Anti-obesity effects of the dual-active adenosine A2A/A3 receptor-ligand LJ-4378.

BACKGROUND AND OBJECTIVES: A2A adenosine receptor (A2AAR)-mediated signaling in adipose tissues has been investigated as a potential target for obesity-related metabolic diseases. LJ-4378 has been developed as a dual-acting ligand with A2AAR agonist and A3 adenosine receptor (A3AR) antagonist activity. The current study aimed to investigate the anti-obesity effects of LJ-4378 and its underlying molecular mechanisms. METHODS: Immortalized brown adipocytes were used for in vitro analysis. A high-fat diet (HFD)-induced obesity and cell death-inducing DFFA-like effector A reporter mouse models were used for in vivo experiments. The effects of LJ-4378 on lipolysis and mitochondrial metabolism were evaluated using immunoblotting, mitochondrial staining, and oxygen consumption rate analyses. The in vivo anti-obesity effects of LJ-4378 were evaluated using indirect calorimetry, body composition analyses, glucose tolerance tests, and histochemical analyses. RESULTS: In vitro LJ-4378 treatment increased the levels of brown adipocyte markers and mitochondrial proteins, including uncoupling protein 1. The effects of LJ-4378 on lipolysis of adipocytes were more potent than those of the A2AAR agonist or A3AR antagonist. In vivo, LJ-4378 treatment increased energy expenditure by 17.0% (P value < 0.0001) compared to vehicle controls. LJ-4378 (1 mg/kg, i.p.) treatment for 10 days reduced body weight and fat content by 8.24% (P value < 0.0001) and 24.2% (P value = 0.0044), respectively, and improved glucose tolerance in the HFD-fed mice. LJ-4378 increased the expression levels of brown adipocyte markers and mitochondrial proteins in interscapular brown and inguinal white adipose tissue. CONCLUSION: These findings support the in vivo anti-obesity effects of LJ-4378, and suggest a novel therapeutic approach to combat obesity and related metabolic diseases.

Evaluation of a PACAP Peptide Analogue Labeled with (68)Ga Using Two Different Chelating Agents.

OBJECTIVE: The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS: For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS: Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS: NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

Polyglutamic acid-based nanocomposites as efficient non-viral gene carriers in vitro and in vivo.

A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ∼ 80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.

Effect of homobifunctional crosslinkers on nucleic acids delivery ability of PEI nanoparticles.

Polyethylenimine (PEI), a widely used cationic polymeric vector with high transfection efficiency, was converted into nanoparticles by introducing ionic and covalent crosslinkers with varying proportion of 1,6-hexanebisphosphate (HP), adipic acid (AA) and 1,4-butane dialdehyde (BA) to obtain a small library of HP-PEI (HPP), AA-PEI (AAP) and BA-PEI (BAP) nanoparticles, respectively. Particles were characterized by spectroscopic technique as well as physicochemical parameters such as size, morphology, surface charge, effect of crosslinking on buffering capacity and DNA binding ability. The entire series of nanoparticles were compared for their cytoxicity and ability to deliver genes in various cell lines. Among various nanoparticles, AAP-3 nanoparticle/DNA complex exhibited higher transfection efficiency (1.5-7.8 folds) than the native PEI (25kDa) and commercially available transfection reagents, such as GenePorter, GenePorter2, Fugene and Superfect, with cell viability >85%. The highest cell viability was observed with BAP nanoparticles (>95%). Importantly, the transfection activity of nanoparticle/DNA complexes was preserved in the presence of serum. Transfection with GFP-siRNA inhibited expression of transfected GFP gene by approximately 81-92%. All nanoparticle types (HPP, AAP and BAP) required a comparable time for entry into cells and subsequent intracellular passage from the cytoplasm to the nucleus. Intravenous delivery of (99)Tc labeled BAP-2/DNA complex to female Balb/c mice revealed the presence of the complex in most of the organs with the highest retention in liver. In conclusion, HPP, AAP and BAP nanoparticles are safe for efficient gene delivery.