Isolated Fat Cells Research Articles

Permissive action of glucocorticoids on catecholamine-induced lipolysis: Direct “in vitro” effects on the fat cell β-adrenoreceptor-coupled-adenylate cyclase system

Exposure of adipose tissue fragments to dexamethasone leads to enhanced lipolytic and cyclic AMP responses of isolated fat cells to isoproterenol. This permissive effect of the steroid is dose-dependent, prevented by the glucocorticoid receptor antagonist RU 38486, maximum after 48 h exposure to 10 nM dexamethasone and affects only the amplitude of the maximal response (+50%). Exposure to dexamethasone also induces an increase in both the number of beta-adrenergic receptors (+30%), and the adenylate cyclase-catalytic activity (+64%) and - responses to GTP (+114%) and isoproterenol (+55%). These data strongly suggest that the permissive effect of glucocorticoids towards lipolysis "in vivo" results at least in part from a glucocorticoid-receptor mediated action of these hormones on the fat cell membranous components involved in the beta-adrenergic control of lipolysis.

Insulin-like effects of vanadate in isolated rat adipocytes.

Vanadate has been shown to have a number of insulin-like effects in various cells, including isolated rat adipocytes. In the present study we compared the activities of vanadate and insulin in isolated fat cells using a number of different assays of insulin-like activity. Both insulin and vanadate stimulated [2-3H]glucose incorporation into fat cell lipid in a dose-dependent manner, but the maximal effect of vanadate was markedly greater than that of insulin. At 10(-2) M vanadate the effect was 3-4 times as great as the maximal effect of insulin. This effect was dependent on specific glucose transport. Combinations of insulin and vanadate were not more effective than vanadate alone. Vanadate also produced antilipolysis with an effect somewhat greater than that of insulin. Using [U-14C]glucose both vanadate and insulin stimulated 14CO2 production and [14C]glucose incorporation into lipid, and again the effect of vanadate was greater than that of insulin. Vanadate had a greater effect on 14CO2 production than on [14C]glucose incorporation into lipid. When [1-14C]glucose was used vanadate again had a significantly greater effect on 14CO2 production than did insulin, but when [6-14C]glucose was used the effects of vanadate and insulin were equal. These results demonstrate that vanadate has insulin-like effects in isolated fat cells, but it selectively stimulates certain pathways to a greater extent than does insulin. The greater effect of vanadate than insulin appears to be primarily on the pentose phosphate shunt, suggesting that this agent may be useful for examination of this intracellular pathway in fat cells.

Effect of age and day time on the adenosine modulation of basal and insulin-stimulated glucose transport in rat adipocytes

1. 1. The relationship between the activity of adenosine metabolizing enzymes 5′nucleotidase (5′N), adenosine kinase (A.T.) and adenosine deaminase (A.D.) with basal and insulin-stimulated glucose transport in isolated fat cells from young and old animals was studied at 08:00 and 16:00 hr. 2. 2. In cells from young animals a larger insulin-stimulation of glucose transport was observed at 16:00 hr than at 08:00 hr. Also at 16:00 hr small changes in 5′N, A.K. and A.D. activities suggest a decrease in adenosine formation. 3. 3. In the cells from old animals no effect of insulin was observed at any time, while a 3–5-fold increase in 5′N indicated a predominance of adenosine formation at both times studied. 4. 4. An inverse relationship was observed in the changes of adenosine metabolism and insulin action.

Dietary influence on the insulin function in the epididymal fat cell of the Wistar rat. III. Effect of the ratio carbohydrate to fat.

Newly weaned male Wistar rats were fed semipurified diets containing sunflowerseed oil or palm oil. The fatty acid compositions of the phospholipid and triacylglycerol fractions either from whole epididymal fat pads or from fat cells isolated from the pads appeared to be nearly similar. Feeding the diets for a period of only 3-4 weeks after weaning was found to be sufficient to produce a difference in insulin response and insulin binding in the epididymal fat cells. A reduction in the dietary fat content from 30 to 15% of the energy had only minor effects on fat pad weight and on the fatty acid composition of the phospholipid- and triacylglycerol fractions isolated from the epididymal fat pads. There was no effect either on body weights or on average fat cell diameter but there was an increase in insulin response in the isolated fat cells, however. The difference in insulin response after a diet rich in polyunsaturated fatty acids (sunflowerseed oil) as compared to one rich in saturated fatty acids (palm oil) remained after the reduction of fat content.

Fluorescence detection of mitochondrial clusters in mammalian white fat cells in vivo.

Rhodamine 123 and epifluorescence microscopy revealed a significant portion of the fat cell's mitochondria existed in the form of clusters or aggregates, whereas the remainder were scattered about the cytoplasm. The aggregates were variable in size and number and apparently bore no fixed relationship to the nucleus or to each other. Mitochondrial clusters were seen in vivo in rat and mouse adipocytes of the mesenteric and epididymal depots, in excised tissue pieces of other depots, and in isolated fat cells. Physiological factors investigated such as species type (rat, mouse, rabbit, dog), sex, age, depot location (superficial vs. deep), fat cell size, hypercholesterolemia, and 24-h fasting had no apparent effect on cluster prevalence or size. Similar aggregates were not visible in several cultured cell lines studied nor in various non-fat cells, capillary endothelial cells, or nerve fibers contained within adipose depots examined. These results indicate that mitochondrial clusters exist naturally in mammalian white fat cells and conclude that they represent a form of cytoplasmic organization whose purposes are not well understood.

Lipolysis in diabetic adipocytes: differences in response to growth hormone and adenosine.

The sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM.

Influences of variation in total energy intake and dietary composition on regulation of fat cell lipolysis in ideal-weight subjects.

Weight-maintaining fat-rich, "prudent," carbohydrate-rich, as well as energy-restricted diets (300 kcal/d) were fed in succession for 7 d to 12 healthy males of ideal body weight under metabolic ward conditions. At the end of each period isolated fat cells were prepared from subcutaneous abdominal adipose tissue and incubated in vitro in the absence or presence of adenosine deaminase, either alone or in combination with various lipolytic or antilipolytic hormones and agents. Variations in total energy intake and dietary composition had characteristic and specific effects on fat cell lipolysis in vitro. High carbohydrate and prudent diets resulted in low rates of nonstimulated glycerol release and impaired insulin action in the presence of adenosine deaminase (320 mU/ml). High-fat and energy restricted diets were characterized by high rates of nonstimulated glycerol release. Sensitivity of antilipolysis to insulin and prostaglandin E2 was 10 to 200 times lower respectively on energy-restricted than on fat-rich diets. The effects of alpha 2- and beta-adrenergic catecholamines and of N6-phenylisopropyladenosine were not affected by the preceding diets.

Influence of age and obesity on protein metabolism in rat epididymal adipose tissue and liver

3H-leucine incorporation and translation of ribosome-bound mRNA were examined in adipocytes, adipose and liver tissues of male Sprague Dawley and Fischer rats ranging in age from 1.5–24 months and 6.32 months, respectively. 3H-leucine incorporation into isolated fat cell protein and mRNA within polysomes, isolated from adipose and liver tissue homogenates, were translated in a liver pH 5 enzyme cell-free system. Protein synthesis and translation of ribosome-bound mRNA declined significantly with age in the liver and adipose tissue, by 40% at 18 months in Sprague Dawley rats, and by 50% at 24 months in Fischer rats. In the very old rats of both strains (24 to 32 months), there was a paradoxical enhancement (18.30%) in protein synthesis and protein synthetic activity of the ribosomes in adipose and liver tissues compared to the mature rats. This enhancement was associated with an apparent increase in fat cell number. The biphasic responses in tissue protein synthetic activity of the ribosomes coincided with a fractional shift from polysomea to monosomes. The paradoxical effects of extreme aging could relate to recruitment of newly differentiated, highly active adlpocytes in the fat depot. The recruitment may be triggered by cell enlargement associated with chronic obesity and/or extreme aging.

