Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein.
To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 +/- 0.33 x 10(-8) cm(2)/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 +/- 0.12 x 10(-7) cm(2)/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 +/- 0.3 microM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 +/- 3.0 microM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system. (+info)
Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.
The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP. (+info)
Loss of regulation of lipogenesis in the Zucker diabetic (ZDF) rat.
We present here a study on the role of leptin in the regulation of lipogenesis by examining the effect of dietary macronutrient composition on lipogenesis in the leptin receptor-defective Zucker diabetic fatty rat (ZDF) and its lean litter mate (ZL). Animals were pair fed two isocaloric diets differing in their fat-to-carbohydrate ratio providing 10 and 30% energy as fat. Lipogenesis was measured in the rats using deuterated water and isotopomer analysis. From the deuterium incorporation into plasma palmitate, stearate, and oleate, we determined de novo synthesis of palmitate and synthesis of stearate by chain elongation and of oleate by desaturation. Because the macronutrient composition and the caloric density were controlled, changes in de novo lipogenesis under these dietary conditions represent adaptation to changes in the fat-to-carbohydrate ratio of the diet. De novo lipogenesis was normally suppressed in response to the high-fat diet in the ZL rat to maintain a relatively constant amount of lipids transported. The ZDF rat had a higher rate of lipogenesis, which was not suppressed by the high-fat diet. The results suggest an important hormonal role of leptin in the feedback regulation of lipogenesis. (+info)
Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains.
The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels. (+info)
Augmentation of myocardial transfection using TerplexDNA: a novel gene delivery system.
Gene therapy is a potential new strategy for the treatment of cardiovascular disease. The most efficacious method of gene delivery remains a key hurdle to effective gene therapy. We present the application of a novel, nonviral gene delivery system (TerplexDNA) to augment myocardial transfection. The hearts of New Zealand white rabbits were injected with reporter genes, luciferase cDNA or beta-galactosidase cDNA, either as naked plasmid DNA or plasmid DNA complexed with stearyl-poly(L-lysine)-low density lipoprotein (TerplexDNA). Three day left heart myocardial cell lysates produced 44571 +/- 8730 RLU (RLU = total light units/mg protein) for the TerplexDNA luciferase rabbits versus 1638 +/- 567 RLU for the naked luciferase rabbits (P = 0.002). Thirty days after injection, myocardial lysates produced 677 +/- 52 RLU for the TerplexDNA luciferase hearts versus 18 +/- 3 RLU for the naked luciferase hearts (P = 0.002). Histologic analysis of the hearts transfected with beta-galactosidase showed that TerplexDNA increased the area and depth of transfection compared with the naked plasmid DNA alone. The hearts of Sprague-Dawley rats were injected in a similar fashion and analyzed at 1, 3, 5, 10, 15, 25 and 30 days after injection. The naked luciferase injected hearts showed transient elevation of luciferase activity to day 5 but fell back to baseline levels after that time-point. The TerplexDNA luciferase injected hearts had significantly elevated luciferase activity to 30 days. The Terplex gene delivery system significantly augments myocardial transfection compared with a naked plasmid DNA system alone. The advantage in transfection efficiency appears to be related to the unique properties of the TerplexDNA carrier molecule. The TerplexDNA delivery system represents a novel means to augment transfection of the myocardium. (+info)
Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets.
Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components. (+info)
Oxidative and drug-induced alterations in brush border membrane hemileaflet fluidity, functional consequences for glucose transport.
Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na(+)-dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na(+)-dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A(2)C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity. (+info)
Dietary effects of the esters of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, and stearic acids on food intake, weight gain, plasma glucose, and tissue lipid in the male white rat.
Eight saturated fatty acid esters were fed to male white rats for 30 days in a 1/81 fractional factorial experiment in which diets contained 12-38% of their total energy as lipid. Marked increases in food intake, feed efficiency, and weight gain were achieved when lipid provided 36% of diet energy, and when that lipid was more than half caproate, caprate, myristate, and/or stearate. Caproate was the only saturated fatty acid to increase plasma glucose levels. The feeding of stearate or caprylate decreased plasma and liver cholesterol. Caprate increased liver fat. The short-chain fatty acids (butyrate to myristate) increased the concentration of fat in the carcass. (+info)