Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE₂ (KH₂-PGE₂) which has a longer half-life in blood. We examined the effects of PGE₂ infusion and alterations in lipolysis , and of clotting, prolonged storage and hemolysis , on KH₂-PGE₂ immunoreactivity in unextracted human plasma and serum samples. Indeed KH₂-PGE₂ levels rose several hundred fold during infusions of PGE₂ at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH₂-PGE₂ levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH₂-PGE₂ levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH₂-PGE₂ levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH₂-PGE₂ measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores or from disrupted membrane phospholipids . Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Effects of infusion of some prostaglandins in essential fatty acid-deficient and normal rats

Infusion of 1 mg/kg per day of prostaglandin E(1) (PGE(1)) for 2 and 7 wk failed to correct the dermal signs of essential fatty acid (EFA) deficiency in rats despite the known conversion of EFA to certain prostaglandins. PGE(1) caused no significant changes in serum cholesterol, triglycerides, or phospholipids or in liver neutral lipids in EFA-deficient or normal rats. In normal rats epinephrine-induced lipolysis was greater in fat pads from infused than from untreated rats. The effect on epinephrine-induced lipolysis was greater after the 7 wk infusion than after the 2 wk infusion. The 7 wk infusion also lowered plasma free fatty acid (FFA) concentrations. Infusion of PGE(2) and PGF(2alpha) in combination for 4 wk had no significant effect on either dermal signs of EFA deficiency, lipolysis, or plasma FFA concentrations.     

Effects of prostaglandin E1 on lipolysis and plasma free fatty acids in the fasted rat

Contrary to published reports, prostaglandin E(1) (PGE(1)) in vitro and in vivo inhibited fasting lipolysis in rats. Adipose tissue lipolysis was inhibited when the tissue was incubated in the presence of PGE(1) and when the compound was administered intravenously. A biphasic plasma free fatty acid (FFA) response was obtained in fasted rats after intravenous injection of 80 micrograms of PGE(1) per kg body weight; plasma FFA concentrations were lowered at 7 min, elevated at 15 min, and at normal concentrations at 30 min. The FFA depression at 7 min was independent of the animal's nutritional state, but the rebound at 15 min did not occur in fed rats. The plasma FFA rebound in fasted rats at 15 min may be a consequence of rapid inactivation of PGE(1), followed by unopposed activity of factors which enhance fasting lipolysis.     

Role of growth hormone in lipid mobilization stimulated by prolonged muscular exercise in the rat.

The effects of prolonged muscular exercise (swim in tepid water for 60 min) on blood glucose, plasma FFA and R-GH were studied in a group of normal rats and the effect on blood glucose and plasma FFA in a group of hypophysectomized rats. The data obtained showed that, whereas in normal rats plasma FFA rose after muscular exercise, plasma R-GH fell sharply. In Hypophysectomized rats, however, muscular exercise did not increase plasma FFA levels.     

The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.     

Physiological circulating levels of secretin-like immunoreactivity in the human do not stimulate free fatty acid production

An in vivo study was carried out to establish whether infused secretin, which achieves physiological levels of secretin-like immunoreactivity (SLI), promotes lipolysis. Six healthy volunteers received two infusions after separate 8 h overnight fasts. The paired infusions of either 500 ml of normal saline or 150 C.U. of porcine secretin in 500 ml of normal saline were infused at a constant rate of 1.38 ml/min. Venous blood was sampled at 0, 1, 2, 3, 4, 5 and 6 h after the infusion started. Mean plasma concentrations of SLI were significantly higher after infusion of saline with secretin in comparison to infusion of saline alone but remained within the physiological range. Mean serum free fatty acid (FFA) and 3-hydroxybutyrate concentrations rose significantly with time during both infusions but the mean FFA and 3-hydroxybutyrate concentrations did not differ significantly between infusions at each time of assessment. We conclude that a lipolytic role for secretin has not been shown to be of importance in relation to the in vivo rise in FFA concentrations observed in the fasting normal subject.     

