Pyruvate dehydrogenase activities and rates of lipogenesis during the fed-to-starved transition in liver and brown adipose tissue of the rat.

The percentages of pyruvate dehydrogenase complex (PDH) in the active form (PDHa) in two lipogenic tissues (liver and brown adipose tissue) in the fed state were 12.0% and 13.4% respectively. After acute (0.5 h) insulin treatment, PDHa activities had increased by 77% in liver and by 234% in brown fat. Significant decreases in PDHa activities were observed in both tissues by 5 h after the removal of food. The patterns of decline in PDHa activities in the two lipogenic tissues were similar in that the major decreases in activities were observed within the first 7 h of starvation. The significant decreases in PDHa activities observed after starvation for 6 h were accompanied by decreased rates of lipogenesis. Hepatic and brown-fat PDHa activities after acute (30 min) exposure to exogenous insulin were less in 6 h-starved than in fed rats, but the absolute increases in PDHa activities over the 30 min exposure period were similar in fed and 6 h-starved rats. Increases in PDHa activities were paralleled by increases in lipid synthesis in both tissues. Re-activation of PDH in response to insulin treatment or chow re-feeding after 48 h starvation occurred more rapidly in brown adipose tissue than in liver. The results are discussed in relation to the importance of the activity of the PDH complex as a determinant of the total rate of lipogenesis during the fed-to-starved transition and after insulin challenge or re-feeding.     

Influence of alterations in meal frequency on lipogenesis and body fat content in the rat.

Rats were allowed to eat only 2 hr per day (meal-fed) or were fed ad libitum (nibbler) for 12 wk; another group of animals was meal-fed for 3 wk and then fed ad libitum (converted I) while the fourth group of rats (converted II) was meal-fed for 3 wk, allowed to nibble for the next 3 wk, meal-fed from the 6th to 9th wk and then returned to ad libitum feeding for the last 3 wk. Body fat gain and food efficiency was increased in converted I rats. The lipogenic capacity of adipose tissue from meal-fed rats was greater than observen meal-fed rats were reverted to ad libitum feeding whereas lipogenic activity increased rapidly when ad libitum fed rats were switched to meal-feeding.     

Activities of cardiac and hepatic pyruvate dehydrogenase complex are decreased after surgical stress

The work investigated the effects of surgical stress on the activities of cardiac and hepatic pyruvate dehydrogenase complex (active form, PDHa) in fed rats. PDHa activities in heart and liver were decreased within 4h of surgery with maximum inhibition at 24h after surgery. PDHa activities remained low until the fourth (liver) and eighth (heart) post-operative days. The decreased activities found at 4h and 24h after surgery were associated with increased plasma fatty acid concentrations, and inhibition of lipolysis resulted in reactivation of the enzyme complex. The results are discussed with reference to the control of pyruvate dehydrogenase activities by the oxidation of fat fuels and multisite phosphorylation in stress states, and its possible importance in glucose conservation after surgery and trauma.     

Ischaemia and tissue pyruvate dehydrogenase activities in the rat: a comparison of the effects of cervical dislocation and pentobarbital anaesthesia

The work defined the relationship between [long-chain acylcarnitine] and PDHa activities in hearts, kidneys and livers of rats sampled after cervical dislocation or pentobarbital anaesthesia. Although tissue [long-chain acylcarnitine] correlated with fatty acid availability or its mitochondrial oxidation in anaesthetized rats, this was not the case for hearts or kidneys of rats sampled after cervical dislocation. Cardiac [long-chain acylcarnitine] and PDHa activities were higher in rats killed by cervical dislocation. Metabolite changes within the hearts were consistent with tissue hypoxia and the effects of cervical dislocation were mimicked in hearts of pentobarbital-anaesthetized rats by 20s ischaemia. Renal and hepatic PDHa activities were unaffected by this short period of ischaemia. The susceptibility of cardiac PDHa to hypoxia or ischaemia may explain the variability in activities often observed within or between laboratories.     

Meal-feeding studies in mice: effects of diet on blood lipids and energy expenditure

To identify optimal study-design conditions to investigate lipid metabolism, male, C57BL/6J mice (age, 59 +/- 3 days) were allotted to eight groups, with six animals per group that were stratified by three factors: diet type (high fat [HF]: 60% of energy from fat versus that of a standard rodent diet, 14% fat, fed for 7 weeks), feeding regimen (ad libitum [ad lib] versus meal fed), and metabolic state (data collected in fasted or fed states). Serum free fatty acids (FFA) and triacylglycerols (TAG) concentrations, and energy expenditure (EE) were assessed. Mice gained 0.30 +/- 0.11 g of body weight/day when allowed ad lib access to HF diet, similar weight when meal-fed the HF or ad lib-fed the standard diet (0.10 +/- 0.03 g/day), and no weight when meal-fed the standard diet (0.01 +/- 0.02 g/day). Fed-state TAG concentration was 88 to 100% higher (P < 0.02) than that of the fasted state, except when animals were ad lib-fed the HF diet. When the standard diet was meal fed, FFA concentration was 30% higher in the fasted compared with the fed state (P = 0.003). Mice had 33% higher postprandial EE when either diet was meal fed (P = 0.01). Mice adapted to meal feeding developed transitions in metabolism consistent with known physiologic changes that occur from fasting to feeding. When fed the standard diet, a 6-h per day meal-feeding regimen was restrictive for normal growth. These data support use of a meal-feeding regimen when HF diets are used and research is focused on metabolic differences between fasted and fed states. This protocol allows study of the metabolic effects of an HF diet without the confounding effects of over-consumption of food and excess body weight gain.     

Effect of feeding pattern on the sensitivity of hepatic carnitine palmitoyl-transferase to inhibition by malonyl-CoA in the rat

1. The feeding pattern influences the inhibitory effects of malonyl-CoA on carnitine palmitoyltransferase-I. 2. The sensitivity of liver carnitine palmitoyltransferase-I to malonyl-CoA is increased in rats meal-fed when compared to rats fed ad libitum. 3. Moreover, liver carnitine palmitoyltransferase-I of meal-fed rats remains more sensitive to inhibition by malonyl-CoA during a 24 hour fast than liver carnitine palmitoyltransferase-I of rats previously fed ad libitum.     

Evidence that the stimulation of lipogenesis in the mammary glands of starved lactating rats re-fed with a chow diet is dependent on continued hepatic gluconeogenesis during the absorptive period. Effects of a gluconeogenic inhibitory, mercaptopicolinic acid, in vivo.

The rapid stimulation of lipogenesis in mammary gland that occurs on re-feeding starved lactating rats with a chow diet was decreased (60%) by injection of mercaptopicolinic acid, an inhibitor of hepatic gluconeogenesis at the phosphoenolpyruvate carboxykinase step. Mercaptopicolinate had no effect on lipogenesis in mammary glands of fed lactating rats. The inhibition of lipogenesis persisted in vitro when acini from mammary glands of re-fed rats treated with mercaptopicolinate were incubated with [1-14C]glucose. Mercaptopicolinate added in vitro had no significant effect on lipogenesis in acini from starved-re-fed lactating rats. Mercaptopicolinate prevented the deposition of glycogen and increased the rate of lipogenesis in livers of starved-re-fed lactating rats, whereas it had no significant effect on livers of fed lactating rats. Administration of intraperitoneal glucose restored the rate of mammary-gland lipogenesis in re-fed rats treated with mercaptopicolinate to the values for re-fed rats. Hepatic glycogen deposition was also restored, and the rate of hepatic lipogenesis was stimulated 5-fold. It is concluded that stimulation of mammary-gland lipogenesis on re-feeding with a chow diet after a period of starvation is in part dependent on continued hepatic gluconeogenesis during the absorptive period. Possible sources of the glucose precursors are discussed.     

Adaptive changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats

A number of metabolic factors and the activity of a number of enzymes were determined in meal-fed (animals fed a single daily 2 hr meal) and nibbling (ad libitum-fed) rats. The dependency of the observed adaptive changes on the ingestion of carbohydrate was studied by feeding diets high in carbohydrate or fat. Glucose-6-phosphate dehydrogenase and NADP-malic dehydrogenase were more active in adipose tissue from high carbohydrate meal-fed rats than in tissue from ad libitum-fed rats. The activity in adipose tissue of isocitric dehydrogenase, 6-phosphogluconate dehydrogenase, and NAD-malic dehydrogenase did not increase significantly in response to meal-feeding the high carbohydrate diet. No increase in lipogenesis or enzyme activity could be demonstrated in adipose tissue from rats meal-fed a high fat diet. Lipase activity of adipose tissue was increased by high carbohydrate meal-feeding and decreased by feeding a high fat diet. The in vitro uptake of palmitate-1-(14)C by adipose tissue was depressed by a high fat diet and enhanced in rats meal-fed a high carbohydrate diet. Diaphragm or slices of liver from high fat-fed rats oxidized palmitate-1-(14)C more rapidly than did tissue from ad libitum-fed animals. Evidence is presented for the quantitative importance of citrate as a source of extramitochondrial acetyl CoA in adipose tissue of meal-eating and ad libitum-fed rats. The relationship of extramitochondrially formed citrate to the NAD-malic dehydrogenase-malic enzyme system in adipose tissue is discussed.     

Ornithine decarboxylase and polyamines in liver and kidneys of rats on cyclical regimen of protein-free and protein-containing diets. Relationship to deoxyribonucleic acid synthesis in liver

1. The activity of ornithine decarboxylase in the liver and kidneys of rats maintained on a cyclical regimen of protein-free and protein-containing diets was investigated. There was a daily activation of the enzyme in response to the feeding of protein after 3 days feeding of protein-free diet. 2. The activation of ornithine decarboxylase in the liver and kidneys of rats re-fed on protein was demonstrable throughout 16 cycles of alternating 3-day periods of protein-free and protein-containing diets. The magnitude of the activation in the kidneys diminished from 20-fold stimulation in the first cycle to 5-fold stimulation (compared with animals fed with protein-free diet) in the later cycles of protein re-feeding. The activation of the enzyme in liver was decreased from 20-fold stimulation in the first cycle to approx. 10-fold stimulation in later cycles. 3. The concentration of spermidine was increased by approx. 50% in the liver of animals during cycling from protein-free to protein-containing diets. Spermine was unchanged, and putrescine was maintained at a low concentration approx. one-fifth to one-tenth that of spermidine after protein re-feeding. 4. The incorporation of [(3)H]thymidine into liver DNA was increased 10-fold in animals re-fed with protein compared with animals receiving protein-free diets. 5. The activation of ornithine decarboxylase by re-feeding of protein was inhibited 90% by the injection of propane-1,3-diamine during re-feeding. The stimulation of DNA synthesis was inhibited 60% by multiple injections of propane-1,3-diamine during the re-feeding of protein.     

Infusion of human insulin-like growth factor I (IGF-I) into malnourished rats reduces hepatic IGF-I mRNA abundance

To determine whether the serum level of IGF-I influences its hepatic synthesis through negative feedback regulation, we infused 200 micrograms/d of human IGF-I subcutaneously into young male rats eating either an energy-restricted or ad lib diet. In energy-restricted rats, a two-fold increase in serum IGF-I concentration produced a 41% increase in growth rate at the end of one week, and a 30% decrease in steady state hepatic IGF-I mRNA and 56% drop in serum GH at the end of two weeks. In ad lib fed rats, the increased serum IGF-I concentration neither enhanced growth rate nor significantly reduced hepatic IGF-I mRNA abundance or serum GH levels. These data suggest that the abundance of hepatic IGF-I mRNA in energy-restricted rats is controlled, in part, by serum IGF-I levels via negative feedback regulation.     

Regulation of renal and hepatic pyruvate dehydrogenase complex on carbohydrate re-feeding after starvation. Possible mechanisms and a regulatory role for thyroid hormone.

The work investigated the mechanisms for modulation of renal and hepatic pyruvate dehydrogenase complex (PDH) activities after carbohydrate re-feeding of 48 h-starved rats, and identified a regulatory role for tri-iodothyronine. Glucose re-feeding decreased blood concentrations of lipid fuels in both euthyroid and hyperthyroid rats. This treatment was not associated with re-activation of hepatic PDH in either group of rats, or of renal PDH in hyperthyroid rats (where activity was already high), but it increased renal PDH in euthyroid rats. Dichloroacetate (DCA), an activator of PDH kinase, increased renal PDH activities in euthyroid rats, but not hyperthyroid rats, and effects of glucose re-feeding or hyperthyroidism were no longer apparent. These treatments therefore exert their effects on renal PDH through changes in PDH kinase. DCA re-activation of hepatic PDH was more marked in hyperthyroid than in euthyroid rats, suggesting that, under conditions of inhibited kinase activity, PDH phosphatase is more active in livers of hyperthyroid rats. The limited effect of DCA on hepatic PDH in euthyroid rats was potentiated by glucose re-feeding or insulin, but not by inhibition of lipolysis, demonstrating a direct effect of insulin to increase hepatic PDH phosphatase. Glucose re-feeding, inhibition of lipolysis or insulin administration did not increase hepatic PDH in DCA-treated hyperthyroid rats, indicating that effects of hyperthyroidism and of insulin on PDH phosphatase are not additive.     

Effects of high sucrose diet on insulin-like effects of vanadate in diabetic rats

The insulin-like effects of vanadate were compared in streptozotocin-induced diabetic rats fed on high starch control and high sucrose diets for a period of six weeks. Diabetic rats in both diet groups were characterized by hypoinsulinemia, hyperglycemia (6.8–7.0 fold increase) and significant decreases (p<0.001) in the activities of glycogen synthase, phosphorylase and lipogenic enzymes, ATP-citrate lyase, glucose 6-phosphate dehydrogenase and malic enzyme in liver. There were no diet-dependent differences in these abnormalities. However, the insulin-mimetic agent vanadate was more effective in diabetic rats fed sucrose diet as compared to animals fed control starch diet. Vanadate administration resulted in 30% and 64% decreases in plasma glucose levels in diabetic rats fed control and sucrose diets, respectively. The activities of glycogen synthase (active) and phosphorylase (active and total) were restored significantly by vanadate in control (p<0.05–0.01) and sucrose (p<0.001) diets fed diabetic rats. This insulin-mimetic agent increased the activities of hepatic lipogenic enzymes in control diet fed rats to 38–47% of normal levels whereas in sucrose fed group it completely restored the activities. Sucrose diet caused a distinct effect on the plasma levels of triacylglycerol (4-fold increase) and apolipoprotein B (2.8-fold increase) in diabetic rats and vanadate supplementation decreased their levels by 65–75%. These data indicate that vanadate exerts insulin-like effects in diabetic rats more effectively in sucrose fed group than the animals fed control diet. In addition, vanadate also prevents sucrose-induced hypertriglyceridemia.     

Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.     

Correlation between circadian rhythms of polyamine synthesis and cell proliferation in rat liver.

In rats fed ad libitum, a marked circadian rhythm with a peak at night was observed in the hepatic level of ornithine decarboxylase (ODC) [EC 4.1.1.17], the enzyme for the first step of polyamine synthesis. A similar rhythm was found in the hepatic content of putrescine, but not of spermidine or spermine. The mitotic activity of the liver also exhibited a clear rhythm with a peak in the daytime. The rhythms of both ODC and mitosis were generated by cyclic ingestion of proteinous food, since the peaks shifted when rats were meal-fed and both activities disappeared on starvation or protein deprivation. The close parallel between the rhythms suggested that synthesis of polyamine, especially that of putrescine, was a prerequisite for the rhythmic growth of liver. The dietary induction of hepatic ODC depended on the nutritive value of dietary protein; zein or gelatin was effective only when supplemented with limiting amino acids and there was a good correlation between the hepatic ODC level and the relative growth rate.     

Hepatic pyruvate dehydrogenase kinase activities during the starved-to-fed transition.

Starvation for 48 h elicited a 74% increase in hepatic pyruvate dehydrogenase (PDH) kinase activity, measured directly by 32Pi-incorporation from [gamma-32P]ATP into a synthetic peptide corresponding to the major phosphorylation site on E1. The administration of chow ad libitum to previously-starved rats suppressed hepatic PDH kinase activity by only approx. 20% within 2 h of re-feeding, and the relatively high activity of PDH kinase was associated with continued suppression of PDC complex re-activation. Whereas there was no further decline in PDH kinase activity over the next 2 h, PDC re-activation to the fed value was observed during this time interval. PDH kinase activity decreased to fed values only after 8 h.     

Ornithine decarboxylase activity in insulin-deficient states

The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24–48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.     

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

In the fed state, hyperthyroidism increased glucose utilization indices (GUIs) of skeletal muscles containing a lower proportion of oxidative fibres. Glycogen concentrations were unchanged, but active pyruvate dehydrogenase (PDHa) activities were decreased. Hyperthyroidism attenuated the effects of 48 h of starvation to decrease muscle GUI. Glycogen concentrations and PDHa activities after 48 h of starvation were low and similar in euthyroid and hyperthyroid rats. The increase in glucose uptake and phosphorylation relative to oxidation and storage in skeletal muscle induced by hyperthyroidism may contribute to increased glucose re-cycling in the fed hyperthyroid state and to glucose turnover in the starved hyperthyroid state.     

Dietary whey protein modulates liver glycogen level and glycoregulatory enzyme activities in exercise-trained rats

This study compared the effects of dietary whey protein with dietary casein or soy protein on glycogen storage and glycoregulatory enzyme activities in the liver of sedentary and exercise-trained rats. Male Sprague-Dawley rats (ca. 130 g) were divided into one sedentary and three exercise-trained groups, with eight animals in each group. Casein was provided as the source of dietary protein in the sedentary group while the exercise-trained groups were fed casein, whey, or soy protein. Rats in the exercise-trained groups ran for 30 mins/day, 4 days/week on a motor-driven treadmill. In the exercise-trained rats, animals fed whey protein had higher liver glycogen content than animals in the other two diet groups. Glucokinase activity was significantly higher in rats fed whey protein compared to that in rats fed soy protein, while glucose 6-phosphatase activity was significantly decreased in animals on the whey protein diet compared with those the other two diets. Although 6-phospho-fructokinase activity was significantly lower in the whey protein group than in the soy protein group, we found that fructose 1,6-bisphosphatase activity was significantly higher in the whey group compared with either the casein or soy groups. Pyruvate kinase activity in rats fed the casein diet was significantly higher than in rats fed either the whey or soy protein diets. In addition, hepatic alanine aminotransferase activity and serum alanine level were also increased in the whey protein group compared with the casein or soy protein groups. Taken together, these results demonstrate that the whey protein diet in exercise-trained rats results in significantly higher levels of liver glycogen, because of the combined effects of regulation of rate limiting glycolytic and gluconeogenic enzyme activities and activation of glycogenesis from alanine via alanine amino-transferase.     

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid

Spray-dried milk enriched with n-3 fatty acids from linseed oil (LSO) or fish oil (FO) were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of α linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25–36% and glutathione transferase activity increased to an extent of 34–39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2 to 4.5-fold compared to control. The serum thromboxane B₂ level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas 6-keto-prostaglandin F1α level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase.     

Prevention of insulin resistance by environmental manipulation as young rats mature

This study was initiated in an attempt to see if the insulin resistance associated with maturation in young rats could be prevented by environmental manipulation. Consequently, seven week-old rats were either housed in standard laboratory cages and fed a calorie-restricted diet or placed individually in exercise wheel cages and allowed to eat chow ad lib. A control group of rats was housed in standard laboratory cages from seven weeks to five months of age, and also allowed to eat chow ad lib. When studied at five months of age, the chow-fed rats weighed more (624 +/- 8 g) than either the calorie restricted (479 +/- 9 g) or exercise trained (485 +/- 13 g) rats. Insulin action was compared in the three groups by assessing the steady-state serum glucose (SSSG) and insulin (SSSI) concentrations achieved during a continuous intravenous infusion of glucose and exogenous insulin. The results of these studies indicated that SSSG concentration was significantly higher (P less than 0.001) in chow-fed rats than in the two experimental groups. Since SSSI concentrations were the same in all three groups, lower SSSG concentrations in calorie-restricted and exercise trained rats indicates that insulin-stimulated glucose uptake was preserved in these two groups as compared to the chow-fed population. In an attempt to understand why exercise training and calorie restriction prevented the development of insulin resistance, muscle glycogen synthase activity and muscle capillary density were compared in the three groups of five month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)