Lipolytic activity of Brochothrix thermosphacta on natural triglycerides

Four natural glycerides were used to study the lipolytic characteristics of Brochothrix thermosphacta lipase. Hydrolysis of triglycerides from linseed oil and pork fat adipose tissue with a high percentage of unsaturated fatty acids was two to three times greater than in saturated triglycerides from lamb or beef fat adipose tissues. Hydrolysis was not specific as the percentage of the released fatty acids was similar to that of the starting triglycerides.     

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.     

Alteration of rat adipose tissue lipolytic response to norepinephrine by dietary fatty acid manipulation.

The present study clearly shows that, by feeding rats a semi-synthetic diet of known composition enriched with saturated fatty acids, the epididymal fat pad responsiveness to norepinephrine $\text{in}\text{vitro}$ can be abolished relative to fat pads from animals fed a similar diet but enriched with polyunsaturated fatty acids. Addition of varying concentrations of norepinephrine to the incubation medium produced a clear dose-response relationship in fat pads from animals fed diet enriched with polyunsaturated fatty acids while no effect of norepinephrine was apparent at any dose level in fat tissue from animals fed saturated fatty acids. These changes in lipolytic responsiveness were concurrent with alterations in fatty acid compositions of adipose tissue phospholipids and triglycerides as well as in total tissue contents of phospholipids and cholesterol.     

Lipoprotein lipase in plasma after an oral fat load: relation to free fatty acids.

Lipoprotein lipase (LPL) releases fatty acids from triglyceride-rich lipoproteins for use in cellular metabolic reactions. How this hydrolysis, which occurs at the vascular endothelium, is regulated is poorly understood. A fatty acid feedback system has been proposed by which accumulation of fatty acids impedes LPL-catalyzed hydrolysis and dissociates the enzyme from its endothelial binding sites. We examined this hypothesis in humans who were subjected to an oral fat tolerance test of a mixed-meal type. Plasma triglycerides, free fatty acids, and LPL activity were measured before and repeatedly during a 12-h period after intake of the fat load. Since soybean oil with a high content of linoleic fatty acid was the source of triglycerides, a distinction could be made between endogenous free fatty acids (FFA) and FFA derived directly from lipolysis of postprandial triglyceride-rich lipoproteins. Mean LPL activity was almost doubled (P less than 0.01) 6 h after intake of the oral fat load. The rise in LPL activity was accompanied by an increase of plasma triglycerides and linoleic free fatty acids (18:2 FFA), but not of total plasma FFA, which instead displayed a heterogeneous pattern with essentially unchanged mean levels. The postprandial response of LPL activity largely paralleled the postprandial responses of 18:2 FFA and triglycerides. The highest degree of parallelism was seen between postprandial 18:2 FFA and LPL activity levels. Furthermore, the integrated response (area under the curve, AUC) for plasma measurements of LPL correlated with the AUC for 18:2 FFA (r = 0.40, P less than 0.05), but not with the AUC for plasma triglycerides (r = 0.21, ns). The high degree of parallelism and significant correlation between postprandial plasma LPL activity and 18:2 FFA support the hypothesis of fatty acid control of endothelial LPL during physiological conditions in humans.     

Lipid composition of liver peroxisomes isolated from untreated and clofibrate-treated mice and rats

Peroxisomes were isolated from liver tissue of control and clofibrate-treated adult male NMRI mice and Sprague-Dawley rats. Phospholipids, cholesterol, triglycerides and free fatty acids were measured in the peroxisomes. The fatty acid profiles of the phosphatidylethanolamine, the phosphatidylcholine, the triglyceride and the free fatty acid fractions were also analyzed. Phosphatidylethanolamine was the dominating phospholipid in peroxisomes from untreated animals. The fatty acid profiles of phosphatidylethanolamine, free fatty acids and triglycerides were similar for untreated mice and rats but differences between the species were observed in the pattern derived from phosphatidylcholine. Phosphatidylcholine was the most abundant phospholipid after clofibrate treatment. Clofibrate treatment caused an increase in the concentrations of phospholipids and unsaturated long-chain fatty acids and a decrease in the concentrations of triglycerides, free fatty acids, cholesterol and shorter saturated fatty acids.     