Plasma free fatty acid transport during prolonged glucose consumption and its relationship to plasma triglyceride fatty acids in man

Plasma free fatty acid (FFA) transport in human subjects has been studied during the course of prolonged ingestion of different amounts of glucose. Compared with the fasting state, hypocaloric glucose intake resulted in marked suppression of net transport of FFA with no change in (fractional) turnover rate. There was no further suppression of net transport of FFA when the intake was increased to isocaloric or hypercaloric levels, but there was a significant increase in the (fractional) turnover rate, indicating an enhancement of clearance mechanisms. During the 20-24-hr period of fasting after isocaloric glucose consumption, the (fractional) turnover rate quickly fell to that found in the fasting individual, whereas net transport remained suppressed for much longer. This suggested that ingestion of glucose maintains an influence on lipolysis longer than on esterification. During this period of fasting after glucose administration, the contribution of plasma FFA to circulating triglyceride fatty acids increased with time and was positively and significantly correlated with the changes in the net transport of plasma FFA.     

Continuous 24-h nicotinic acid infusion in rats causes FFA rebound and insulin resistance by altering gene expression and basal lipolysis in adipose tissue

Nicotinic acid (NA) has been used as a lipid drug for five decades. The lipid-lowering effects of NA are attributed to its ability to suppress lipolysis in adipocytes and lower plasma FFA levels. However, plasma FFA levels often rebound during NA treatment, offsetting some of the lipid-lowering effects of NA and/or causing insulin resistance, but the underlying mechanisms are unclear. The present study was designed to determine whether a prolonged, continuous NA infusion in rats produces a FFA rebound and/or insulin resistance. NA infusion rapidly lowered plasma FFA levels (>60%, P < 0.01), and this effect was maintained for ≥5 h. However, when this infusion was extended to 24 h, plasma FFA levels rebounded to the levels of saline-infused control rats. This was not due to a downregulation of NA action, because when the NA infusion was stopped, plasma FFA levels rapidly increased more than twofold (P < 0.01), indicating that basal lipolysis was increased. Microarray analysis revealed many changes in gene expression in adipose tissue, which would contribute to the increase in basal lipolysis. In particular, phosphodiesterase-3B gene expression decreased significantly, which would increase cAMP levels and thus lipolysis. Hyperinsulinemic glucose clamps showed that insulin's action on glucose metabolism was improved during 24-h NA infusion but became impaired with increased plasma FFA levels after cessation of NA infusion. In conclusion, a 24-h continuous NA infusion in rats resulted in an FFA rebound, which appeared to be due to altered gene expression and increased basal lipolysis in adipose tissue. In addition, our data support a previous suggestion that insulin resistance develops as a result of FFA rebound during NA treatment. Thus, the present study provides an animal model and potential molecular mechanisms of FFA rebound and insulin resistance, observed in clinical studies with chronic NA treatment.     

Different lipolytic effects of theophylline and dibutyryl cyclic AMP in vivo and in vitro.

Both dcAMP and theophylline are known to promote lipolysis in vitro by increasing intracellular cAMP. Although theophylline stimulates FFA mobilization in vivo as well, a report of low circulating FFA levels in the rat given dcAMP suggested that dcAMP may inhibit lipolysis in the intact animal. To explore this possibility, a comparison of the in vitro and in vivo lipolytic effects of theophylline and dcAMP was made in the young dog. Circulating glycerol and FFA levels rose following the administration of theophylline. While glycerol and FFA fell slightly in puppies given dcAMP, only the FFA change was significant. Epinephrine infusions given alone produced sustained elevations of glycerol and FFA. When theophylline was given in conjunction with ongoing epinephrine infusions, plasma glycerol and FFA levels remained high. On the other hand, epinephrine-stimulated lipolysis was markedly inhibited by dcAMP, as shown by pronounced falls of glycerol and FFA from the elevated levels found with epinephrine alone. In vitro studies involving fragments of puppy adipose tissue reveal that epinephrine, theophylline, and dcAMP promoted glycerol release. In contrast to the in vivo observations, lipolysis was also stimulated by combinations of both epinephrine and theophylline as well as by epinephrine and dcAMP. Thus, theophylline stimulates lipolysis in vitro and in vivo in the puppy. In contrast, dcAMP stimulates lipolysis in vitro but inhibits this action in the intact animal. This important difference in the two pharmacologic agents suggests the need for caution when using them in in vivo studies involving the action of cAMP.     

