Aspects of adipose-tissue metabolism in foetal lambs.

1. The mean volume of adipocytes, the rates of fatty acid and acylglycerol glycerol synthesis from various precursors (in vitro), the rates of oxidation of acetate and glucose (in vitro) and the activities of lipoprotein lipase and various lipogenic enzymes were determined for perirenal adipose tissue from foetal lambs during the last month of gestation. 2. The fall in the rate of growth of perirenal adipose tissue during the last month of gestation is associated with a diminished capacity for fatty acid synthesis and lipoprotein lipase activity, but no change in the rate of acylglycerol glycerol synthesis was observed. There was no fall in the activities of cytosolic acetyl-CoA synthetase or the NADP-linked dehydrogenases, suggesting that the decrease in the rate of fatty acid synthesis was due to an impairment at the level of acetyl-CoA carboxylase or fatty acid synthetase. 3. The rate of fatty acid synthesis from acetate was greater than that from glucose. The rate of fatty acid synthesis from glucose per adipocyte of foetal lambs was similar to that of young sheep. The characteristic metabolism of adipose tissue of the adult sheep is thus present in the foetus, despite the relatively large amounts of glucose in the foetal 'diet'.     

Insulin-like effects of fatty acid synthesis in liver of hamsters infected with plerocercoids of the tapeworm, Spirometra mansonoides

Elevated serum lipids are associated with infections of laboratory rodents with plerocercoids of Spirometra mansonoides. The effect of infection with these larval tapeworms on triglyceride degradation and hepatic de novo fatty acid synthesis was investigated in Syrian hamsters. Serum lipoprotein electrophoresis revealed a consistent elevation in very low density lipoproteins in the infected animals. Lipoprotein lipase activity was enhanced in the infected animals. After seven days of plerocercoid infection the activity of acetyl-CoA carboxylase (E.C. 6.4.1.2) was significantly elevated after 6, 12 and 18 hours of fasting. Fatty acid synthetase was significantly increased after 0, 6, 12, 18 and 24 hours of fasting. Therefore, a chronic insulin-like activity on lipid metabolism of hamsters is associated with plerocercoid infection.     

Metabolism of sheep adipose tissue during pregnancy and lactation. Adaptation and regulation.

1. Changes in the mean volume, the rate of fatty acid and acylglycerol glycerol synthesis, the activity of lipoprotein lipase and the numbers and affinities of insulin receptors of subcutaneous adipocytes are reported for sheep at different stages of pregnancy and lactation. In addition, the serum concentrations of insulin, progesterone, prolactin, choriomammotropin, somatotropin, glucose, acetate, L-lactate, glycerol and unesterified fatty acids are reported for these sheep. 2. A switch from lipid accumulation to net lipid mobilization accompanied by a decline in the capacity for lipid synthesis, occurred at the onset of the last third of pregnancy. Net lipid mobilization continued during lactation. 3. The changes that occurred in the serum concentrations of the various hormones listed above are discussed in relation to their possible roles in the modulation of adipose tissue metabolism in sheep during pregnancy and lactation. The observations are compared with those from previous studies on the hormonal control of adipose tissue metabolism in the rat during pregnancy and lactation.     

Further studies on the effects of dehydroepiandrosterone on hepatic metabolism in BHE rats

Two experiments were conducted to determine the effects of dehydroepiandrosterone (DHEA) on de novo fatty acid synthesis and oxygen consumption in BHE rats fed a 65% glucose diet. In Experiment 1, starved glucose-refed rats were injected ip with 120 mg of DHEA/kg body wt and hepatic de novo fatty acid synthesis was measured. DHEA-treated rats synthesized less fatty acid in response to starvation refeeding than nontreated rats. In Experiment 2, weanling rats were fed the glucose diet for 4 weeks. One-hundred twenty milligrams of DHEA/kg were injected daily for 3 weeks. Body weight gain, epididymal fat pad weight, and carcass lipid were less in the DHEA-treated rats than in the control rats. Mitochondrial respiration was less and liver size was greater in DHEA-treated rats compared with control rats. Whole body oxygen consumption was increased in DHEA-treated rats, suggesting that this steroid might be stimulating futile energy cycles involving lipid and protein turnover possibly through its effect on glucocorticoid and thyroid hormone function.     

Effect of adrenaline on the activity of lipolytic enzymes in calves

The research of adrenalin influence on the lipid synthesis and split intensity and fat acid oxidation was carried out in two groups of one-month old calves of black-white breed. The animals were injected by adrenalin (1 mg/kg) during 3 days. The specimens of the musculus quadriceps extensor femoris were used for biochemical researches. The increase of the triacylglycerol lipase activity and oxidation intensity of [1-14C] palmitic and [1-14C] stearic acids and the reduction of lipoprotein lipase activity and lipid synthesis from acetic acid intensity were found during researches. So, the reciprocal dependence between lipid synthesis and fatty acid oxidation as well as the connection between processes of lipolysis and fatty acid beta-oxidation, influenced by adrenalin, were observed in the skeletal muscles of the cattle.     