Specific identification of free and esterified fatty acids in tissue sections

Synopsis The histochemical method of Adamset al. (1966) for demonstrating triglycerides in tissue sections was applied to kidneys exhibiting a wide variety of disease states. It became apparent, as would be expected, that the existing method demonstrates not only triglycerides but also free fatty acids in the same section. Even though the presence of free fatty acids could be detected in the control sections, their existence made it impossible to identify triglycerides with certainty.A modification is described which employs a potassium hydroxide-dioxan mixture to saponify and extract selectively free fatty acids from tissue sections. Fatty acids in free form can be demonstrated separately, in parallel sections, from those esterified as triglyceride. This modified technique was applied to frozen sections of formalin-fixed human and rat tissues, revealing distinct and highly characteristic distribution patterns for these two forms of fatty acid.     

Regulation of lipid flux between liver and adipose tissue during transient hepatic steatosis in carnitine-depleted rats

Rats with carnitine deficiency due to trimethylhydrazinium propionate (mildronate) administered at 80 mg/100 g body weight per day for 10 days developed liver steatosis only upon fasting. This study aimed to determine whether the transient steatosis resulted from triglyceride accumulation due to the amount of fatty acids preserved through impaired fatty acid oxidation and/or from up-regulation of lipid exchange between liver and adipose tissue. In liver, mildronate decreased the carnitine content by approximately 13-fold and, in fasted rats, lowered the palmitate oxidation rate by 50% in the perfused organ, increased 9-fold the triglyceride content, and doubled the hepatic very low density lipoprotein secretion rate. Concomitantly, triglyceridemia was 13-fold greater than in controls. Hepatic carnitine palmitoyltransferase I activity and palmitate oxidation capacities measured in vitro were increased after treatment. Gene expression of hepatic proteins involved in fatty acid oxidation, triglyceride formation, and lipid uptake were all increased and were associated with increased hepatic free fatty acid content in treated rats. In periepididymal adipose tissue, mildronate markedly increased lipoprotein lipase and hormone-sensitive lipase activities in fed and fasted rats, respectively. On refeeding, carnitine-depleted rats exhibited a rapid decrease in blood triglycerides and free fatty acids, then after approximately 2 h, a marked drop of liver triglycerides and a progressive decrease in liver free fatty acids. Data show that up-regulation of liver activities, peripheral lipolysis, and lipoprotein lipase activity were likely essential factors for excess fat deposit and release alternately occurring in liver and adipose tissue of carnitine-depleted rats during the fed/fasted transition.     

Fatty liver produced by dietary deficiencies: its pathogenesis and potentiation by ethanol

In a study of the pathogenesis of hepatic fat accumulation under experimental conditions mimicking chronic alcoholism, rats were fed a low-fat diet, deficient in amino acids and choline, containing either ethanol or isocaloric amounts of carbohydrate. Dietary deficiencies alone produced a moderately fatty liver after 24 days. The combination of ethanol and dietary deficiencies resulted in enhanced lipid accumulation, which was apparent after only 11 days. In an investigation of the origin of hepatic triglyceride fatty acids, the experiment was repeated after the adipose lipids had been marked by the feeding of oils containing characteristic fatty acids (linseed oil, containing linolenate, or coconut oil, containing laurate and myristate). In all animals, the fatty acid composition of the hepatic triglycerides differed markedly from that of adipose tissue; it had a larger percentage of endogenously synthesized fatty acids and a five times smaller percentage of the marker fatty acids. In addition, ethanol feeding resulted in a greater retention of the marker fatty acids in the adipose tissue. Thus, the deposition of hepatic triglycerides produced by the feeding of deficient diets is markedly potentiated by ethanol; the triglyceride fatty acids accumulated under these conditions appear to originate, for the most part, not from mobilization of depot fat, but from endogenous synthesis.     

Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglyceride by livers of carbohydrate-fed rats

High carbohydrate diets enhance the hepatic output of very low density lipoprotein triglycerides. The fatty acids of these triglycerides could come from exogenous sources (i.e., diet or adipose tissue) or from de novo fatty acid synthesis in the liver. The role of exogenous free fatty acids was evaluated in rats fed Purina Chow or diets containing 10% fructose for up to 14 wk. In carbohydrate-fed rats, serum triglycerides were twice normal, and VLDL accounted for about 60% of the increases. Pre-beta-lipoprotein was increased and alpha- and beta-lipoprotein were decreased. Phospholipid and cholesterol levels were unchanged. Livers were perfused with glucose and free fatty acids. Perfusate free fatty acids rose from 180 to 1800 micro eq/liter as the infused acids increased from 0 to 992 micro eq/3 hr; simultaneously, net free fatty acid uptake rose from < 1 to 18 micro eq/g/hr and triglyceride output by the liver doubled. However, rates of secretion of triglyceride became constant, and triglyceride accumulated in liver at uptakes of free fatty acids > 13 micro eq/g/hr. More lauric and myristic acid appeared in the perfusate than was infused, suggesting the hepatic discharge of free fatty acids. Livers of fructose-fed rats secreted twice as much oleate-(14)C-labeled triglyceride as controls at all levels of free fatty acid uptake. The ratios of the specific activities of perfusate triglyceride to free oleate-(14)C were unaffected by diet and were about 0.6 and 1.0 at low and high triglyceride secretion rates, respectively. Thus, carbohydrate feeding did not result in altered uptakes of free fatty acids or preferential secretion of triglycerides containing endogenously synthesized fatty acid. Instead, the increased secretion of triglyceride was accomplished by enhanced formation of VLDL triglyceride from exogenous free fatty acids.     

Splanchnic and hepatic triglyceride secretion during hypercaloric intravenous glucose infusion in conscious swine.

We have validated a radiochemical technique for measuring the rate of secretion of plasma triglycerides from the liver and/or splanchnic region during the consumption of glucose under isotopic steady-state conditions. Values obtained with this technique correlated closely with those based on transhepatic or transsplanchnic chemical gradients (r = 0.95). Likewise, values for secretion of triglycerides obtained with the radiochemical technique correlated closely with those obtained for extrahepatic or extrasplanchnic triglyceride clearance. Values for mean net splanchnic and hepatic secretion of plasma triglyceride fatty acids, transported essentially in very low density lipoproteins, were 1.9 and 2.0 mumoles/min.kg body wt0.75, respectively, about one-half of the rate of transport of free fatty acids. However, the fraction of triglyceride fatty acids of plasma very low density lipoproteins that was derived from plasma free fatty acids averaged 9% and that derived from glucose, though increasing with time, reached only 2% after constant intravenous infusion of radioglucose for 5 hr. Porcine hepatic secretion of plasma triglycerides is large in the glucose-fed state, and the secreted triglyceride fatty acids evidently are derived from stored fat or glycon.     

Histochemical and thin layer chromatographic analyses of neutral lipids in Echinostoma revolutum metacercariae cultured in vitro

Excysted metacercariae of Echinostoma revolutum cultured in vitro in the defined medium, NCTC 135 supplemented with 20% hens' egg yolk, doubled their mean relative body area and showed significant sucker growth within 14 days. Histochemical Oil Red O staining showed neutral fat mainly in the excretory system of excysted metacercariae and in adults grown in the domestic chick. In vitro cultured worms showed neutral fat in the intestine, parenchyma, and excretory system. As detected by TLC the major neutral lipid fractions were free fatty acids for excysted metacercariae; free sterols for adults grown in chicks; and triglycerides, free fatty acids, and free sterols for cultured worms. Excysted metacercariae excreted free fatty acids into a nonnutrient incubation medium, whereas cultured worms excreted diglycerides, triglycerides, and free fatty acids into the medium.     