Muscle triglyceride utilization during exercise: effect of training

The respiratory exchange ratio (RER) is lower during exercise of the same intensity in the trained compared with the untrained state, even though plasma free fatty acids (FFA) and glycerol levels are lower, suggesting reduced availability of plasma FFA. In this context, we evaluated the possibility that lipolysis of muscle triglycerides might be higher in the trained state. Nine adult male subjects performed a prolonged bout of exercise of the same absolute intensity before and after adapting to a strenuous 12-wk program of endurance exercise. The exercise test required 64% of maximum O2 uptake before training. Plasma FFA and glycerol concentrations and RER during the exercise test were lower in the trained than in the untrained state. The proportion of the caloric expenditure derived from fat, calculated from the RER, during the exercise test increased from 35% before training to 57% after training. Muscle glycogen utilization was 41% lower, whereas the decrease in quadriceps muscle triglyceride concentration was roughly twice as great (12.7 +/- 5.5 vs. 26.1 +/- 9.3 mmol/kg dry wt, P less than 0.001) in the trained state. These results suggest that the greater utilization of FFA in the trained state is fueled by increased lipolysis of muscle triglyceride.     

Fatty acid cycling in the fasting rat

Adipose tissue lipolysis and fatty acid reesterification by liver and adipose tissue were investigated in rats fasted for 15 h under basal and calorigenic conditions. The fatty acid flux initiated by adipose fat lipolysis in the fasted rat is mostly futile and is characterized by reesterification of 57% of lipolyzed free fatty acid (FFA) back into adipose triglycerides (TG). About two-thirds of FFA reesterification are carried out before FFA release into plasma, whereas the rest consists of plasma FFA extracted by adipose tissue. Thirty-six percent of the fasting lipolytic flux is accounted for by oxidation of plasma FFA, whereas only a minor fraction is channeled into hepatic very low density lipoprotein-triglycerides (VLDL-TG). Total body calorigenesis induced by thyroid hormone treatment and liver-specific calorigenesis induced by treatment with beta, beta'-tetramethylhexadecanedioic acid (Medica 16) are characterized by a 1.7- and 1.3-fold increase in FFA oxidation, respectively, maintained by a 1.5-fold increase in adipose fat lipolysis. Hepatic reesterification of plasma FFA into VLDL-TG is negligible under both calorigenic conditions. Hence, total body fatty acid metabolism is regulated by adipose tissue as both source and sink. The futile nature of fatty acid cycling allows for its fine tuning in response to metabolic demands.     

Effect of glucose on plasma glucagon and free fatty acids during prolonged exercise

The effects of glucose ingestion on the changes in blood glucose, FFA, insulin and glucagon levels induced by a prolonged exercise at about 50% of maximal oxygen uptake were investigated. Healthy volunteers were submitted to the following procedures: 1. a control test at rest consisting of the ingestion of 100 g glucose, 2. an exercise test without, or 3. with ingestion of 100 g of glucose. Exercise without glucose induced a progressive decrease in blood glucose and plasma insulin; plasma glucagon rose significantly from the 60th min onward (+45 pg/ml), the maximal increase being recorded during the 4th h of exercise (+135 pg/ml); plasma FFA rose significantly from the 60th min onward and reached their maximal values during the 4th h of exercise (2177 +/- 144 muEq/l, m +/- SE). Exercise with glucose ingestion blunted almost completely the normal insulin response to glucose. Under these conditions, exercise did not increase plasma glucagon before the 210th min; similarly, the exercise-induced increase in plasma FFA was markedly delayed and reduced by about 60%. It is suggested that glucose availability reduces exercise-induced glucagon secretion and, possibly consequently, FFA mobilization.     

