No abstract

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

Cytochrome P450 (CYP) is a gene superfamily of hemoproteins that catalyze the oxidation of lipophilic substrates to more water-soluble products. Among them, the human CYP3A subfamily contains mainly three isoforms, CYP3A4, CYP3A5, and CYP3A7. 1) CYP3A4 is the predominant isoform expressed in adult human liver and intestine, 2,3) and plays a significant role in the metabolism of endogenous and exogenous compounds. 4) CYP3A5 is expressed polymorphically throughout development in the liver.5,6) CYP3A7 is a major CYP isoform in the human fetal and newborn liver. 7)The levels of expression of CYP3A isoforms are enhanced by treatment with various agents, such as rifampicin (RIF), phenobarbital, clotrimazole, and dexamethasone (DEX). [8][9][10] Unlike the fetal liver in most experimental animals, the human fetus supports the oxidative metabolism of a wide range of drugs and other xenobiotics. 11) Toxicological and pharmacological studies designed to assess the safety and efficacy of candidate drugs and chemicals in the human fetus depend greatly on extrapolation from animal models. However, it has been difficult to estimate alterations of drug metabolism by inducers of drug-metabolizing enzymes in the human fetus due to the problem of differences among species.12) Species differences in the inducibility of drug-metabolizing enzymes 13,14) result in important differences in the metabolism of drugs and potential carcinogens. The induction of CYP3A in human-derived cell lines has been studied as a model of human tissues.15) As research with human subjects, especially when pregnant, should be restricted due to ethical considerations, human fetal in vitro models have been evaluated with increasing frequency to find those that are most suitable for predicting human responses in vivo.The HepG2 cell line was established from a liver tumor biopsy specimen from a 15-year-old Caucasian male subject.16) This cell line has features of hepatoblast-like cells, because the cells express CYP3A7 17) and secrete a-fetoprotein (AFP) 16) into the cell culture medium, both proteins that are found predominantly in human fetal hepatocytes.7) However, it is unknown whether HepG2 is a useful cell line as a human fetal liver (HFL) model for experiments regarding drug metabolism, because there have been no comparisons of gene expression between HFL and HepG2 cells, and limited data are available regarding comparison of the responses of HepG2 and HFL cells to inducers.In the present study, to characterize the expression and induction of CYP in HFL cells, the basal gene expression patterns and inducibility of CYP, especially CYP3A isoforms, were compared to those in HepG2 cells. MATERIALS AND METHODS ChemicalsDulbecco's phosphate-buffered saline (PBS) was from Dainippon Pharmaceutical Co. (Osaka, Japan). Dulbecco's modified Eagle's medium and Williams' medium E were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Penicillin-streptomycin-neomycin antibiotic mixture and SuperScript First-Strand Synthesis System for reverse transcription-po...

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.