synthesized TM5441. or HFD (60% of total calories from fat) for 10?weeks, and TM5441 (20?mgkg?1 oral gavage) was given daily with the initiation of HFD. Important Results TM5441 prevented HFD\induced body weight gain and systemic insulin resistance. TM5441 normalized HFD\induced dysregulated JNK and Akt phosphorylation, suggesting that it prevents the insulin resistance of adipocytes. TM5441 also attenuated the macrophage infiltration and improved manifestation of pro\inflammatory cytokines, such as inducible nitric oxide synthase, induced from the HFD. In addition, TM5441 prevented the HFD\induced down\rules of genes involved in mitochondrial biogenesis and function, suggesting that it may prevent adipocyte swelling and dysregulation CACNA1H by keeping mitochondrial fitness. Summary and Implications Our data suggest that TM5441 may become a novel restorative agent for obesity and obesity\related metabolic disorders. AbbreviationsATGLadipose triglyceride lipaseCoxcytochrome c oxidaseFASfatty acid synthaseFFAfree fatty acidGTTglucose tolerance testH&Ehaematoxylin and eosinHFDhigh\fat dietHSLhormone\sensitive lipaseiNOSinducible nitric oxide synthaseITTinsulin tolerance testKOknockoutMCP\1monocyte chemotactic protein\1mtDNAmitochondrial DNANDnormal dietPAI\1plasminogen activator inhibitor\1PGC1PPAR coactivator\1Tfammitochondrial transcription factor ATGtriglycerideTM52755\chloro\2\[(2\[4\(diphenylmethyl)piperazin\1\yl]\2\oxoethoxyacetyl) amino]benzoateTM54415\chloro\2 [(2\[3\(furan\3\yl)phenyl]amino\2\oxoethoxy) acethyl]amino benzoic acidUCPuncoupling proteinWATwhite adipose tissue Tables of Links has not yet been explored. While the exact mechanism involved in PAI\1\induced insulin resistance is still elusive, mitochondrial dysfunction has been proposed to play an important role in the development of obesity and metabolic disorder (Patti and Corvera, 2010). A recent report showing that adipose\specific crif1 deficiency reduces mitochondrial oxidative phosphorylation and obesity (Ryu for 10?weeks. Food was freely available, except when fasted prior to the glucose and insulin tolerance assessments (ITTs). Body weight and calorie intake were measured once each week during the experimental periods. TM5441 was synthesized by Dr Toshio Miyata, and its characteristics and specificity were as described previously (Boe insulin stimulation and analysis of insulin signalling in adipose tissue, mice were fasted overnight, anaesthetized and then injected via the inferior vena cava with Humulin? (10?Ukg?1, Eli Lilly, Indianapolis, IN, Disulfiram USA). Epididymal white adipose tissue (WAT) from the left side was removed 4?min after insulin injection as described previously (Jiang for 15?min at 4C, and the serum in the supernatant was collected. Fasted plasma triglyceride (TG), glycerol, free fatty acid (FFA) and total cholesterol were measured using an EnzyChrom? colorimetric assay kit (BioAssay Systems, Hayward, CA, USA). For fasted plasma PAI\1 and insulin measurements, commercial ELISA kits (R&D Systems) were used according to the manufacturer’s instruction. Real\time quantitative reverse transcription PCR The expression of mRNAs was assessed by real\time quantitative reverse transcription PCR using a SYBR Green PCR Grasp Mix kit (Applied Biosystems, Foster City, CA, USA) with an ABI 7300 real\time PCR thermal cycler (Applied Biosystems). The mRNA expression levels of the test genes were normalized to 18S rRNA levels. The primer sequences are listed in Table?1. Table 1 Primer sequences at 4C Disulfiram for 15?min. The concentration of protein was decided using the Bradford methods (Bio\Rad Laboratories, Hercules, CA, USA), and aliquots of tissue homogenates were mixed with sample buffer made up of SDS and \mercaptoethanol and heated at 95C for 5?min. The samples were then applied to an SDS\PAGE gel and separated by electrophoresis. The proteins were transferred onto a PVDF membrane (GE Healthcare BioSciences Co., Piscataway, NJ, USA) in a transblot chamber with Tris buffer. The membrane was blocked for 1?h at room temperature with 5% skimmed milk in TBS\Tween 20 buffer, followed by an overnight incubation at 4C in a 1:1000 dilution of the indicated antibodies. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti\phospho\Akt (Ser473), anti\Akt, anti\phospho\hormone\sensitive lipase (HSL) (Ser563), anti\HSL antibody, adipose triglyceride lipase (ATGL) antibody, anti\phosphor\JNK (Thr183/Tyr185) and anti\JNK. Anti\PAI\1 and anti\\tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti\uncoupling protein (ucp)\1 was purchased from Abcam (Cambridge, MA, USA) and anti\\actin was purchased from Sigma\Aldrich (St Louis, MO, USA) for immunoblotting. The membrane was then washed and incubated with peroxidase\conjugated secondary antibody for 1?h at room temperature. The washes were repeated, and the membrane was developed with an enhanced chemiluminescence detection reagent (GE Healthcare BioSciences Co.) Disulfiram according to the manufacturer’s instructions. Positive immunoreactive bands were quantified using a densitometer (LAS\3000, FUJIFILM Corporation, Tokyo, Japan), normalized by \tubulin, and compared with each control. Statistical analysis The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis value?0.05 was considered significant. Results TM5441 prevents HFD\induced obesity and systemic insulin resistance in mice To generate a diet\induced obesity model, 10\week\old mice were placed on an HFD made up of 60% calories from fat for 10?weeks. Rapid increase of body weight was observed from 4?weeks after the initiation of HFD compared with ND containing 18% calories from fat (Physique?1A). The weight gain was manifested.