Synergistic effect of vespa amino acid mixture on lipolysis in rat adipocytes

The effect of Vespa amino acid mixture (VAAM) on the release of lipolytic products was examined in isolated rat adipocytes. Concentrations of 112.5 to 225 ppm of VAAM showed significantly greater release of non-esterified fatty acids (NEFA) and glycerol than the same concentrations of casein amino acid mixture (CAAM). The integrated relative release of NEFA and glycerol was lower in response to individual administration of amino acids comprising VAAM than to VAAM itself. Further, amino acids mixtures deficient in a single amino acid comprising VAAM showed significantly lower release of lipolytic products than VAAM. These data suggest that the synergistic effect of VAAM on the release of lipolytic products is a function of concurrent exposure to the unique composition of amino acids found in VAAM as compared to the effect of exposure to the same individual un-mixed amino acids or to a mixture lacking one of the amino acids comprising VAAM.     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

Summary Hormone stimulated lipolysis of mouse and rabbit adipocytes as measured by both free fatty acid and glycerol release, is proportionally elevated with increase in the adipocyte cAMP level up to 1 nmole/g. The correlation coefficients are 0.94 and 0.97 for FFA/cAMP and glycerol/cAMP respectively. Increments in cAMP greater than 1 nmole/g show no correlation with increase in lipolysis. The release of lipolytic products, glycerol and free fatty acids, from white adipocytes in response to ACTH, epinephrine or morepinephrine was measured using radiochemical assays in short term incubation systems, with cAMP levels measured at the same time and from the same cell sample. Under the conditions studied, epinephrine is a more effective lipolytic hormone than ACTH in mouse adipocyte, and ACTH is more effective than epinephrine in rabbit adipocyte. The effect of catecholamines on the rabbit adipocyte is not modified by phentolamine (10 μM), but it is potentiated by 1-methyl-3-isobutyl xanthine (0.1 mM). The results suggest that cAMP mediates the action of these lipolytic hormones in white adipocytes of mouse and rabbit.     

Uptake of the components of phenylalanylphenylalanine and maltose by intestinal epithelium

The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na⁺ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (K_t) is greater in the absence of Na⁺. For Phe-Phe, the V decreases in the absence of Na⁺ whilst K_t is not influenced by the Na⁺ concentration. The different effect of Na⁺ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-¹⁴C]Phe-Phe and Phe-[G-³H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids.Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase.These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Glucose‐dependent Insulin Modulation of Oscillatory Lipolysis in Perifused Rat Adipocytes

Objective: We showed glucose‐dependent lipolytic oscillations in adipocytes that are modulated by free fatty acids (FFAs). We hypothesized that the oscillations are driven by oscillatory glucose metabolism that leads to oscillatory formation of α‐glycerophosphate (α‐GP), oscillatory removal of long‐chain coenzyme A (LC‐CoA) by α‐GP to form triglycerides, and oscillatory relief of LC‐CoA inhibition of triglyceride lipases. This study examined the effect of insulin on this hypothesis. Research Methods and Procedures: Samples were collected every minute from perifused rat adipocytes during the basal state followed by insulin (±glucose) or isoproterenol (±insulin; n = 4 each). Results: Insulin caused a significant increase in glycerol release (18%), with a concomitant significant decrease in FFA release (38%). Without glucose, insulin had no effect on glycerol release while still decreasing FFA release (35%). Insulin (5 μU/mL) attenuated the response of lipolysis to isoproterenol (～3‐fold increase with isoproterenol vs. 2‐fold increase with insulin + isoproterenol). However, 1 mU/mL insulin amplified the lipolytic response (～5‐fold increase in glycerol release with insulin + isoproterenol), with a concomitant increase in FFA reesterification (no increase in FFA release compared with isoproterenol alone). Discussion: We interpret these results to be due to insulin's ability to increase glucose uptake and conversion to α‐GP, thus removing LC‐CoA inhibition of triglyceride lipases. While the physiological importance of lipolytic oscillations remains to be determined, we hypothesize that such an oscillation may play an important role in the delivery of FFAs to the liver, β cells, and other tissues.     

Conserved amino acid networks involved in antibody variable domain interactions

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody V_H and V_L function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which V_H and V_L domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody V_H and V_L domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in V_H and V_L are at or adjacent to the V_H–V_L interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the V_H domain, but not the V_L domain, residue pairs at the variable‐constant domain interface (V_H–C_H1 interface) are also strongly conserved. The same network of conserved V_H positions involved in interactions with both the V_L and C_H1 domains is found in camelid V_HH domains, which have evolved to lack interactions with V_L and C_H1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Metabolic costs of amino acid and protein production in Escherichia coli

Escherichia coli is the most popular microorganism for the production of recombinant proteins and is gaining increasing importance for the production of low-molecular weight compounds such as amino acids. The metabolic cost associated with the production of amino acids and (recombinant) proteins from glucose, glycerol and acetate was determined using three different computational techniques to identify those amino acids that put the highest burden on the biosynthetic machinery of E. coli. Comparing the costs of individual amino acids, we find that methionine is the most expensive amino acid in terms of consumed mol of ATP per molecule produced, while leucine is the most expensive amino acid when taking into account the cellular abundances of amino acids. Moreover, we show that the biosynthesis of a large number of amino acids from glucose and particularly from glycerol provides a surplus of energy, which can be used to balance the high energetic cost of amino acid polymerization.     