The 1990 Borden Award Lecture. Dietary regulation of fatty acid and triglyceride metabolism.

The level of circulating triacylglycerols is determined by the balance between their delivery into the plasma and their removal from it. Plasma triacylglycerols are derived either from dietary fat as chylomicrons or from endogenous hepatic synthesis as very low density lipoproteins. Their removal occurs through the action of lipoprotein lipase after which the fatty acids are either stored in adipose tissue or oxidized, primarily in skeletal muscle and heart. The composition of the diet has been shown to influence many of these processes. Hepatic fatty acid synthesis and triacylglycerol secretion are affected by the quantity and composition of dietary fat, carbohydrate, and protein. Polyunsaturated but not saturated fats reduce hepatic fatty acid synthesis by decreasing the amount of the lipogenic enzymes needed for de novo fatty acid synthesis. Dietary fish oils are particularly effective at reducing both fatty acid synthesis and triacylglycerol secretion and as a result are hypotriacylglycerolemic, particularly in hypertriacylglycerolemic individuals. In addition, dietary fish oils can increase the oxidation of fatty acids and lead to increased activity of lipoprotein lipase in skeletal muscle and heart. It appears that the hypotriacylglycerolemic effect of dietary fish oils is mediated by effects on both synthesis and removal of circulating triacylglycerols.     

Acetate and arachidonate incorporations into lipids of ovine chorioallantoic tissue at day 24 of pregnancy

Incorporation of acetate and arachidonic acid into lipid classes was examined in chorioallantoic membranes obtained from sheep at Day 24 of pregnancy. Conceptus tissues were incubated in vitro with 5 mM acetate, 0.042 mM arachidonate, 0.45 muCi [1-14C]acetate, and 5.0 muCi [5,6,8,9,11,12,14,15-3H]arachidonate for 3 and 6 h. After incubation, tissue lipid fractions were extracted, isolated, and examined for radiolabel incorporations. Medium was extracted and analyzed for radiolabeled metabolites. Metabolic pathways commonly associated with fatty acid metabolism were confirmed to be present. Acetate was utilized for de novo synthesis of free cholesterol and free fatty acid. Fatty acids containing radiolabel from both acetate and arachidonate were mainly esterified in phospholipid and triglyceride, major lipid classes found in chorioallantoic tissue. Labeled metabolites of acetate were not sufficient for analytical measurement in medium. Metabolites of arachidonic acid from lipoxygenase and cyclooxygenase pathways were determined in medium after incubation. Results suggest that, within Day 24 ovine chorioallantoic tissue, utilization of exogenous arachidonate and de novo lipogenesis from acetate function in a parallel and anabolic mode appropriate for membrane expansion.     

Differentiation and function of rat adipocyte precursor cells in primary culture

Precursor cells to adipocytes were purified from the epididymal fat pads of small rats and studied in primary culture. A culture system in which substrate and cofactors were not rate-limiting for complete adipocyte conversion was used by utilizing an agarose feeding-layer. Detachment of cells from the culture dish was prevented by addition of a viscous layer of culture medium, containing methyl cellulose. This system allowed quantitation and definite characterization of formed adipocytes, defined as cells accumulating a lipid droplet >20 micro m in diameter. The cells could be subcultured but then gradually lost their adipocyte conversion ability. Age of the donor depressed the adipocyte conversion which, however, never seemed to stop completely. Prostaglandin E(1) and F(2alpha) had no definite effect in the physiological concentration range while indomethachin possibly had a weak inhibitory effect. Insulin, heparin, and isobutylmethylxanthine increased adipocyte formation. Development of characteristic adipocyte functions with time was examined. Lipoprotein lipase activity was very low in the isolated precursor cells before culture, but developed in culture at confluence and was a thousand-fold higher within a few days. At this peak lipoprotein lipase activity was 50-fold higher than in mature adipocytes from the same donor animal. Triglyceride synthesis from glucose peaked in parallel but never reached the value of mature adipocytes and very little fatty acid was synthesized. Hormone-sensitive glycerol release developed at confluence and reached the level of activity of mature adipocytes. This study and previous work have indicated a role for the cyclase system in the development of adipocytes from precursor cells. Dibutyrylcyclic AMP caused an enhancement of lipoprotein lipase activity and adipocyte conversion. In suspension media, the nucleotide caused inhibition. These results are compatible with an effect of the nucleotide, not directly on lipoprotein lipase and cell determination, but via events taking place at confluence associated with cell to cell interactions. In comparison with previously described cells from an established cell line which undergo adipose conversion (3T3 cells), the cells described in the present work, like adipocytes, showed more metabolic activity in pathways for fatty acid incorporation from exogenous lipid sources (lipoprotein lipase activity) than from de novo synthesis. Furthermore, host-factors could be followed such as in the age- and site-dependence of adipocyte formation. Physiological stimuli such as insulin, lipid substrate, and heparin had effects on adipocyte formation. It was therefore concluded that this cell preparation has a potential of yielding information of physiological significance in studies of the regulation of adipocyte multiplication.-Bj?rntorp, P., M. Karlsson, P. Pettersson, and G. Sypniewska. Differentiation and function of rat adipocyte precursor cells in primary culture.     

