Labeling Of Triglycerides Research Articles

Novel application of the “doubly labeled” water method: measuring CO2production and the tissue-specific dynamics of lipid and protein in vivo

The partitioning of whole body carbon flux between fat and lean compartments affects body composition. We hypothesized that it is possible to simultaneously determine whole body carbon (energy) balance and the dynamics of lipids and proteins in specific tissues in vivo. Growing C57BL/6J mice fed a high-fat low-carbohydrate diet were injected with a bolus of "doubly labeled" water (i.e., (2)H2O and H2(18)O). The rate of CO2 production was determined from the difference between the elimination rates of 2H and 18O from body water. The rates of synthesis and degradation of triglycerides extracted from epididymal fat pads and of proteins extracted from heart muscle were determined by mathematically modeling the 2H labeling of triglyceride-bound glycerol and protein-bound alanine, respectively. We found that mice were in positive carbon balance (approximately 20% retention per day) and accumulated lipid in epididymal fat pads (approximately 9 micromol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to approximately 25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (approximately 10% of the pool per day), consistent with the fact that the heart muscle mass did not change. In total, these findings demonstrate a novel application of the doubly labeled water method. Utilization of this approach, especially in unique rodent models, should facilitate studies aimed at quantifying the efficacy of interventions that modulate whole body carbon balance and lipid flux while in parallel determining their impact on (cardiac) muscle protein turnover. Last, the simplicity of administering doubly labeled water and collecting samples allows this method to be used in virtually any laboratory setting.

Metabolism in Frog Retinal Pigment Epithelium of Docosahexaenoic and Arachidonic Acids Derived from Rod Outer Segment Membranes

Frog photoreceptor cells shed about 10% of their rod outer segments (ROS) every 4th day. Packets of these membranes are phagocytized and digested by retinal pigment epithelial (RPE) cells. Large amounts of lipids must be processed daily, especially docosahexaenoic acid (22:6n-3, DHA), the major fatty acid of these membranes. To study the metabolism of ROS lipids in RPE cells, RPE-eyecups were incubated with [3H]DHA-, [3H]arachidonic acid (AA)-, or [2-3H]glycerol-labeled ROS membranes for 2 hr, followed by a chase for up to 8 hr. Lipid extracts of RPE cells and incubation media were resolved into classes and quantitated for radioactivity. In RPE cells, the relative proportion of DHA and AA label in triglycerides (TG) increased dramatically with incubation time, although the substrate ROS membranes did not contain labeled TG. Other RPE lipids showed prominent reductions or relative little change. The percentage of radioactivity in free fatty acids (FFA) was low (< 3%) in RPE cells. In the chase media, the majority (60-80%) of DHA and AA label was found in FFA, with little radioactivity in TG or phospholipids. When RPE cells were incubated with [3H]glycero-labeled ROS membranes which contained 22% of the label in TG, the most rapid reduction in relative radioactivity appeared in TG. We conclude that DHA and AA are released from phagocytized ROS membranes and are rapidly incorporated into RPE cellular lipids, primarily TG. This lipid class is very active metabolically, since TG derived from ROS are rapidly hydrolysed. Free DHA and AA of ROS origin are released from RPE cells. We suggest that RPE triglycerides are the source of FFA in the interphotoreceptor matrix.

Metabolism of pyrenyl fatty acids in baby hamster kidney fibroblasts. Effect of the acyl chain length.

Biosynthetic labeling of cellular lipids with a fluorescent pyrenyl fatty acid (PyrxFA) moiety was studied in order to assess the usefulness of this approach in the introduction of fluorescent lipid molecules to living cells for transport and metabolic studies. PyrxFAs containing 4-14 aliphatic carbons were added to the culture medium of baby hamster kidney (BHK) fibroblasts, and their incorporation to various lipid species was monitored by thin layer and high pressure liquid chromatography. The results show that the length of the acyl chain has a remarkable effect on the efficiency of incorporation as well as distribution of the label between lipid species. Accordingly, PyrxFAs can be divided into two groups: the short chain ones including the pyrene butyrate and hexanoate derivatives, which show only modest incorporation to phospholipids and negligible labeling of triglycerides and cholesterol esters, and the long chain PyrxFAs including pyrene octanoate, decanoate, dodecanoate, and myristate derivatives, which incorporate efficiently both to phospholipids, mainly phosphatidylcholine (PC) and -ethanolamine (PE), and neutral lipids, i.e. di- and triglycerides and cholesterol esters. Positional analysis showed that the longer PyrxFAs are esterified preferentially to the sn-1 position of the glycerol moiety of PC and PE while the shorter ones are found exclusively in the sn-2 position indicating that the longer PyrxFAs mimic natural saturated fatty acids whereas the shorter ones may be recognized as polyunsaturated fatty acids by the acylating enzymes. Reverse phase chromatography of PC and PE revealed the presence of a variety of labeled molecular species among which the palmitate and oleate containing species were the major ones. Reverse phase analysis with simultaneous monitoring at the monomer and excimer channels showed the presence of tri- and diglyceride species with either 1 or 2 pyrenyl acyl residues. Analysis of the total cellular fatty acids demonstrated that PyrxFAs are shortened, probably by beta-oxidation in peroxisomes, up to pyrene butyrate. It is concluded that metabolic labeling with PyrxFAs is a promising alternative for studies on intracellular lipid traffic, especially because it allows introduction of fluorescent phospholipid species of very different hydrophobicity into intracellular membrane(s).