Duration of coffee- and exercise-induced changes in the fatty acid profile of human serum.

Prolonged moderate exercise increases the concentration of nonesterified fatty acids (NEFA) and the ratio of unsaturated to saturated (U/S) NEFA in human plasma. The present study examined the duration of these effects and compared them with the effects of coffee ingestion. On separate days and in random order, seven men and six women 1) cycled for 1 h, 2) ingested coffee containing 5 mg caffeine/kg body mass, 3) ingested coffee followed by exercise 1 h later, and 4) did nothing. Blood samples were drawn at 0, 1, 2, 4, 8, 12, and 24 h. Serum was analyzed for lactate, glucose, glycerol, individual NEFA, triacylglycerols, total cholesterol, and HDL cholesterol. Exercise elevated the U/S NEFA and the percentage of oleate, while decreasing the percentages of palmitate and stearate, at the end of exercise but not subsequently. Consumption of coffee triggered a lower lipolytic response with no alterations in U/S or percentages of individual NEFA. These findings may prove useful in discovering mechanisms mediating the effects of exercise training on the fatty acid profile of human tissues.     

Nonesterified fatty acids and lipid peroxidation

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidaton of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.     

Effect of exogenous amino acids,glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the -amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

Metabolic characteristics of human subcutaneous abdominal adipose tissue after overnight fast

Subcutaneous abdominal adipose tissue is one of the largest fat depots and contributes the major proportion of circulating nonesterified fatty acids (NEFA). Little is known about aspects of human adipose tissue metabolism in vivo other than lipolysis. Here we collated data from 331 experiments in 255 healthy volunteers over a 23-year period, in which subcutaneous abdominal adipose tissue metabolism was studied by measurements of arterio-venous differences after an overnight fast. NEFA and glycerol were released in a ratio of 2.7:1, different (P < 0.001) from the value of 3.0 that would indicate no fatty acid re-esterification. Fatty acid re-esterification was 10.2 ± 1.4%. Extraction of triacylglycerol (TG) (fractional extraction 5.7 ± 0.4%) indicated intravascular lipolysis by lipoprotein lipase, and this contributed 21 ± 3% of the glycerol released. Glucose uptake (fractional extraction 2.6 ± 0.3%) was partitioned around 20-25% for provision of glycerol 3-phosphate and 30% into lactate production. There was release of lactate and pyruvate, with extraction of the ketone bodies 3-hydroxybutyrate and acetoacetate, although these were small numerically compared with TG and glucose uptake. NEFA release (expressed per 100 g tissue) correlated inversely with measures of fat mass (e.g., with BMI, r(s) = -0.24, P < 0.001). We examined within-person variability. Systemic NEFA concentrations, NEFA release, fatty acid re-esterification, and adipose tissue blood flow were all more consistent within than between individuals. This picture of human adipose tissue metabolism in the fasted state should contribute to a greater understanding of adipose tissue physiology and pathophysiology.     

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis> ATCC 6013

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography–mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone–butanol–ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

     

Minimal Defined Media for Vegetative Growth of the Acrasian Polysphondylium pallidum WS-320*

SYNOPSIS. Polysphondylium pallidum WS-320 grows indefinitely as vegetative amebae in a liquid medium where (a) substrates comprise sucrose, glycerol, acetate, lactate, citrate, and glutamate; (b) essential nutrients (riboflavin, lysine, glycine, and possibly several other amino acids that may be essential) are supplied. The growth thus supported (2 × 10⁶ cells/ml) is more than doubled by provision of a mixture of crude fatty acids, an acid hydrolysate of casein supplemented with B vitamins, purines, pyrimidines, and fat-soluble antioxidants.     

Cell-associated nonesterified fatty acid levels and their alteration during lipolysis in the isolated mouse adipose cell

A rapid and flexible method has been developed for measuring cell-associated, probably intracellular, nonesterified fatty acids (CAFA) in isolated mouse adipose cells. A variety of lipolytic agents as well as various concentrations of epinephrine elevate CAFA levels in rough proportion to their stimulation of glycerol and fatty acid release. Insulin reduces epinephrine-elevated CAFA levels. A detailed, quantitative study of the relationship among lipolytic activity, CAFA levels, and the extracellular molar ratio of fatty acids to albumin has been carried out. Epinephrine-elevated CAFA levels rise linearly with, while epinephrine-stimulated lipolytic activity is independent of, fatty acid to albumin ratios below 2-3. As the ratio increases from 3 to 5, CAFA levels continue to increase, whereas lipolytic activity decreases. Above ratios of 5, fatty acid release almost completely ceases; CAFA levels increase dramatically with residual glycerol release. A temperature-dependent efflux of epinephrine-elevated CAFA can be elicited through blockade of stimulated lipolysis with propranolol, but only in the presence of extracellular fatty acid to albumin ratios below 3. These observations suggest that during stimulated lipolysis, a fatty acid gradient exists between the cell and extracellular serum albumin and that CAFA represent the intracellular component of this gradient. In addition, these observations support the concept that intracellular fatty acids play a role in the feedback regulation of adipose cell function as extracellular fatty acids accumulate during the lipolytic response.