Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Cyclic AMP-dependent protein kinase activity and lipolysis in adipose tissue. Effect of fasting, oligomycin and iodoacetamide

The release of glycerol into the medium, the concentration of cAMP, and the cAMP-dependent protein-kinase activity were studied in adipocytes and in fat-pads obtained from epididymal adipose tissue of rats under different conditions of feeding. An increase in the tissue concentration of cAMP and in the protein-kinase activity was observed in vivo at 48 and 96 h of fasting. A diminished release of glycerol was found in adipocytes from rats fasted for 48 h, in the absence of glucose, and the maximum concentration of cAMP was inferior to that of fed rats. Oligomycin and iodoacetamide, in the presence of epinephrine and glucose, produce a diminution in the values of the parameters studied. No significant differences were observed, however, in the responses of tissue obtained from fed and fasting rats to these compounds. The present results confirm previous observations and show the dependence of the lipolytic process on carbohydrate metabolism.     

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.     

Effects of epinephrine on glucose transport and metabolism in adipose tissue of normal and hypothyroid rats

Epinephrine increases the oxidation of glucose in adipose tissue even when its lipolytic effects are markedly reduced or abolished by propranolol, nicotinic acid, ouabain, or thyroidectomy. In order to locate the site(s) at which epinephrine stimulates glucose utilization, we studied the effects of epinephrine on the oxidation of various metabolites of glucose. Epinephrine neither increased the production of (14)CO(2) from 1- or 3-(14)C-pyruvate nor affected pyruvate conversion to glyceride-glycerol. To assess the possibility that epinephrine might accelerate the entry of glucose into adipocytes, we studied the accumulation of the nonmetabolized sugar l-arabinose in the intracellular water of adipose tissue. Epinephrine increased arabinose penetration into adipocytes to a degree comparable with that caused by 0.1 mU/ml of insulin. Virtually identical results were obtained in tissues from thyroidectomized rats in which the lipolytic effects of epinephrine were significantly reduced. It is concluded that epinephrine increases glucose oxidation by promoting its entry into adipose tissue and that the effect is independent of lipolysis.     

Comparison of the lipolytic activity of adipocytes isolated from white adipose tissue in Zucker and Wistar rats. In vitro effects of 3,5,3'-triiodo-L-thyronine

The in vitro effects of 3,5,3'-triiodo-L-thyronine (L-T3) on (-) epinephrine stimulated lipolytic activity were studied in adipocytes isolated from white adipose tissue of Zucker and Wistar male rats. Basic lipolytic activity was small and nearly insensitive to epinephrine in Zucker genetically obese rats. On the other hand, lipolytic activity was stimulated by epinephrine in Zucker lean rats and Wistar rats in the same way. There was no synergistic interaction between epinephrine and L-T3 on lipolytic activity, since the released glycerol levels were nearly the same. These data corroborate the hypothyroid status of Zucker genetically obese rats.     

Regulation of cytoplasmic acetyl-CoA and acetoacetyl-CoA synthetases by dexamethasone phosphate in rat liver and adipose tissue.

The activity of cytoplasmic acetyl-CoA synthetase and acetoacetyl-CoA synthetase in both liver and adipose tissue was measured in five groups of rats : fed, fasted, fasted and re-fed, fasted, re-fed and injected with dexamethasone during the re-feeding period, fed and injected with dexamethasone. Fasting was found to have a strong inhibitory effect on the activity of the two enzymes in both adipose tissue and liver, which could be abolished by re-feeding. In adipose tissue, dexamethasone prevented this re-establishment of normal enzyme activity but was without effect in the liver. It can therefore be concluded that dexamethasone inhibits the synthesis of acetyl-CoA and acetoacetyl-CoA synthetases in adipose tissue.     

The role of calcium ion in epinephrine activation of lipolysis.

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.     

The effect of 3-mercaptopicolinic acid on phosphoenolpyruvate carboxykinase (GTP) in the rat and guinea pig.

In vitro, 3-mercaptopicolinic acid inhibited phosphoenolpyruvate carboxykinase activity in supernatant fractions of liver, kidney cortex, and adipose tissue obtained from fasted rats. 3-Mercaptopicolinic acid also inhibited enzymatic activity in the mitochondrial and supernatant fractions of liver obtained from fasted guinea pigs. In the fasted rat, the oral administration of 3-mercaptopicolinic acid increased liver carboxykinase activity even though the blood glucose concentrations decreased. Kidney cortex carboxykinase decreased while adipose tissue enzyme was unchanged. In the fasted guinea pig, the oral administration of 3-mercaptopicolinic acid lowered blood glucose concentrations but had no effect on liver mitochondrial or supernatant carboxykinase activity. The elevation in rat liver enzymatic activity appears to be due to protein synthesis, since the concurrent administration of cycloheximide prevents the increase in enzyme activity. 3-Mercaptopicolinic acid appears to be noncompetitive with respect to Mn²⁺.     

Inhibition of free fatty acid mobilization by colchicine

Segments of epididymal adipose tissue from normal male rats were incubated with micromolar concentrations of colchicine for different periods of time up to 4 hr, and the mobilization of free fatty acids (FFA) was measured during a subsequent reincubation. Although pretreatment with colchicine did not alter basal unstimulated FFA release, mobilization of FFA in the presence of epinephrine or theophylline was reduced. However, neither lipolysis, as judged by glycerol production, nor cyclic AMP accumulation was impaired under the same conditions. To assess the possibility that colchicine might limit production of fatty acids by accelerating the entry and metabolism of glucose into adipocytes, the metabolism of glucose by adipose tissue was studied. Pretreatment with colchicine did not affect uptake of glucose nor its oxidation to CO(2), although colchicine-treated tissues did have slightly more [(14)C]glucose incorporated into the glyceride moiety of triglyceride. When adipose tissues pretreated with colchicine were incubated in an albumin-free medium, no reduction in FFA production by colchicine was observed. Because no FFA release occurs in albumin-free media, this experiment suggests that colchicine-induced inhibition of FFA mobilization results from impaired extrusion of FFA from adipose cells.     

Methionine restriction effects on 11 -HSD1 activity and lipogenic/lipolytic balance in F344 rat adipose tissue

Methionine restriction (MR) limits age-related adiposity in Fischer 344 (F344) rats. To assess the mechanism of adiposity resistance, the effect of MR on adipose tissue (AT) 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD1) was examined. MR induced 11beta-HSD1 activity in all ATs, correlating with increased tissue corticosterone. However, an inverse relationship between 11beta-HSD1 activity and adipocyte size was observed. Because dietary restriction controls lipogenic and lipolytic rates, MR's effects on lipogenic and lipolytic enzymes were evaluated. MR increased adipose triglyceride lipase and acetyl-coenzyme A carboxylase (ACC) protein levels but induced ACC phosphorylation at serine residues that render the enzyme inactive, suggesting alterations of basal lipolysis and lipogenesis. In contrast, no changes in basal or phosphorylated hormone-sensitive lipase levels were observed. ACC-phosphorylated sites were specific for AMP-activated protein kinase (AMPK); therefore, AMPK activation was evaluated. Significant differences in AMPKalpha protein, phosphorylation, and activity levels were observed only in retroperitoneal fat from MR rats. No differences in protein kinase A phosphorylation and intracellular cAMP levels were detected. In vitro studies revealed increased lipid degradation and a trend toward increased lipid synthesis, suggesting the presence of a futile cycle. In conclusion, MR disrupts the lipogenic/lipolytic balance, contributing importantly to adiposity resistance in F344 rats.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

Dietary protein and the control of fatty acid synthesis in rat adipose tissue

Fatty acid synthesis in adipose tissue normally proceeds at a high rate when fasted animals are refed a diet containing carbohydrate, protein, and low levels of fat. This study investigated the effect of omitting protein from the refeeding diet. Rats were fasted for 48 hr and refed either a protein-free diet or a balanced diet, and the rate of fatty acid synthesis from glucose, pyruvate, lactate, and aspartate was measured. Refeeding the animals a diet devoid of protein resulted in a low rate of fatty acid synthesis from each of these substrates as well as a reduction in carbon flow over the citrate cleavage pathway. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-malate dehydrogenase, and ATP-citrate lyase were also reduced in epididymal fat pads from these rats. On the other hand, adipose tissue phosphoenolpyruvate carboxykinase activity was five times as great as that in tissue from animals refed a balanced diet. This difference could be eliminated if actinomycin D was injected coincident with refeeding. Refeeding rats diets high in carbohydrate is not, therefore, capable of inducing high rates of fatty acid synthesis in adipose tissue in the absence of dietary proteins. Thus, liver and adipose tissue respond differently to dietary protein.     

Involvement of proteolytic enzymes in the lipotropic effect of the pituitary polypeptide hormones

The influence of proteinase inhibitors on the lipolytic effect of the pituitary polypeptide hormones and epinephrine in an isolated adipose tissue of rabbits and rats has been studied. Neither of proteinase inhibitors changed the basal rate of lipolysis. Trasylol, a serine proteinase inhibitor, suppressed completely growth hormone (GH) effect and partially reduced the effect of adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH) but did not change the effect of epinephrine. Bacitracin proved ineffective with regard to the effect of polypeptide hormones. Pepstatin, an acid proteinase inhibitor, partially blocked the stimulation of lipolysis by ACTH without affecting the effect of GH and beta-LPH. The influence of proteinase inhibitors on the ACTH effect in rat adipose tissue was similar to that found in rabbit tissue. The Trasylol-induced inhibition of the hormone-stimulated lipolysis decreased to a considerable extent after GH or ACTH incubation with rabbit plasma or partial GH digestion with pepsin. This decrease was not observed when plasma serine proteinases were blocked during GH incubation with plasma. The results demonstrate an involvement of some proteolytic enzymes in the realization of the polypeptide hormone lipolytic effect and permit to suppose the requirement of preliminary activation of the hormones by means of proteolytic modification.     

Body fat-lowering effect of conjugated linoleic acid is not due to increased lipolysis

The ability of conjugated linoleic acid (CLA) to reduce adiposity may be due to changes in energy expenditure and/or direct effects on adipocyte lipid metabolism. The aim of the present work was to analyse if CLA supplementation modifies lipolytic activity in adipose tissue from hamsters fed on high-fat diet. Hamsters were divided into two groups and fed on diets supplemented with either 0.5% linoleic acid (control) or 0.5% trans-10,cis-12 CLA. After 6 weeks, animals were fasted overnight and adipose tissues were dissected and weighed. Adipocytes were isolated by collagenase digestion and incubated in Krebs-Ringer bicarbonate buffer with or without several agents acting at different levels of the lipolytic cascade. Adipocyte diameters were measured by microscopy. Adipose tissue DNA content was assessed by spectrophotometry. Animals fed on CLA diet showed significantly reduced adipose tissue mass. No differences between both groups was found for basal lipolysis, lipolytic effects of isoproterenol, forskolin, dibutyryl-cAMP and isobutylmethylxanthine, and pD2 for isoproterenol. A similar total DNA amount was found in adipose tissue of both groups, showing that CLA diet had no effect on total cell number per fat pad. Although DNA content per gram tissue, an indirect reverse index of cell size, was significantly increased in CLA fed hamsters, microscopy did not reveal differences in medium mature adipocyte diameter, nor in cell size distribution between both groups. These results suggest that adipose tissue size reduction induced by trans-10,cis-12 CLA intake is not due to changes in lipolysis. Reduced preadipocyte differentiation into mature adipocytes may account for this fat-lowering effect.     

Evidence for a role of somatostatin in lipid metabolism of liver and adipose tissue

We have studied the effects of somatostatin on lipid metabolism in liver and adipose tissue of fasted mice. The animals were injected subcutaneously with 8 micrograms somatostatin and killed 5 min after injection. In vivo incorporation of [14C]acetate into triglycerides in both tissues and into hepatic cholesterol was significantly enhanced by somatostatin. Concomitantly, a decrease of triglyceride lipase activity was observed, which corresponds well with the variation undergone by cyclic AMP-protein kinase system. In addition, a marked increase of serum cholesterol levels was observed. Additionally, in vitro experiments were also performed by employing 2.4 X 10(-6) M somatostatin. The results showed that the direct effect of somatostatin on liver seems to be a decrease in acetate uptake. The results obtained with the adipose tissue were similar to those obtained in in vivo conditions. On the other hand, when somatostatin was administered in vivo, the ability to incorporate ortho[32P]phosphate into phospholipids was enhanced in both tissues. Likewise in the in vitro experiments with [14C]acetate, the somatostatin seems to act by decreasing the ortho[32 P]phosphate uptake in liver. While in adipose tissue the somatostatin only caused a strong increase in the specific activity of phosphatidylcholine. These data demonstrate in fasted mice that somatostatin is able to counteract the lipolytic manifestations of the fasted state.     

The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.     

A single bout of exercise induces beta-adrenergic desensitization in human adipose tissue

This study was designed to assess whether physiological activation of the sympathetic nervous system induced by exercise changes adipose tissue responsiveness to catecholamines in humans. Lipid mobilization in abdominal subcutaneous adipose tissue was studied with the use of a microdialysis method in 11 nontrained men (age: 22. 3 +/- 1.5 yr; body mass index: 23.0 +/- 1.6). Adipose tissue adrenergic sensitivity was explored with norepinephrine, dobutamine (beta(1)-agonist), or terbutaline (beta(2)-agonist) perfused during 30 min through probes before and after 60-min exercise (50% of the maximal aerobic power). The increase in extracellular glycerol concentration during infusion was significantly lower after the exercise when compared with the increase observed before the exercise (P < 0.05, P < 0.02, and P < 0.01, respectively, for norepinephrine, dobutamine, and terbutaline). In a control experiment realized without exercise, no difference in norepinephrine-induced glycerol increase between the two infusions was observed. To assess the involvement of catecholamines in the blunted beta-adrenergic-induced lipolytic response after exercise, adipose tissue adrenergic sensitivity was explored with two 60-min infusions of norepinephrine or epinephrine separated by a 60-min interval. With both catecholamines, the increase in glycerol was significantly lower during the second infusion (P < 0.05). The findings suggest that aerobic exercise, which increased adrenergic activity, induces a desensitization in beta(1)- and beta(2)-adrenergic lipolytic pathways in human subcutaneous adipose tissue.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.     

In vivo human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue

We studied eight normal-weight male subjects to examine whether the lipolytic rate of deep subcutaneous and preperitoneal adipose tissues differs from that of superficial abdominal subcutaneous adipose tissue. The lipolytic rates in the superficial anterior and deep posterior subcutaneous abdominal adipose tissues and in the preperitoneal adipose tissue in the round ligament were measured by microdialysis and (133)Xe washout under basal, postabsorptive conditions and during intravenous epinephrine infusion (0.15 nmol. kg(-1). min(-1)). Both in the basal state and during epinephrine stimulation, the superficial subcutaneous adipose tissue had higher interstitial glycerol concentrations than the two other depots. Similarly, the calculated glycerol outputs from the superficial depot were significantly higher than those from the deep subcutaneous and the preperitoneal depots. Thus, it is concluded that the lipolytic rate of the superficial subcutaneous adipose tissue on the anterior abdominal wall is higher than that of the deep subcutaneous adipose tissue on the posterior abdominal wall and that of the preperitoneal adipose tissue in the round ligament.     

Impaired hormonal regulation of lipolysis and the interference with adenosine in adipose tissue from hyperglycemic sand rats in vitro

Sand rats (Psammomys obesus) developed in response to different food intake various states of hyperglycemia and hyperinsulinism. 12 normo- and 10 hyperglycemic animals were selected by means of a weekly control of plasma glucose and plasma insulin over a period of 12 weeks after separation from the mother. During this time also the development of body weight gain was checked. In both groups of rats the hormonal regulation of glycerol release by incubated adipose tissue was investigated. In any case, the fat tissue from hyperglycemic sand rats showed a lower lipolytic responsiveness to noradrenaline stimulation than that of their normoglycemic controls. This correlates well with previous results in hyperglycemic sand rats in which the catecholamine-stimulated cAMP production was disturbed (Knospe and K?hler 1981). Degradation of released adenosine by addition of adenosine deaminase significantly enhanced the noradrenaline action on glycerol release in both groups of sand rats. Even though the noradrenaline-stimulated lipolytic activity of adipose tissue from normo- and hyperglycemic animals was enhanced in the presence of adenosine deaminase, the hormone resistance of adipose tissue from hyperglycemic sand rats was nevertheless not abolished. The theophylline-mediated adenosine receptor blockade gave further evidence that particularly endogenous adenosine released during incubation of adipose tissue from sand rats inhibited the noradrenaline action on lipolysis. The antilipolytic action of insulin on glycerol release is negligibly low in normoglycemic as well as hyperglycemic sand rats. The degradation of adenosine by adenosine deaminase failed to improve the insulin action. Adenosine addition completely blocked the stimulating effects of noradrenaline on glycerol release.(ABSTRACT TRUNCATED AT 250 WORDS)