Intermediary metabolism of adipose tissue.

Metabolism of ruminant adipocytes involves the synthesis and mobilization of lipids. Rates of lipid synthesis from the uptake of preformed fatty acids (via lipoprotein lipase) and de novo synthesis of fatty acids are related to the energy balance. Acetate is the major carbon source for fatty acid synthesis with NADPH originating from the pentose cycle and the isocitrate cycle. Ruminant adipose tissue lacks the ability to utilize for lipogenesis those substrates that generate mitochondrial acetyl CoA because of an absence of ATP citrate-lyase and NADP-malate dehydrogenase. Lipid mobilization in ruminant adipocytes is apparently regulated via cAMP levels and a summary of the compounds investigated for lipolytic responses is presented. The control of lipid synthesis and mobilization is interrelated in ruminant adipose tissue. The coordinated manner in which these two functions are regulated is examined with regard to adipocyte responses to insulin and epinephrine. In both lipid synthesis and lipid mobilization, ruminant adipocytes are uniquely different from nonruminant adipose tissue. The physiological significance and possible basis for these species differences in adipose metabolism are discussed.     

Recombinant human bone morphogenetic protein-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts

Molecular mechanisms of lipid synthesis and their controls in hepatic stellate cells are not known. We have previously proposed that, in contrast to other fat storing cells, hepatic stellate cells are not involved in energy storage, but they represent a particular cell population specialized in storage of lipid-soluble substances, the major one being probably retinol. In agreement with this hypothesis, induction of the lipocyte phenotype in stellate cells is not under the control of insulin, but responds to retinoids and other molecules that modify the gene expression program in these cells. In the present study we have monitored the activity of the two major enzymes involved in lipid synthesis during the induction of the lipocyte phenotype in hepatic stellate cells: glycerol-3-phosphate dehydrogenase (GPDH) that mediates the de novo lipid synthesis, and lipoprotein lipase that mediates incorporation of plasma lipids. In early stages of lipocyte induction, both pathways of lipid synthesis are activated. When lipocytes have already constituted the lipid droplets, lipoprotein lipase pathway is downregulated, while GPDH activity remains high. Adult liver has been reported to lack lipoprotein lipase, but under stress, lipase activity was detected around and at the surface of the intrahepatic vasculature. We have now shown that the lipase activity can be induced in the hepatic stellate cells, located in the Disse's space. The high lipoprotein lipase activity under acute induction of lipocyte phenotype, followed by the low activity under conditions of metabolic equilibrium, are in compass with the increased activity of this enzyme under stress, and its low activity in adult liver parenchyma under normal conditions.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

Adaptations of adipose tissue metabolism and number of insulin receptors in pregnant sheep

The endocrine control of adipose tissue metabolism during pregnancy in sheep has been investigated. The number of insulin receptors of sheep adipocytes was increased during pregnancy. There was no apparent change in the concentration of serum insulin during pregnancy in sheep while the rise in serum progesterone concentration was smaller and more gradual than in rats. Net lipid deposition in adipocytes occurred during the first 55 days of pregnancy, probably due primarily to increased lipoprotein lipase activity. Net deposition of lipid had ceased by mid-pregnancy while by 125 days of pregnancy, the rate of fatty acid synthesis in adipose tissue was decreased and the serum fatty acid concentration had risen, suggesting the onset of net lipid mobilization in the tissue. Results are compared with those from other studies with rats; it would appear that different mechanisms regulate lipid deposition during pregnancy in sheep and rats.     

Lipid uptake by insect oocytes

Approximately 30-40% of the dry weight of an insect egg consists of lipid, mostly triacylglycerol (TAG). Although this lipid is essential for the energy needed by the developing embryo, little is known about the mechanism that leads to the accumulation of TAG in the insect egg. Insect oocytes can readily synthesize TAG from free fatty acids (FFAs) and glycerol, however, de novo synthesis of FAs by the oocyte is marginal. Hence, FAs have to be imported from the fat body or the diet. Insect hemolymph contains two lipoproteins that transport lipids, lipophorin and vitellogenin. Both are taken up via endocytosis by the oocyte, however, this provides only about 10% of the egg's lipid reserves. The rest is unloaded from circulating lipoprotein particles at the oocyte surface in the form of diacylglycerol (DAG), the major lipid transport form in insects, or as FFA. The mechanism of lipoprotein unloading at the oocyte surface is currently unclear. Possible roles of the lipid transfer particle (LTP), FA transporters, and lipoprotein lipase activity are discussed.     