Effects of dietary fat and zinc on adiposity,serum leptin and adipose fatty acid composition in C57BL/6J mice

Zinc (Zn) has been implicated in altered adipose metabolism, insulin resistance and obesity. The objective of this study was to investigate the effects dietary Zn deficiency and supplementation on adiposity, serum leptin and fatty acid composition of adipose triglycerides and phospholipid in C57BL/6J mice fed low-fat (LF) or high-fat (HF) diets for a 16 week period. Weanling C57BL/6J mice were fed LF (16% kcal from soybean oil) or HF (39% kcal from lard and 16% kcal from soybean oil) diets containing 3, 30 or 150 mg Zn/kg diet (ZD = Zn-deficient, ZC = Zn control and ZS = Zn-supplemented, respectively). HF-fed mice had higher fat pad weights and lower adipose Zn concentrations than the LF-fed mice. The ZD and ZS groups had a reduced content of fatty acids in adipose triglycerides compared to the ZC group, suggesting that zinc status may influence fatty acid accumulation in adipose tissue. Serum leptin concentration was positively correlated with body weight and body fat, and negatively correlated with adipose Zn concentration. Dietary fat, but not dietary Zn, altered the fatty acid composition of adipose tissue phospholipid and triglyceride despite differences in Zn status assessed by femur Zn concentrations. The fatty acid profile of adipose triglycerides generally reflected the diets. HF-fed mice had a higher percentage of C20:4 n-6, elevated ratio of n-6/n-3, lower ratio of PUFA/SAT and reduced percentage of total n-3 fatty acids in adipose phospholipid, a fatty acid profile associated with obesity-induced risks for insulin resistance and impaired glucose transport. In summary, the reduced adipose Zn concentrations in HF-fed mice and the negative correlation between serum leptin and adipose Zn concentrations support an interrelationship among obesity, leptin and Zn metabolism.     

The role of lipolysis in human orosensory fat perception

Taste perception elicited by food constituents and facilitated by sensory cells in the oral cavity is important for the survival of organisms. In addition to the five basic taste modalities, sweet, umami, bitter, sour, and salty, orosensory perception of stimuli such as fat constituents is intensely investigated. Experiments in rodents and humans suggest that free fatty acids represent a major stimulus for the perception of fat-containing food. However, the lipid fraction of foods mainly consists of triglycerides in which fatty acids are esterified with glycerol. Whereas effective lipolysis by secreted lipases (LIPs) liberating fatty acids from triglycerides in the rodent oral cavity is well established, a similar mechanism in humans is disputed. By psychophysical analyses of humans, we demonstrate responses upon stimulation with triglycerides which are attenuated by concomitant LIP inhibitor administration. Moreover, lipolytic activities detected in minor salivary gland secretions directly supplying gustatory papillae were correlated to individual sensitivities for triglycerides, suggesting that differential LIP levels may contribute to variant fat perception. Intriguingly, we found that the LIPF gene coding for lingual/gastric LIP is not expressed in human lingual tissue. Instead, we identified the expression of other LIPs, which may compensate for the absence of LIPF.     

Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.

Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzyme in vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.     

Biochemistry of radioiodinated free fatty acids

Summary Radioiodinated free fatty acids have been developed to study myocardial metabolism non-invasively in man. In the present study the distribution of radiolabeled lipids in the myocardium and in arterial and coronary sinus blood was evaluated following injection of three commonly used iodinated fatty acids in fasted (n = 5) and lactate loaded (n = 3) dogs. Five minutes after simultaneous i.v. injection of radioiodinated 17-I-heptadecanoic acid (IHDA),15-(p-I-phenyl) pentadecanoic acid (IPPA) and 15-(p-I-phenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) a biopsy specimen and samples of arterial and coronary sinus blood were taken. After extraction and TLC the relative distribution of radioactivity in the aqueous phase (containing the oxidation products), pellet and organic phase was calculated. The organic phase was further divided into phospholipids, diglycerides, free fatty acids, triglycerides and cholesterolesters. Seventy two percent of IHDA was oxidized, 36% of IPPA and 7% of DMIPPA. The organic phase consisted primarily of triglycerides and phospholipids. The ratios of triglycerides to phospholipids were about the same for IHDA, IPPA and DMIPPA (0.58, 0.65 and 0.50, respectively). Free IHDA in tissue samples was low (4%) and elevated for IPPA and DMIPPA, (17% and 37%). During lactate loading triglycerides were higher for all three fatty acids. For IHDA and IPPA this increase was paralleled by a decrease in the aqueous phase, in case of DMIPPA the aqueous phase remained the same. Five minutes after injection most of the organic phase of both arterial and coronary sinus blood consisted of the injected fatty acids, the aqueous phase contained oxidation products. There were only minor differences during lactate loading. During the evaluation of scintigraphic patterns of the radioiodinated fatty acids under normal conditions (eg at rest) and during elevated lactate levels (eg during exercise) the differences in distribution must therefore be considered.     

Effects of postnatal age and diet on the fatty acid composition of plasma lipid fractions in preterm infants

Diet and postnatal age effect the fatty acid composition of plasma and tissue lipids. This work was designed as a transversal study to evaluate the changes in the fatty acid composition of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in preterm infants (28-35 weeks gestational age), fed human milk (HM) and milk formula (MF) from birth to 1 month of life. Sixteen blood samples were obtained from cord, and 19 at 6-8 h after birth, 14 at 1 week and 9 at 4 weeks from HM-fed infants and 18 at 1 week and 14 at 4 weeks from MF-fed ones. Groups had similar mean birth weight, gestational age and sex ratio. The MF provided 69 kcal/dl and contained 16% of linoleic acid and 1.3% of alpha-linolenic acid on the total fat. Plasma lipid fractions were extracted and separated by thin-layer chromatography and fatty acid methyl esters were quantitated by gas liquid chromatography. In plasma phospholipids, linoleic acid (18:2 omega 6) continuously increased from birth to 1 month of age, but no changes were seen as related to type of diet; polyunsaturated fatty acids greater than 18 carbon atoms of both the omega 6 and omega 3 series (PUFA omega 6 greater than 18 C and omega 3 greater than 18 C) dropped from birth to 1 week and continued to decrease in MF-fed infants until 1 month; eicosatrienoic (20:3 omega 6), arachidonic (20:4 omega 6) and docosahexaenoic (22:6 omega 3) were the fatty acids implicated. In cholesteryl esters palmitoleic (16:1 omega 7) and oleic (18:1 omega 9) acids decreased from birth to 1 month and linoleic acid increased and arachidonic acid dropped, especially in MF fed infants. In triglycerides, palmitic, palmitoleic and stearic acid (18:0) decreased during the first month of life; oleic acid remained constant and linoleic acid increased in all infants, but arachidonic acid decreased only in those fed formula. Free fatty acids showed a similar behavior in fatty acids and in plasma triglycerides. Preterm neonates seem to have special requirements of long-chain PUFA and adapted MF should contain these fatty acids in similar amounts to those of HM to allow the maintenance of an adequate tissue structure and physiology.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Seasonal variation and food deprivation in common vampire bats (Chiroptera: Phyllostomidae).

The aim of this study was to investigate the effects of seasonal variation and fasting on fat reserves of the common vampire bat Desmodus rotundus. Plasma free fatty acids (FFA), along with lipid content of the liver and muscles, and fatty acids from the carcass were obtained from bats fed bovine blood and from whom food was subsequently withheld for 24 and 48 h. Animals were caught during both dry and rainy seasons. In general, fat tissue stores were not significantly influenced by seasonal variation. Lipid content of liver, muscles, and carcass decreased during some food deprivation periods, although the concomitant increase expected in plasma FFA was not observed. Lipid metabolism is hypothesized as being continued by the tissues themselves. In addition, free access to food sources (e.g., domestic livestock) throughout the year is believed to contribute to the low seasonal variations in fat reserves observed in the common vampire bat.     

Fat mobilization in vitro and in vivo in the genetically obese Zucker rat "fatty"

Fat mobilization was studied in vitro with epididymal fat pad tissue and also with cell suspensions from epididymal, retroperitoneal, and subcutaneous fat from the obese mutant "fatty" (fafa) and control rats of four different ages. Fat mobilization per cell in response to epinephrine was well above normal in young "fatties"; in older "fatties" the output per cell fell to near normal, but the much greater number of fat cells per rat indicated that the fat mobilizing capacity of the older "fatty" is well above normal. The "fatty" showed normal reactions to epinephrine in vivo: hyperglycemia, glycogenolysis, lipolysis with elevated free fatty acids and glycerol, and increased entry of free fatty acids into muscle and liver. Response was at least as great in "fatty" as in control animals. Metabolic indices-levels of circulating free fatty acids, glycerol, and in some cases glucose and lipid-determined at various ages in fed "fatties" and controls, and at intervals during prolonged fasting (70 days), were consistent with a picture of excessive adipose tissue lipolysis, excessive reesterification in the adipose tissue, fat mobilization in excess of need, and return of the excess to the adipose tissue via lipoproteins.     

Effects of cloprostenol administration on neutral lipid and prostaglandin F metabolism by porcine luteal tissue

The effects of Cloprostenol administration on porcine luteal lipid metabolism, progesterone production, and prostaglandin F production were examined in 32 pigs at day 12 of the estrous cycle. Pigs were killed between 0 and 18 hours after treatment. Recovered luteal tissue was incubated at 0 C and at 37 C in the absence and presence of dibutyryl cyclic AMP and indomethacin. Net release of progesterone from luteal tissue was depressed within 1 hour after Cloprostenol treatment whereas net release of prostaglandin F was accelerated 4 hours after Cloprostenol treatment. Inclusion of dibutyryl cyclic AMP in the incubation media did not alter progesterone production by did enhance prostaglandin F production at 0 and 1 hour after Cloprostenol treatment. Inclusion of indomethacin in the incubation media completely inhibited the Cloprostenol-induced acceleration of luteal PGF production. Cloprostenol treatment increased luteal triglycerides and decreased luteal free cholesterol and cholesterol esters within 1 hr after treatment. Arachidonic acid percentages in free fatty acids and triglycerides were also increased within 1 hr after treatment. When 37 C and 0 C incubations were compared, luteal accumulation of free fatty acids was maximum at 1 hr after Cloprostenol treatment. accumulation of triglycerides in luteal tissie was comparatively uniform at all times examined during the first 18 hr after Cloprostenol treatment. Comparison of 37 C and 0 C incubations further revealed that luteal triglycerides were active in accumulation of arachidonic acid. Inclusion of dibutyryl cyclic AMP and/or indomethacin in the incubation media did not alter luteal lipid contents or fatty acid compositions. Blood plasma progesterone was depressed at 4 hours after Cloprostenol whereas 13,14-dihydro-15-keto-prostaglandin F2a was elevated at 18 hours after treatment. Blood plasma free fatty acids increased 330 percetn at 4 hours and free fatty acid compositions also changed at this time. In both luteal tissue and blood plasma, changes in steroid and fatty acid metabolism occurred prior to changes in prostaglandin metabolism, suggesting that Cloprostenol induced functional luteal regression prior to altering prostaglandin metabolism.