Adiponectin inhibits KISS1 gene transcription through AMPK and specificity protein-1 in the hypothalamic GT1-7 neurons.

Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.

Globular adiponectin regulates energy homeostasis through AMP-activated protein kinase-acetyl-CoA carboxylase (AMPK/ACC) pathway in the hypothalamus.

Adiponectin is a newly researched adipokine which participates in the regulation of energy homeostasis. AMP-activated protein kinase (AMPK) represents an energy sensor that responds to hormone and nutrition status in vivo and exerts a regulatory effect in the hypothalamus and multiple peripheral tissues. We investigated the possible mechanisms involved in appetite regulation by adiponectin in vitro with GT1-7 cells, a mouse immortalized hypothalamic neuron. The results showed that adiponectin increased the phosphorylation of AMPK, activated AMPK phosphorylated and inactivated acetyl-CoA carboxylase (ACC), and subsequently increased expression of agouti-related peptide (AgRP) mRNA. Our results also indicated that adiponectin had no effect on signal transducer and activator of transcription (STAT3). Together these findings suggest that adiponectin regulated energy homeostasis through the AMPK/ACC pathway but not the JAK/STAT3 pathway in the hypothalamus.

[Construction of GSK-3beta-targeting RNAi adenovirus vector and the influence of Wnt/beta-catenin pathway in proliferation of human thyrocytes].

OBJECTIVE: To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3beta (GSK-3beta) and to observe its gene knockdown effect on the expression of GSK-3beta, and to explore the effect of Wnt/beta-catenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector. METHODS: An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3beta gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney (HEK) 293A cells to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3beta specific RNAi adenovirus. The GSK-3beta gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups: infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of beta-catenin. RESULTS: The GSK-3beta expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST (all P<0.05). The expression of beta-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group (both P<0.05). BrdU assay suggested that the proliferation rates 1, 3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST (all P<0.05). CONCLUSION: RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently. The Wnt/beta-catenin pathway plays an important role in the regulation of proliferation of human thyrocytes.

[Effects of captopril on myocardial tissue energy metabolism and inflammation in rats with diabetic cardiomyopathy].

OBJECTIVE: To study the protective mechanism of captopril in diabetic cardiomyopathy by means of DNA microarray. METHODS: Rat models of diabetic cardiomyopathy were divided into test and control groups (n=5), and the rats in the test group were given oral captopril (1.5 mg/kg b.w.) for 15 weeks. DNA microarray was prepared by blotting the PCR products of 4 000 rat cDNAs onto a specially treated glass slides. The probes were prepared by labeling the mRNA from the myocardial tissue of both control and test groups with Cy3-d UTP and Cy5-d UTP separately through reverse transcription. The arrays were then hybridized against the cDNA probes and the fluorescent signals scanned. RESULTS: The expression of genes in relation to fatty acid b oxidation, mitochondrial proton-electron coupling and oxidative phosphorylation, and that of dithiolethione-inducible gene-1 were up-regulated, while the dimethylarginine dimethylaminohydrolase gene expression was obviously lowered in the test group in comparison with those of the control group. CONCLUSION: Captopril may protect the myocardial tissue through improving myocardial energy supply and depressing inflammatory reaction.

[Expression of energy metabolism-related genes in myocardial tissues of rats with diabetic cardiomyopathy].

OBJECTIVE: To study the pathogenesis of diabetic cardiomyopathy using DNA microarray. METHODS: Experimental rats were grouped into a diabetic cardiomyopathy and a normal control group. The cDNA probes were prepared by labeling the mRNA extracted from normal tissue and tissue of diabetic cardiomyopathy with Cy3-dUTP and Cy5-dUTP respectively through reverse transcription. DNA microarray were constructed by spotting PCR products of 4 000 rat cDNAs onto a specially treated glass slides, and were then hybridized against the cDNA probes followed by fluorescent signals scanning. RESULTS: The expression of energy metabolism-related genes were lower in the tissues of diabetic cardiomyopathy than in normal tissue. CONCLUSION: The energy metabolism disorder may play an important role in the pathogenesis of diabetic cardiomyopathy.