An ultramicrochemical test for mycoplasmal contamination of cultured cells

Summary A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-¹⁴C]adenine and ribose-1-phosphate to [8-¹⁴C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosinephosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contaminations in which most of the mycoplasma particles are attached to cell membranes. This investigation was partly supported by FUNGO (The Netherlands), INSERM (France) (A. T. P. 36.76.68), and DGRST (France) (No. 75.50.004).     

On the presence of adenosine 3′, 5′ -cyclic monophosphate in moss (Funariahygrometrica)

Partially purified nucleotide fraction of moss containing [14C]-labelled putative adenosine 3′, 5′ -cyclic monophosphate (cAMP) and marker authentic [3H] -cAMP was characterized by chemical deamination and also by the enzymatic hydrolysis with beef heart cyclic nucleotide phosphodiesterase. A significant conversion of marker authentic [3H] -cAMP into [3H] -inosine 3′, 5′ -cyclic monophosphate (cIMP) and [3H] -5′ adenosine monophosphate was observed by respective treatments. In contrast, the [14C] -labelled putative cAMP from control and theophylline-treated moss tissue was insensitive to chemical deamination and enzymatic hydrolysis. Apparently, the [14C] -labelled product which comigrates with authentic [3H] -cAMP does not represent true cAMP. Both the methods employed for characterization of the labelled putative cAMP were sensitive enough to detect picomole quantities of authentic [3H] -cAMP. Lack of detectability of prelabelled [14C] -cAMP in our preparations implies that the tissue may contain authentic cyclic AMP below the picomole levels. Thus, the attributed physiological role to adenosine 3′, 5′ -cyclic monophosphate in moss tissue appears somewhat skeptical.     

Catabolism of adenine nucleotides in suspension-cultured plant cells

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.     

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin fibroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Relationship Between [8-14C]Adenosine Transport and Growth Inhibition in Neurospora crassa Strain ad-8

The kinetic parameters of [8-(14)C]adenosine transport by a general nucleoside uptake system were studied in germinated conidia of the ad 8 strain of Neurospora crassa. The apparent K(m) for adenosine uptake by this system was found to be 6.2 muM. The apparent K(i) values for other nucleosides competing with adenosine for uptake were measured by using Dixon plots. Nucleosides which were efficient competitive inhibitors of adenosine transport were found to inhibit severely the rate of growth of strain ad-8 on adenosine-supplemented medium. Xanthosine and thymidine did not inhibit [8-(14)C]adenosine uptake as severely as other nucleosides, nor did they cause significant inhibition of ad-8 growth rate on adenosine.     

Capillary transport of adenosine

We tested the hypothesis that capillary exchange of adenosine is influenced by the ability of endothelial cells (ECs) to take up adenosine. Triple-indicator diffusion experiments were performed by injecting [14C]adenosine, [3H]9-beta-D-arabinofuranosylhypoxanthine ( [3H]araH), and radioiodinated serum albumin (RISA) into the arterial perfusate of isolated nonworking guinea pig hearts. Tracer appearance in venous effluent was observed over time. The early extraction of [14C]adenosine was much higher than that of [3H]araH. Extracted [3H]araH returned to the vascular space, but [14C]adenosine did not. Quantitative analysis of the curves by using a mathematical model indicates that approximately half of the extracted adenosine enters ECs and is metabolized. The remainder enters the interstitium and is taken up by myocytes, ECs, or other cells and is metabolized. We conclude that uptake of adenosine by ECs represents a significant influence on the capillary exchange of adenosine.     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Intracellular formation of analogs of cyclic AMP. Studies with brain slices labeled with radioactive derivatives of adenine and adenosine.

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N6-etheno[14C] adenosine nor 1,N6-etheno[14C] adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-3H] adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chloro-cyclic AMP was not obtained. N6-Benzyl[14C] adenosine was converted only to intracellular monophosphates and significant formation of radioactive N6-benzylcyclic AMP was not detected during a subsequent incubation. 2'-Deoxy-[8-14C] adenosine was converted to both intracellular radioactive 2'-deoxy-adenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2'-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Metabolism of S-adenosylhomocysteine and S-tubercidinylhomocysteine in neuroblastoma cells.