Hormonal and metabolic response to physical exercise, fasting and cold exposure in the rat. Effects on ketogenesis in isolated hepatocytes

Four groups of rats were subjected to the following conditions: (1) 48 h fasting, (2) 48 h of 4 degrees C cold exposure, (3) 5 h treadmill running, (4) 48 h fasting with 4 degrees C cold exposure. The groups were compared to fed control rats in order to study hormonal and metabolic responses in blood and tissue samples. Isolated hepatocytes were used to evaluate the rate of ketogenesis. Decreases in liver glycogen and increases in blood free fatty acids (FFA) confirmed that glycogenolysis and lipolysis occur in these situations of metabolic stress. Increases in the glucagon/insulin plasma ratio were also noted. Plasma catecholamine levels were only enhanced after running and after cold exposure. Production of blood ketone bodies was stimulated more by running and by fasting than by cold exposure. The low ketone body production observed after cold exposure seems to be linked to increases liver glycogen levels and decreased FFA availability. Liver cells isolated after cold exposure exhibited higher ketogenesis than these isolated after running. This difference in ketogenic capacity could result both from the longer hormonal stimulation by high glucagon/insulin plasma ratios and from the metabolic state of the liver.     

Effect of beta1- and beta2-adrenergic stimulation on energy expenditure,substrate oxidation,and UCP3 expression in humans

In humans, beta-adrenergic stimulation increases energy and fat metabolism. In the case of beta1-adrenergic stimulation, it is fueled by an increased lipolysis. We examined the effect of beta2-adrenergic stimulation, with and without a blocker of lipolysis, on thermogenesis and substrate oxidation. Furthermore, the effect of beta1-and beta2-adrenergic stimulation on uncoupling protein 3 (UCP3) mRNA expression was studied. Nine lean males received a 3-h infusion of dobutamine (DOB, beta1) or salbutamol (SAL, beta2). Also, we combined SAL with acipimox to block lipolysis (SAL+ACI). Energy and substrate metabolism were measured continuously, blood was sampled every 30 min, and muscle biopsies were taken before and after infusion. Energy expenditure significantly increased approximately 13% in all conditions. Fat oxidation increased 47 +/- 7% in the DOB group and 19 +/- 7% in the SAL group but remained unchanged in the SAL+ACI condition. Glucose oxidation decreased 40 +/- 9% upon DOB, remained unchanged during SAL, and increased 27 +/- 11% upon SAL+ACI. Plasma free fatty acid (FFA) levels were increased by SAL (57 +/- 11%) and DOB (47 +/- 16%), whereas SAL+ACI caused about fourfold lower FFA levels compared with basal levels. No change in UCP3 was found after DOB or SAL, whereas SAL+ACI downregulated skeletal muscle UCP3 mRNA levels 38 +/- 13%. In conclusion, beta2-adrenergic stimulation directly increased energy expenditure independently of plasma FFA levels. Furthermore, this is the first study to demonstrate a downregulation of skeletal muscle UCP3 mRNA expression after the lowering of plasma FFA concentrations in humans, despite an increase in energy expenditure upon beta2-adrenergic stimulation.     

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.     

Influence of carnitine supplementation on muscle substrate and carnitine metabolism during exercise