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Regulation of lipoprotein lipase synthesis and 3T3-L1 adipocyte metabolism by recombinant interleukin 1

When fully differentiated 3T3-L1 adipocytes were exposed to purified, recombinant murine interleukin 1 (rIL-1), a dose-dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to an effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate [35S]methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporation into acid precipitable protein; however, after a 17 h exposure of the 3T3-L1 cells to recombinant interleukin 1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several-fold. When the effect of Il-1 on the major metabolic pathways of the adipocyte was investigated, lipolysis as measured by glycerol release from the cells was markedly enhanced after a 17 h incubation with the hormone, while no effect was observed on de novo fatty acid synthesis. These effects on the metabolism of the adipocytes occur at concentration on a basis of molecules per cell, similar (only a 3-fold difference) to those required for stimulation of [3H]thymidine incorporation into mouse thymocyte DNA, suggesting that IL-1 may be a physiologically significant effector of adipocyte metabolism.     

Sampling the lipogenic hepatic acetyl-CoA pool in vivo in the rat. Comparison of xenobiotic probe to values predicted from isotopomeric distribution in circulating lipids and measurement of lipogenesis and acetyl-CoA dilution

The acetylation of xenobiotics may provide a means for sampling hepatic cytosolic acetyl-CoA in vivo for isotopic studies of lipogenesis. Here, we test the accuracy of acetylated-sulfamethoxazole (SMX) in representing the true precursor pool for hepatic lipogenesis by comparison to a mathematical technique for estimating acetyl-CoA enrichment using the mass isotopomer distribution in circulating lipids. We then go on to measure hepatic fatty acid synthesis in intact rats using stable and radioisotopes. Specific activities and enrichments of SMX-acetate (the latter determined by high performance liquid chromatography-mass spectrometry) were monitored during fasting and refeeding. The dilution rate of hepatic acetyl-CoA relative to infused 13C- or 14C-acetates was 0.158-0.200 mmol/kg body weight/min during fasting, and did not increase significantly in rats refed with intravenous glucose at 25-30 mg/kg/min or refed ad libitum with chow, suggesting little additional input of acetate units. Plasma beta-hydroxybutyrate specific activity was much lower than SMX-acetate. The isotopomeric frequency distributions in circulating very low density lipoprotein (VLDL)-palmitate and VLDL-stearate were used to estimate the enrichment of the true precursor, hepatic acetyl-CoA, from a model based on the binomial distribution. The calculated acetyl-CoA values (7.28 +/- 0.49 molar percent excess (n = 16] based on isotopomeric frequencies were very close to measured SMX-acetate enrichments (7.44 +/- 0.41 molar percent excess (n = 21] and values within individual animals (n = 14) correlated very well (r2 = 0.90, p less than 0.0001). The contribution of VLDL-fatty acid by the de novo lipogenic pathway was similar using the stable isotope approach or radioisotopes (only 1-2% in fasted or intravenous glucose refed rats, 5% in chow refed). Combining fractional de novo lipogenesis values with absolute de novo lipogenesis rates allows estimation of total VLDL-triglyceride synthesis. In conclusion, the xenobiotic acetylation technique provides continuous access to the lipogenic hepatic acetyl-CoA pool in vivo and permits measurement of fatty acid synthesis. Isotopomer ratios in secreted lipids provide another method for estimating true precursor acetyl-CoA enrichments.     

Changes in lipoprotein lipase activity in the adipose tissue, heart and liver of continuously irradiated rats

Adult male Wistar rats were continuously irradiated for 30 days on an experimental field from a 60Co source or radiation. Lipoprotein lipase activity was determined in their adipose tissue, heart and liver at intervals of 1, 3, 7, 14, 21 and 30 days from the beginning of irradiation and triacylglycerol, total cholesterol, phospholipid and non-esterified fatty acid concentrations were determined in their serum. Throughout the whole of the study, lipoprotein lipase activity was lower in the adipose tissue and higher in the heart of irradiated rats than in the controls. In the liver it was low 3 days from the onset of irradiation; at the other intervals it was variable and differed only non-significantly from the controls. Serum lipid concentrations were raised in irradiated rats--triacylglycerol from the 7th day, phospholipids from the 14th day and non-esterified fatty acids throughout the whole period of irradiation. In keeping with the high triacylglycerol values in the serum of irradiated rats, lipoprotein lipase activity in their adipose tissue was low.     

Post-heparin triacylglycerol lipases in ovine plasma

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.