Satoshi Kimura

Promoter Analysis of the Mouse Sterol Regulatory Element-binding Protein-1c Gene

Recent data suggest that sterol regulatory-binding protein (SREBP)-1c plays a key role in the transcriptional regulation of different lipogenic genes mediating lipid synthesis as a key regulator of fuel metabolism. SREBP-1c regulates its downstream genes by changing its own mRNA level, which led us to sequence and analyze the promoter region of the mouse SREBP-1c gene. A cluster of putative binding sites of several transcription factors composed of an NF-Y site, an E-box, a sterol-regulatory element 3, and an Sp1 site were located at −90 base pairs of the SREBP-1c promoter. Luciferase reporter gene assays indicated that this SRE complex is essential to the basal promoter activity and confers responsiveness to activation by nuclear SREBPs. Deletion and mutation analyses suggest that the NF-Y site and SRE3 in the SRE complex are responsible for SREBP activation, although the other sites were also involved in the basal activity. Gel mobility shift assays demonstrate that SREBP-1 binds to the SRE3. Taken together, these findings implicate a positive loop production of SREBP-1c through the SRE complex, possibly leading to the overshoot in induction of SREBP-1c and its downstream genes seen in the livers of refed mice. Furthermore, reporter assays using larger upstream fragments indicated another region that was inducible by addition of sterols. The presence of the SRE complex and a sterol-inducible region in the same promoter suggests a novel regulatory link between cholesterol and fatty acid synthesis.

Elimination of Cholesterol Ester from Macrophage Foam Cells by Adenovirus-mediated Gene Transfer of Hormone-sensitive Lipase

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol.

Function of the N-terminal region of the phosphate-sensing histidine kinase, SphS, in Synechocystis sp. PCC 6803

In Synechocystis sp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS in Escherichia coli indicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi availability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Pi availability.

Activation of glycogenolysis by the reduction in the extracellular calcium concentration in verapamil-prefused rat liver

In an attempt to elucidate the role of Ca2+ flux in the initial events of hepatic glycogenolysis, extracellular Ca2+ concentration was manipulated in rat liver perfused with Ca2+ antagonistic drugs. After the liver had been perfused with a buffer containing verapamil and 1 mM CaCl2, either the addition of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N-tetraacetic acid to the perfusate or the replacement of the perfusate with Ca2+-free buffer caused a rapid increase in glucose output as well as 45Ca2+ efflux. Substitution of diltiazem, but not 5-20 mM LaCl2, for verapamil also stimulated glucose output and 45Ca2+ efflux. However, when Ca+-free buffer was used throughout the experiment, any modes of verapamil or diltiazem perfusion were without significant effects on glucose output or Ca2+ efflux. The increases in glucose output and 45Ca2+ efflux were not affected by either 20 microM phentolamine or 300 microM ouabain, but they were inhibited significantly by 10-100 microM trifluoperazine. These results indicate that rapid decline in the extracellular Ca2+ concentration in verapamil- or diltiazem-perfused liver initiates the change in Ca2+ equilibrium on or across plasma membrane and activates glycogenolysis through a Ca2+-dependent mechanism.