Using yeast two-hybrid screen, we previously isolated HELZ2 (helicase with zinc finger 2, transcriptional coactivator) that functions as a coregulator of peroxisome proliferator-activated receptorγ (PPARγ). To further delineate its molecular function, we here identified thyroid hormone receptor-associated protein3 (THRAP3), a putative component of the Mediator complex, as a protein stably associating with HELZ2 using immunoprecipitation coupled with mass spectrometry analyses. In immunoprecipitation assays, Thrap3 could associate with endogenous Helz2 as well as Pparg in differentiated 3T3-L1 cells. HELZ2 interacts with the serine/arginine-rich domain and Bcl2 associated transcription factor1-homologous region in THRAP3, whereas THRAP3 directly binds 2 helicase motifs in HELZ2. HELZ2 and THRAP3 synergistically augment transcriptional activation mediated by PPARγ, whereas knockdown of endogenous THRAP3 abolished the enhancement by HELZ2 in reporter assays. Thrap3, similar to Helz2, is evenly expressed in the process of adipogenic differentiation in 3T3-L1 cells. Knockdown of Thrap3 in 3T3-L1 preadipocytes using short-interfering RNA did not influence the expression of Krox20, Klf5, Cebpb, or Cebpd during early stages of adipocyte differentiation, but significantly attenuated the expression of Pparg, Cebpa, and Fabp4/aP2 and accumulation of lipid droplets. Pharmacologic activation of Pparg by troglitazone could not fully restore the differentiation of Thrap3-knockdown adipocytes. In chromatin immunoprecipitation assays, endogenous Helz2 and Thrap3 could be co-recruited, in a ligand-dependent manner, to the PPARγ-response elements in Fabp4/aP2 and Adipoq gene enhancers in differentiated 3T3-L1 cells. These findings collectively suggest that Thrap3 could play indispensable roles in terminal differentiation of adipocytes by enhancing PPARγ-mediated gene activation cooperatively with Helz2.

Obesity arises from impaired energy balance, which is centrally coordinated by leptin through activation of the long form of leptin receptor (Leprb). Obesity causes central leptin resistance. However, whether enhanced peripheral leptin sensitivity could overcome central leptin resistance remains obscure. A peripheral metabolic organ targeted by leptin is the liver, with low Leprb expression. We here show that mice fed a high-fat diet (HFD) and obese patients with hepatosteatosis exhibit increased expression of hepatic helicase with zinc finger 2, a transcriptional coactivator (Helz2), which functions as a transcriptional coregulator of several nuclear receptors, including peroxisome proliferator-activated receptor γ in vitro. To explore the physiological importance of Helz2, we generated Helz2-deficient mice and analyzed their metabolic phenotypes. Helz2-deficient mice showing hyperleptinemia associated with central leptin resistance were protected against HFD-induced obesity and had significantly up-regulated hepatic Leprb expression. Helz2 deficiency and adenovirus-mediated liver-specific exogenous Leprb overexpression in wild-type mice significantly stimulated hepatic AMP-activated protein kinase on HFD, whereas Helz2-deficient db/db mice lacking functional Leprb did not. Fatty acid-β oxidation was increased in Helz2-deficeint hepatocytes, and Helz2-deficient mice revealed increased oxygen consumption and decreased respiratory quotient in calorimetry analyses. The enhanced hepatic AMP-activated protein kinase energy-sensing pathway in Helz2-deficient mice ameliorated hyperlipidemia, hepatosteatosis, and insulin resistance by reducing lipogenic gene expression and stimulating lipid-burning gene expression in the liver. These findings together demonstrate that Helz2 deficiency ameliorates HFD-induced metabolic abnormalities by stimulating endogenous hepatic Leprb expression, despite central leptin resistance. Hepatic HELZ2 might be a novel target molecule for the treatment of obesity with hepatosteatosis.

PRIMARY ALDOSTERONISM (PA) has been reported to affect at least 6~10% of patients with essential hypertension [1][2][3][4][5] and is caused mostly by an adrenal adenoma (aldosterone-producing adenoma, APA) or by adrenal hyperplasia (idiopathic hyperaldosteronism, IHA). Although the tumorigenesis of APA remained unclear, somatic mutations of the potassium Characteristics of Japanese aldosterone-producing adenomas with KCNJ5 mutations Abstract. Somatic mutations in KCNJ5 gene have been identified in patients with adrenal aldosterone-producing adenomas (APAs). We previously reported that Japanese patients with APAs had distinct characteristics from patients in Western countries; i.e. they had a high frequency of KCNJ5 mutations and exhibited a frequent association with cortisol cosecretion. Therefore, APAs among Japanese patients may have different features from those in Western countries. We added recent cases, examined 47 cases (43% male) of APAs, including clinicopathological features, KCNJ5 mutations, and the mRNA levels of several steroidogenic enzymes, and compared the results obtained to those reported in other countries. While the prevalence of KCNJ5 mutations is approximately 40% in Western countries, 37 APA cases (78.7%) showed mutations: 26 with p.G151R and 11 with p.L168R. Although a significant gender difference has been reported in the frequency of KCNJ5 mutations in Europe, we did not find any gender difference. However, the phenotypes of Japanese patients with mutations were similar to those of patients in Western countries; patients were younger and had higher plasma aldosterone levels, lower potassium levels, and higher diastolic blood pressure. Reflecting these phenotypes, APAs with mutations had higher CYP11B2 mRNA levels. However, in contrast to APAs in Western countries, Japanese APAs with mutations showed lower CYP11B1, CYP17A1, and CYP11A1 mRNA levels. These findings demonstrated that Japanese APA patients may have distinct features including a higher prevalence of KCNJ5 mutations, no gender difference in the frequency of these mutations, and characteristics similar to the zona glomerulosa.

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Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the −309/−60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at −84/−68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1α expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1α may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1. Angiopoietin-like proteins (Angptls) are a family of proteins structurally similar to angiopoietins. Previous reports on Angptls have identified them as key regulators of circulating triglyceride(TG) levels, implicating them as potential new drug targets for treatment of metabolic syndrome 1,2 ANGPTL8, alternatively called the TD26, lipasin, C19orf80, or RIFL (refeeding induced fat and liver) gene, was originally identified as a novel adipocyte-enriched insulin target gene with a role in lipid metabolism 3-5. (A previously reported identification of Angptl8 as 'Betatrophin, ' a factor controlling the differentiation and proliferation of pancreatic β cells 6 , retracted article), was later determined to be incorrect 7-10. Ren et al. found that the RIFL gene encodes a predicted protein of 22kD with homology to Angptl3 3 , Furthemore, the murine white and brown adipose tissue (WAT and BAT) and liver were highly enriched in the RIFL transcript, and the level increased ~80-fold in WAT and 12-fold in liver, following 8 h refeedings of fasting mice 3. Several studies with Angptl8-deficient or Angptl8-overxpressing rodents have demonstrated that Angptl8, which is homologous to the N-terminal domain of Angptl3, modulates circulating TG clearance by inhibiting lipoprotein lipase (LPL) activity in the presence of Angptl3 3-5,7,8,11. Recent human studies have shown that serum ANGPTL8 levels are increased in subjects with Type 1 diabetes (T1D) 12 , obesity 13 , Type 2 diabet...