Naonobu Yoshizuka

Purification, cDNA Cloning, and Expression of UDP-Gal: Glucosylceramide {beta}-1,4-Galactosyltransferase from Rat Brain

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse β-1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to β-1,4-GlcNAc in glycoproteins.

cDNA cloning and expression of human lactosylceramide synthase

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.

Expression of Uncoupling Proteins in Human Skin and Skin-Derived Cells

Uncoupling protein (UCP) is a mitochondrial membrane protein that uncouples oxidative phosphorylation. The physiological function of major isoforms of UCPs is related to the control of body temperature and reactive oxygen species production. Although skin is an important organ for heat radiation and protection against stress, the expression and function of UCPs in the skin have remained unclear. The expression of UCPs in human skin and its derived cells was researched at the mRNA and protein levels. The effects of norepinephrine (NE) and 9-cis retinoic acid (RA) on UCP expression were also investigated. The expression of UCP1 mRNA was found in the human epidermis and was upregulated in differentiated keratinocytes. UCP1 expression in keratinocytes was synergistically upregulated by NE and RA treatment. Significant expression of UCP2 and UCP3 was observed also in cultured keratinocytes and fibroblasts. By immunohistochemistry, localization of UCP1 was found in the granular layer of the epidermis, sweat glands, hair follicles, and sebaceous glands of various sites in the human body. UCP3 was widely found in the dermis. This showed that UCPs exist in human skin, with their expression being under hormonal control. These findings are in stark contrast with the well-accepted view of UCP1 expression being exclusive to brown adipose tissue.

Rapid Desensitization of Lipolysis in the Visceral and Subcutaneous Adipocytes of Rats

In adipocytes, short and long term stimulation of β adrenergic receptors (βAR) induces the desensitization to catecholamines, leading to a decrease in the intracellular accumulation of cAMP, but the roles played by this in lipolysis is not clear. In this study, we assessed the catecholamine-induced desensitization of lipolysis and compared this in adipocytes isolated from visceral and subcutaneous fat tissues of rats. When adipocytes were pretreated with isoproterenol (ISO), the norepinephrine (NE)-induced lipolysis was significantly reduced dose- and time-dependently. A similar reduction of the lipolytic response was also found in NE-, dobutamine-, terbutaline- or BRL37344-induced lipolysis. The ISO- and each βAR agonist-induced lipolysis in the visceral fat was not only higher than in the subcutaneous fat, but also markedly reduced by ISO- or NE-pretreatment. These results showed that short-term treatment of three subtypes of βAR by each agonist induces a rapid reduction in the lipolytic response to βAR stimulation. This suggests some common mechanism for the rapid desensitization of βAR-agonist-induced lipolysis, in contrast with previous reports on the characteristics of βAR subtypes. In addition, the regional difference of adipose tissue not only in inducing lipolysis but also in rapid desensitization was also apparent.

Macrophage chemotactic factor (MCF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, D6-18, producing high levels of macrophage chemotactic factor (MCF) was established by the emetine-actinomycin D selection method. MCF was found to be present not only in the culture medium but also in the cell lysate of D6-18 cells. The secretion of the MCF from D6-18 cells was effectively inhibited by disodium cromoglycate, which is an inhibitor of the degranulation of mast cells, suggesting that MCF is stored in granules. The MCF of D6-18 cells was purified from the sonicated cell lysate by ion-exchange chromatographies and high-performance liquid chromatography. The amino acid sequence of the purified MCF was revealed to be WLGREDGSE or WLGRQDGSE. The synthetic peptide WLGREDGSE showed chemotactic activity against guinea pig macrophages and human monocytes at the concentration of about 10(-8) M.

Enhancement of lipolytic responsiveness of adipocytes by novel plant extract in rat.

Subcutaneous adipocytes accumulate excess energy as triglycerides, but lipolytic response is less sensitive to catecholamines than visceral adipocytes. Obesity also induces catecholamine resistance of adipocytes. We have searched for crude drugs that could enhance the lipolytic response to noradrenalin. In this study, the lipolysis-promoting activities and action mechanisms of a novel plant extract from Hemerocallis fulva (HE) were investigated in isolated adipocytes from rat subcutaneous fat. HE exhibited no lipolysis-promoting activity alone but markedly promoted lipolysis when combined with noradrenaline; however, this synergistic activity was accompanied by no increase of intracellular cAMP production. This activity of HE was also observed when combined with cAMP analogue and was further enhanced by phosphodiesterase inhibitor. PKA inhibitor could reduce these activities of HE. These results indicate that HE is a novel lipolysis-promoting material that can sensitize the lipolytic response of adipocytes to catecholamine and suggest that HE can amplify the intra-cellular signaling pathway related to PKA or modify the other mechanism-regulating lipase activity. This characteristic material could contribute to improvement of adipose mobility in obesity-related disorder or in subcutaneous adiposity and to suppression of body fat accumulation.