Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase

FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of 58m52u2685h8408/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (kinase F_A/GSK-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">) in rat adipocytes. The cellular activity of kinase F_A/GSK-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> is inhibited to 58u4q/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0"> 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 58u4q/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0"> 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> to 58u4q/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0"> 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-358u4q/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> can be decreased only to 58u4q/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0"> 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Characterization of zinc effect to inhibit osteoclast-like cell formation in mouse marrow culture: Interaction with dexamethasone

The inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was characterized. The bone marrow cells were cultured for 7 days in 58m15246/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-minimal essential medium containing a well-known bone resorbing agent, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25(OH)2D3 (10-8 M) or PGE2 (10-6 M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were enhanced by the presence of dexamethasone (10-9 to 10-6 M). The dexamethasone (10-7 M)-enhanced osteoclast-like cell formation was not inhibited by the presence of zinc sulfate (10-6 M) or zinc-chelating dipeptide (58m15246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-alanyl-L-histidinato zinc; 10-6 M), although the zinc compounds had an inhibitory effect on osteoclastic formation in the absence of the steroid. The effect of dexamethasone was not seen, when the steroid was added at the later stage of culture with bone-resorbing agents. In this case, the inhibitory effect of zinc compounds was clearly revealed. This effect of zinc compounds disappeared in the presence of Ca2+-chelating agent (0.5 mM EGTA). The present study suggests that zinc compounds have an inhibitory effect at the stage of differentiation of preosteoclastic cells in bone marrow cell culture system. (Mol Cell Biochem 166: 145-151, 1997)     

Golgi 58K-like protein in pollens and pollen tubes of Lilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.     

CCK-58: A novel reagent for studying the regulation of cholecystokinin bioactivity

CCK-58 has been shown to be the major circulating form of the hormone in the dog and human. To date, there have been no reports on its biological activity in vivo. We report here that CCK-8 and CCK-58 were equipotent in decreasing gastric motor function after bolus doses and in stimulating protein secretion after continuous infusion in urethane-anesthetized rats. The present results are the first on the in vivo activity of CCK-58, and indicate that because CCK-58 is equipotent to CCK-8, and because it is a major released and circulating form, it may be considered as a major contributor to the expression of cholecystokinin bioactivity.