A practical VLSI characterization and failure analysis system for the IC user : Randy King and John Hiatt. 24 a. Proc. IEEE Reliab. Phys. Symp., 87 (1986)

5-Chloro-6-(ethylamino)-1,3-dihydro-2H-imidazo[4,5-b]pyrazin-2-one (CE-IP), a new compound with bronchodilator, cardiac stimulant and peripheral vasodilatory properties was found to be approximately two to five times more potent than theophylline as an inhibitor of cyclic AMP phosphodiesterase from several sources. CEIP stimulated lipolysis in isolated fat cells and was synergistic with norepinephrine in increasing glycerol release and the accumulation of [14C] cyclic AMP from adenine8-14C pre-labeled fat cells. CEIP vivo produced up to a 10-fold increase in the cyclic AMP content of rat heart.While imidazopyrazines with a secondary amine substituent on position 6 were in general relatively potent phosphodiesterase inhibitors, substituents with a more basic character resulted in decreased enzyme inhibitory activity. 5-Chloro-6-[[2-(dimethyl-amino)ethyl] amino]-1,3-dihydro-2H-imidazo[4,5-b]pyrazin-2-one (CDIP) was less active than theophylline as a phosphodiesterase inhibitor but was nearly as active as theophylline in potentiating norepinephrine-stimulated lipolysis and cyclic AMP accumulation in isolated fat cells. CDIP in vivo has peripheral vasodilatory and bronchodilatory activity but is distinct from theophylline and CEIP in that it does not cause tachycardia.

Insulin-like material in parotid and submaxillary salivary glands of normal and diabetic adult male mice.

Acid-ethanol extracts of homogenates from the parotid and submaxillary salivary glands of normal and streptozocin-induced diabetic adult male mice were investigated for insulin-like material. Extracts of both the parotid and submaxillary glands contained insulin-like immunoreactivity. The values were 156 +/- 72 ng/g wet tissue in the parotid and 104 +/- 36 ng/g wet tissue in the submaxillary gland. Fractionation of this material on Sephadex G-50 (superfine) columns revealed a single peak corresponding to the elution volume of isotopically labeled insulin. Isolated fat cells were stimulated by these extracts to convert [14C]glucose to 14CO2. This effect was blocked by preincubation with anti-insulin serum. It was observed with the avidin-biotin immunocytochemical technique that both the parotid and submaxillary glands of adult male mice possess a population of cells containing an insulin-like material. After intraperitoneal injection of streptozocin there was a marked decrease of insulin-like material extractable from both the parotid and submaxillary glands. However, this beta-cell cytotoxic agent did not completely destroy the salivary cells containing the insulin-like material. These data suggest that both the parotid and submaxillary salivary glands may be extrapancreatic sources of insulin in mice.

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane.

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).

Effect of physical training in humans on the response of isolated fat cells to epinephrine.

Endurance training helps muscle tissue oxidize lipids and therefore helps conserve glycogen. It was thought interesting to find out if, in addition to this preferential use of fatty acids by muscle tissue, there is an increase in the capacity of adipose tissue to mobilize lipids. So the response to epinephrine of collagenase-isolated fat cells obtained after biopsies of fat performed in the periumbilical region of 10 trained marathon runners (T) and 10 sedentary subjects (S), all males, was studied in vitro. Glycerol release, chosen as adipocyte lipolysis indicator, was measured by bioluminescence. Lipolysis was studied with increased epinephrine concentration. This caused a significant increase in lipolysis only in the T subjects. The dose-response curves were significantly different for T and S subjects at 10(-6) M and above (P less than 0.05). To determine the modification mechanisms observed, lipolysis with isoproterenol and epinephrine plus propranolol were studied. Isoproterenol significantly increased lipolysis in both groups. The dose-response curves were significantly different at 10(-7) M (P less than 0.01) and above. In both groups, epinephrine plus propranolol significantly decreased lipolysis without distinction between T and S. It is concluded that in male subjects endurance training increases the sensitivity of subcutaneous abdominal adipose tissue to the lipolytic action of epinephrine; this effect seems to be related to an increased response of the beta-adrenergic pathways.

Insulin action in human adipose tissue in acromegaly.