Glucose uptake in human adipose tissue

One hundred grams of glucose with 50 μCi U- 14C-glucose were given orally to 17 women with widely varying amounts of body fat. Radioactivity and glucose metabolism in vitro were then measured in adipose tissue obtained by needle biopsies in the abdominal and femoral regions after four hours. Radioactivity in triglycerides was then measured in repeated biopsies 1 day, 1 week, and monthly up to 7 months after glucose administration. Glucose label in triglycerides after four hours was higher in abdominal than femoral adipocytes in obese women. It increased slightly during the following week, and then decreased exponentially with a half-life of 12 months in the abdominal region and 19 months in the femoral region. Uptake of glucose carbon in total body fat was estimated from the triglyceride label measured and determinations of body fat mass, and found to be in the order of less than 4% of given glucose. The studies in vitro suggested that much of the glucose taken up in adipose tissue is converted to lactate. If this is the case in vivo, then glucose uptake in adipose tissue might well be of significance for total body glucose homeostasis, particularly in obese subjects, amounting to maximally perhaps one third to one half of the oral glucose given. The majority of this glucose uptake would then, however, leave adipose tissue again as lactate. The shorter half-life of label in abdominal adipocytes is in agreement with findings of increased lipolysis in these adipocytes in vitro.

Plasma triglyceride turnover during use of oral contraceptives

The effect of oral contraceptives on the concentration, production rate, and removal efficiency of plasma triglycerides was studied in premenopausal women using an endogenous labeling of triglycerides with tritiated glycerol. Thirteen subjects were studied while receiving the drug and seventeen normal healthy females served as controls. In some instances the study was made both before and during the drug or repeated after discontinuation of the treatment. The elimination of triglycerides from the circulation was characterized using the Michaelis constant, K m, which gives the plasma concentration at half-maximal removal velocity. The control group showed a polymorphism of plasma triglyceride kinetics being composed of three subgroups which had different maximal turnover rates. Treatment with oral contraceptives was associated with highly significant increases of concentration, rate of production, and efficiency of removal of plasma triglycerides whereas the maximal turnover rate was apparently unaffected. Thus, the increase of plasma triglyceride concentration (average 1.5-fold) observed in women receiving oral contraceptives is accounted for by a much enhanced influx (average 1.9-fold). The influence of the latter is partly compensated by a simulataneous acceleration of triglyceride efflux, however. The effects are produced already by one cycle of treatment but almost no reversion of the changes can be observed at 1 mo after discontinuation of the drug. The triglyceride concentration and production rate were significantly correlated to the duration of intake of contraceptive preparations while the removal efficiency was not.

The incorporation of glucose and palmitic acid into lipids in human arterial intima and media in vitro

Summary The incorporation into lipids of [ 14 C]glucose and [ 14 C]palmitate with and without unlabelled glucose in defined samples of apparently normal, fresh human intima and media was measured in vitro . After incubation of the intima samples with [ 14 C]-glucose about 50 % and 15 % of the label occurred in triglycerides and lecithin, respectively. A moderate degree of activity was found in phosphatidyl inositol and cholesterol and/or diglyceride; in other lipid classes the activity was low with the exception of a varying degree of label in free fatty acids. After incubation with [ 14 C]palmitate the distribution of label was similar but the proportion of label in triglycerides and lecithin was reversed and the degree of labelling of sphingomyelin was higher. After addition of unlabelled glucose the above differences for the [ 14 C]-glucose and [ 14 C]palmitate precursors were partially levelled out. The differences were less pronounced for the media samples. The rates of incorporation of the respective precursors were similar in intima and media both on a dry weight and DNA basis. The distribution of label in triglycerides and lecithin after incubation with [ 14 C]palmitate agrees well with results obtained by others for species susceptible to experimentally induced or spontaneous atherosclerosis but deviates from more atherosclerosis-resistant species. It is possible that this correlation reflects important factors for the formation of atherosclerosis.

Lipid Turnover in an Isolated Population of Irradiated Bone Marrow Cells

In vitro experiments are reported in this paper for the incorporation of ${\rm K}\text{-palmitate-}1\text{-}{\rm C}^{14}$ into triglycerides, diglycerides, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, lysolecithin, and sphingomyelin. At intervals of 1 day or more after total-body irradiation, there was a significant inhibition of the esterification of fatty acids into phospholipids, primarily lecithin. Labeled diglycerides did not increase under these conditions. The labeling of triglycerides in the irradiated marrow continued at essentially the same rate as in the control for the first 2 days after 800 R, after which it, too, was inhibited by ionizing irradiation. We conclude that enzymatic pathways for triglyceride biosynthesis are more resistant to damage by irradiation than are the pathways for the biosynthesis of phosphatides.