The metabolism of the methylase product inhibitor S-adenosylhomocysteine and its 7-deaza analogue S-tubercidinylhomocysteine has been studied in cultured N-18 neuroblastoma cells. The latter compound, designed to resist metabolic degradation, has been shown to be inert under the same conditions where S-adenosylhomocysteine is rapidly and extensively degraded. The product analyses elucidated by high-performance liquid chromatography indicate that the primary route of S-[8-(14)C]adenosylhomocysteine metabolism in these cells leads to adenosine. This product does not accumulate but is rapidly converted to nucleotides or oxypurines by the action of adenosine kinase and adenosine deaminase, respectively. The presence of the potent adenosine deaminase inhibitor coformycin leads to a pronounced inhibition of oxypurine formation, an increase in nucleotide formation, and a slight accumulation of the primary metabolic products adenosine and adenine.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

Preferential Synthesis of Prostaglandin D2 by Neurons and Prostaglandin E2 by Fibroblasts and Nonneuronal Cells in Chick Dorsal Root Ganglia

To determine the type and the relative amount of prostaglandins (PGs) synthesized by various neural tissues, homogenates of meninges, dorsal root ganglia (DRG) capsules, decapsulated DRG, and unsheathed sciatic nerves were incubated with [1-14C]arachidonic acid. Homogenates of cultured cells (meningeal cells, fibroblasts, and nonneuronal or neuronal DRG cells) were used to specify the cells producing particular PGs. The highest synthetic capacity was found in fibroblast-rich tissues (meninges and DRG capsules) and in cultures of meningeal cells or fibroblasts. Two major cyclooxygenase products were formed: [14C]PGE2 and an unusual 14C-labeled compound, Y. The accumulation of compound Y, corresponding probably to 15-hydroperoxy PGE2, was completely impaired by addition of exogenous GSH, which conversely enhanced the synthesis of [14C]PGE2 and promoted the formation of [14C]PGD2. In contrast, decapsulated DRG or unsheathed sciatic nerves displayed a 10-20 times lower capacity to synthesize PGs than fibroblast-rich tissues and produced mainly [14C]PGE2 and [14C]PGD2. In this case, [14C]PGE2 or [14C]PGD2 synthesis was neither enhanced nor promoted by addition of exogenous GSH. Neuron-enriched DRG cell cultures allowed us to specify that [14C]PGD2 is the major prostanoid produced by primary sensory neurons as compared with nonneuronal DRG cells. Because PGD2 synthesis in DRG and more specifically in DRG neurons does not depend on exogenous GSH and differs from PGD2 synthesis in fibroblast-rich tissues, it is concluded that at least two distinct enzymatic processes contribute to PGD2 formation in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase.

Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.     

Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue

Glucose utilization was studied in isolated fat cells prepared from rat adipose tissue which had been cultured for 18 hr in TC 199 medium. When 1% bovine serum albumin (BSA) was in the culture medium, basal rates of (14)CO(2) and [(14)C]triglyceride production from [1-(14)C]glucose were markedly depressed and there was no effect of insulin. With 4% BSA, basal (14)CO(2) production was the same as in cells prepared from fresh tissue and basal triglyceride production was greatly increased. Insulin effect on these cells was minimal. One-minute uptake of [(14)C]2-deoxyglucose was stimulated by 800-1000% in fresh cells and 300-500% in cells cultured with either 1% or 4% BSA. Oxidation of [U-(14)C]glucose showed a much smaller impairment in cultured cells than for [1-(14)C]glucose, suggesting that the pentose phosphate shunt was more severely impaired than glycolysis. Glyceride-glycerol production was increased in cultured cells relative to preculture (fresh) cells. There was no effect of insulin in the culture medium in any of these systems. Rates of free fatty acid and glycerol release were markedly increased in cultured cells, especially when insulin was present in the culture medium. The acute antilipolytic effect of insulin was retained, so that insulin in the test incubation decreased lipolysis by 40-80%. Nevertheless, cell-associated fatty acids were increased in cultured cells and FFA/albumin ratios in the medium often reached potentially toxic levels. The reduction in pentose phosphate shunt activity, lipogenesis, and insulin effect resembles other models of insulin insensitivity. The impaired metabolism is probably due to an intracellular defect. A possible toxic role of either intracellular or extracellular fatty acids cannot be excluded. This system should be a useful model in which to study the cellular mechanisms of insulin insensitivity in adipocytes.-Bernstein, R. S. Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue.