation in the SHR-SP stroke model is analyzed for the first time using a multiexponential method. The power of a detailed analysis of MRI relaxation times is confirmed by the correspondence between the revealed changes in T(2A), T(2B), A and B, and the known T(2)W and DWI results about blood-brain barrier functionality.

The neuroprotective activity of the glycine receptor antagonist GV150526: an in vivo study by magnetic resonance imaging.

The neuroprotective activity of GV150526 (3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt), a selective glycine receptor antagonist of the NMDA receptor, has been evaluated by magnetic resonance imaging (MRI) in a rat model of middle cerebral artery occlusion. The aim of the work was to evaluate, using an in vivo method, whether GV150526 was able to reduce the extent of ischemic brain damage when administered both before and after (6 h) middle cerebral artery occlusion. GV150526 was administered at a dose of 3 mg/kg i.v. T2-weighted (T2W) and diffusion weighted (DW) images were acquired at 6, 24 and 144 h after the establishment of the cerebral ischemia. Substantial neuroprotection was demonstrated at all investigated time points when GV150526 was administered before the ischemic insult. The ischemic volume was reduced by 84% and 72%, compared to control values, when measured from T2W and DW images, acquired 24 h after middle cerebral artery occlusion. Administration of the same dose of GV150526, 6 h post-ischemia, also resulted in a significant (p < 0.05) neuroprotection. The ischemic volume was reduced by 48% from control values when measured from T2W images and by 45% when measured from DW images. No significant difference was found between volumes of brain ischemia obtained by either MRI or triphenyltetrazolium chloride staining. These data confirm the potential neuroprotective activity of the glycine receptor antagonist GV150526 when administered either before or up to 6 h after ischemia.

Acute-phase proteins before cerebral ischemia in stroke-prone rats: identification by proteomics.

BACKGROUND AND PURPOSE: A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. METHODS: Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. RESULTS: Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, alpha(2)-HS-glycoprotein, kallikrein-binding protein, alpha(1)-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. CONCLUSIONS: In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

Chemical shift imaging at 4.7 tesla of brown adipose tissue.

In vivo distinction between small deposits of brown adipose tissue (BAT) and surrounding tissues may be difficult. In this article, we propose an experiment paradigm, based on techniques of chemical shift magnetic resonance imaging (CSI), which can improve the methods presently available for the study of BAT. Male rats were examined in an imager-spectrometer equipped with a 4.7 T magnet. Proton spectra of isolated BAT deposits showed that both fat and water protons contributed significantly to the genesis of the magnetic resonance signal. An equivocal definition of BAT deposits was obtained by three (respectively, spin-echo, water-selective, and fat-selective) images. The spin-echo (SE), T1-weighted image provided the best anatomical description of the structures. The images selective for fat-protons displayed the degree of lipid accumulation in each area. The images selective for water-protons provided an internal control of adipose tissue localization. The proposed paradigm allows an unequivocal definition of BAT deposits and appears particularly useful in studies where experimental manipulation (i.e., cold acclimation or drug treatment) produces changes in this issue.

The microvascular system in ischemic cortical lesions.

In the rat, normal blood flow can be restored in the territory of the occluded artery after an arterial occlusion. This event has been attributed to changes in the collateral vessels supplying the territory of the occluded artery. Since only a limited amount of data is available about the plasticity of the microvascular system after a cortical ischemic lesion, in the present study we have evaluated whether the restoration of blood flow to normal levels in the territory of the middle cerebral artery after permanent ischemia is due only to flow through preexisting collateral vessels or also to the development of new microvessels. Middle cerebral artery occlusion was performed in 45 rats. After 24 h of ischemia, magnetic resonance imaging was used to select 16 rats with cortical lesions of similar size and location. After 2 weeks, vascular corrosion casts were obtained from 8 rats by injection of low-viscosity resin and observed by scanning electron microscopy. A correlative light and electron microscopy study was performed using the remaining 8 rats. Two different patterns of vascular modifications were found, one dorsal and one ventral to the lesion. The dorsal portion of the lesion was vascularized by collateral arteries originating from the anterior or posterior cerebral arteries. Collateral trunks showed a meandering course, mainly in the occipital pole. In the ventral portion of the lesion a complex microvascular system was found characterized by an intense vascular proliferation. The arterioles showed a parallel, candelabrum-like pattern with dichotomic branching. Contraction rings were frequently seen. The capillaries showed a sinusoid-like structure, with a large lumen and a continuous endothelium with many micropinocytotic vesicles. A peripheral ring-shaped venous sinus was composed of a network of flat vessels. These results give the first comprehensive description of the microvascular modifications in a focal model of infarct and suggest that the restoration of blood flow to normal levels described in the territory of the middle cerebral artery after permanent ischemia may be due not only to flow through collateral vessels but also to the development of a new vascular system originating mainly from branches of the middle cerebral artery before the occlusion point.

Evaluation of frog gastric mucous secretion by 7T magnetic resonance imaging.

This work describes a new method to evaluate the action of drugs on gastric mucus secretion by 7 T MRI. A polyethylene probe was inserted into the stomach of frogs, a water washing cycle was performed, air was inflated into the stomach, and the probe was removed. Drugs were administered topically by mixing them with the water used for stomach washing, and a series of images was collected. In untreated animals, a mucous layer emitting a high intensity signal covered the gastric mucosa. Atropine administration strongly reduced the mucous layer thickness. After pilocarpine administration, MRI images showed that the mucous layer was thicker than in controls. After metacholine administration, the thickness of the mucous layer was increased in comparison with both controls and pilocarpine-treated animals, in accord with the expected drug-induced increase of mucous secretion. Quantitative analysis of the mucous layer thickness on MRI images confirmed the above findings. In conclusion, these results indicate that MRI at 7 T is a sensitive in vivo screening test to evaluate the effect of drugs on gastric mucus secretion in absence of surgical manipulation, thereby enabling morphofunctional studies that cannot be performed by conventional methods.