The mechanisms underlying insulin resistance in acromegaly were investigated. Adipose tissue was obtained from nine patients with acromegaly who had in vivo insulin resistance and from 14 matched healthy control subjects. Receptor binding and the antilipolytic effect of insulin were determined in isolated fat cells. Insulin-induced glucose oxidation at a physiological hexose concentration was investigated in fat segments. In fat cells obtained from acromegaly patients after an overnight fast, insulin binding at low hormone concentrations was significantly reduced by 20-30%, insulin-induced antilipolysis was unchanged, but glucose oxidation was unresponsive to insulin. Since it has recently been observed that glucose feeding may rapidly modify insulin action in human adipocytes, fat cells were also obtained 60 min after an 100-g oral glucose load. In this situation, insulin binding at low hormone concentrations was further reduced to one-half of that in the control group, and the sensitivity of insulin-induced antilipolysis was markedly decreased in acromegaly. It is concluded that, in the fasting state, the action of insulin on glucose utilization but not on lipolysis is impaired in adipose tissue of acromegalic patients because of a postreceptor defect. After glucose ingestion, the resistance to insulin in acromegaly is further enhanced and antilipolysis is also impaired. Altered coupling between receptor and effector alone or in combination with an additional decrease in receptor binding may explain the enhancement of insulin resistance. These mechanisms may be essential factors in the pathogenesis of insulin resistance in acromegaly.

Long-term in vitro effects of insulin on insulin binding and glucose transport

The long-term in vitro effects of insulin on insulin binding and glucose transport were studied using rat adipocytes in a time-dependent manner. Isolated fat cells were incubated with insulin (100 ng/ml) for 4, 8 and 24 h in a TCM 199 medium, at 37 degrees C, and then insulin binding (37 degrees C, 60 min) and 3-O-methylglucose transport (37 degrees C, 2 s) were determined. Decreased insulin binding was demonstrated in the cells incubated with insulin for 8 h, and Scatchard analysis revealed that receptor number was decreased to 61.7% of that of control cells. Thus, insulin-induced down regulation of receptors was evident after 8 h incubation with insulin. On the other hand, 8 h incubation with insulin resulted in markedly increased basal (i.e., in the absence of insulin) glucose transport up to 246% of control values. In the cells incubated with insulin for 24 h, maximally insulin-stimulated glucose transport was significantly increased up to 248% of control value. Thus, these results suggested that insulin-induced down regulation of receptors appeared to be coupled with increased cell-surface glucose transporters, and that there was a time-lag between down regulation of insulin receptors and increase of available glucose transporters in the plasma membrane.

Evolution de la capacité d'estérification des acides gras dans les adipocytes de rat avant le sevrage : effet de la suralimentation lactée

During the suckling period, lipid storage in rat adipose tissue arises from exogenous triglycerides, as milk contains a large amount of lipid. In the present work, we studied the effect of overfeeding during the suckling period on the developmental pattern of the fatty acid esterification pathway in fat cells. Early overfeeding was induced by restricting litter size; at 2 days of age, the pups were randomly distributed to form litters of 4 (overfed group) or 8 (controls). During the suckling period the activity of lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), glycerophosphate acyl transferase (GPAT) and acyl CoA ligase were measured in isolated fat cells from the inguinal adipose tissue. As early as 10 days of age, plasma triglyceride levels were increased in overfed pups as compared to the controls and, in these animals, the adipose tissue was overdeveloped, mainly due to fat cell hypertrophy. At that age, overfeeding induced a significant increase in LPL and GPAT activities, but acyl CoA ligase and GPDH were not modified in overfed pups. Fat cell hypertrophy appeared to be responsible for the increased GPAT activity in the overfed pups. On the contrary, the effect of overfeeding on LPL activity was not only related to fat cell enlargement, as the enzyme activity continued to increase in the overfed rats when expressed per unit of cell surface area. These results show that overfeeding during the suckling period rapidly induced an increase in the fat storage capacity of the adipose tissue. This suggests that circulating triglyceride levels could regulate directly or through hormonal control the activity of the fatty acid esterification pathway in fat cells.

Influence of fasting and refeeding on the antilipolytic effect of insulin in human fat cells obtained from obese subjects.

The antilipolytic effect of insulin was investigated in obese subjects before and after 7 days of total fasting, and 1 h after oral refeeding with 100 g glucose. Isolated fat cells were prepared from subcutaneous gluteal adipose tissue and incubated in vitro. Specific insulin receptor binding and insulin inhibition of basal and isoprenaline-stimulated lipolysis were determined. During the fasting period, a 15% increase (P less than 0.05) in high-affinity insulin binding and a concomitant 3-4-fold increase in insulin sensitivity were noted, and there was a marked enhancement of the maximum insulin-induced inhibition of basal lipolysis, from 4 to 10 mumol of glycerol/10(7) cells/2 h. The maximum insulin-induced inhibition of isoprenaline-induced lipolysis was similar before and after fasting, about 10 mumol/10(7) cells/2 h. Glucose refeeding induced a 30% decrease (P less than 0.02) in high-affinity insulin binding and a 20-60-fold decrease (P less than 0.01) in the sensitivity of the antilipolytic effect of insulin under basal conditions and in the presence of isoprenaline. The maximum antilipolytic effect of insulin, however, was not altered by glucose refeeding. Thus, in the basal state, maximum antilipolytic effect was larger after refeeding as compared with that before fasting. The high-affinity insulin binding and insulin sensitivity were significantly lower after refeeding than before fasting. Before the fasting period, neither the insulin binding nor the antilipolytic effect of the hormone was altered by oral glucose. It is concluded that fasting and glucose refeeding are associated with marked alterations in the antilipolytic effect of insulin on human fat cells of obese subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-stimulated phosphoinositide metabolism in isolated fat cells.

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.

Insulin resistance of fat cells from spontaneously diabetic KK mice. Analysis of insulin-sensitive phosphodiesterase.

In an attempt to determine the mechanism of insulin resistance in the presence of obesity, we examined effects of insulin on insulin-sensitive phosphodiesterase (PDE) in spontaneously diabetic KK mice. Isolated fat cells prepared from epididymal adipose tissue were incubated, with or without insulin, for 10 min. In the case of subcellular fractionation, only membrane-bound PDE was activated by insulin, as was noted in the case of rat fat cells. The specific activity was decreased in KK mice compared with control C57BL/6 mice. The dose-response curve, expressed as a percent of the maximal insulin effect, shifted to the right and the increase of ED50 indicated a decreased insulin sensitivity in the KK mice. The maximal insulin effect did not change, either when expressed as a percent of the basal enzyme activity or when expressed on a per cell basis. Specific binding of [125I]-insulin in fat cells increased in KK mice and curvilinear Scatchard plots showed an increase of the high-affinity sites. These data indicate that impairment of PDE activation in fat cells of KK mice relates to postreceptor defects and the uncoupling may result in a decreased sensitivity.

Nature of the inhibitory effect of collagenase on phosphodiesterase activity.

The level of phosphodiesterase (PDE) activity is lower in collagenase-isolated human fat cells than in adipose tissue fragments. The inhibition is not species-specific since collagenase also inhibits PDE in rat adipose tissue and bovine heart. In subcellular fractions from isolated fat cells, the PDE activities were lowest in the plasma membrane-enriched fractions and highest in the cytosolic fractions. This is opposite to PDE in subcellular fractions obtained from adipose tissue fragments. In dose-response experiments, collagenase inhibited particulate PDE to a much larger extent than it inhibited soluble PDE. The extracellular activities of PDE were completely eliminated by collagenase. Repeated washings or reincubation of the isolated fat cells did not restore the PDE activity. A purified collagenase with low specific protease activity reduced the PDE activity in isolated fat cells to a lesser extent than did a collagenase with high specific protease activities. Collagen and several protease inhibitors were ineffective in preventing the reduction of PDE after exposure to collagenase. It is concluded that nonspecific proteases in the collagenase preparations used for fat cell isolation interact with particulate and soluble PDE causing an irreversible inhibition of PDE activity in isolated fat cells. Of the various forms of PDE, plasma membrane-associated PDE seems most sensitive to collagenase.

The influence of a new compound, PATP, on glucose transport in the isolated fat cell

A newly synthesized compound, N-(4-pyridylcarbonyl amino) 1, 2, 3, 6 tetrahydropyridine (PATP) was earlier found to elevate the blood glucose level in rats. This suggested that the compound might be accomplishing this by blocking glucose transport into tissue cells. This hypothesis has now been examined in the isolated fat cell system using a modification of the method to improve the accuracy of measurements made. This study indicated that PATP is a competitive inhibitor of glucose transport and metabolism (K I = 0.89 mM) but could not unequivocally prove that the effect was on transporter action alone. That the compounds action was at this level, however was shown by its ability to inhibit the uptake of the transported but non-metabolized sugar, 3-0-methyl glucose (K I = 3 mM). PATP is a non-phenolic inhibitor of glucose transport unrelated in structure to the sugar or to another more potent inhibitor, phloretin.