We examined 1) the effect of L-carnitine supplementation on free fatty acid (FFA) utilization during exercise and 2) exercise-induced alterations in plasma levels and skeletal muscle exchange of carnitine. Seven moderately trained human male subjects serving as their own controls participated in two bicycle exercise sessions (120 min, 50% of VO2max). The second exercise was preceded by 5 days of oral carnitine supplementation (CS; 5 g daily). Despite a doubling of plasma carnitine levels, with CS, there were no effects on exercise-induced changes in arterial levels and turnover of FFA, the relation between leg FFA inflow and FFA uptake, or the leg exchange of other substrates. Heart rate during exercise after CS decreased 7-8%, but O2 uptake was unchanged. Exercise before CS induced a fall from 33.4 +/- 1.6 to 30.8 +/- 1.0 (SE) mumol/l in free plasma carnitine despite a release (2.5 +/- 0.9 mumol/min) from the leg. Simultaneously, acylated plasma carnitine rose from 5.0 +/- 1.0 to 14.2 +/- 1.4 mumol/l, with no evidence of leg release. Consequently, total plasma carnitine increased. We concluded that in healthy subjects CS does not influence muscle substrate utilization either at rest or during prolonged exercise and that free carnitine released from muscle during exercise is presumably acylated in the liver and released to plasma.     

The effect of temperature on the degradation of triglycerides by a pseudomonad isolated from milk: free fatty acid accumulation as a balance between rates of triglyceride hydrolysis and fatty acid consumption

The lipolysis of triglyceride by a lipolytic pseudomonad, LS107d2, and the net levels of free fatty acids (FFA) in milk cultures of LS107d2 have been studied as a function of temperature. The metabolism of triolein results in the accumulation of oleic acid at 4°C but, at higher temperatures, triolein is hydrolysed without detectable oleate accumulation. Temperature also has a profound effect on the levels of FFA in whole milk cultures of LS107d2, and on the temporal pattern of changes in FFA levels. The results are consistent in demonstrating that FFA consumption plays a significant role in determining the net level of FFA in milk, even in the presence of very high rates of lipolysis. Thus the net levels of FFA as a result of the growth of LS107d2 in milk are the result of a dynamic balance between rates of FFA production (by lipolysis) and subsequent consumption. Temperature can dramatically affect the levels of FFA in milk cultures, and these changes are proposed to be due to opposing influences on FFA consumption and production, affecting the balance between them.     

Time-dependent effects of fatty acids on skeletal muscle metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin-stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin-stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA-induced insulin resistance.     

Homocysteine suppresses lipolysis in adipocytes by activating the AMPK pathway

Hyperhomocysteinemia (HHcy) is an independent risk factor for coronary artery disease. Emerging evidence suggests that HHcy is also associated with adipocyte tissue dysfunction. One of the principal functions of adipose tissue is to provide energy substrate via lipolysis. In the present study, we investigated the effects of homocysteine (Hcy) on lipolysis in adipocytes. We found that Hcy inhibited release of glycerol and fatty acids, two typical indicators of the lipolytic response, in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner under both basal and isoproterenol-stimulated conditions. In differentiated 3T3-L1 adipocytes, decreased glycerol and free fatty acid (FFA) release was associated with elevation of intracellular TG content. Further studies showed that Hcy-mediated antilipolytic responses were independent of the cyclic AMP-PKA and MEK-ERK1/2 pathways. However, Hcy increased phosphorylation levels of AMP-activated protein kinase (AMPK) and its downstream enzyme acetyl-CoA carboxylase. Compound C, an AMPK inhibitor, abolished Hcy-induced reduction of glycerol and FFA release under both basal and isoproterenol-stimulated conditions. Furthermore, AMPKα1 siRNA reversed Hcy-inhibited glycerol release. Supplementation of exogenous Hcy in the diet for 2 wk lowered circulating glycerol and FFA levels. Moreover, Hcy supplementation was associated with elevated leptin levels and reduced adiponectin levels in plasma. These results show that Hcy inhibits lipolysis through a pathway that involves AMPK activation.     

Heparin, lipoproteins, and oxygenated fatty acids in blood: a cautionary note

We measured 16 nonesterified oxygenated fatty acid derivatives (oxylipids) in plasmas from seven human subjects. Two arterial samples from each subject were analyzed, drawn approximately 2h apart. We observed a marked increase in levels of most oxylipids in the second sample, as high as 470-fold. Between the first and second samples, subjects received approximately 800-1000 IU of heparin to prevent clotting in intravascular catheters. We postulate that heparin activated lipoprotein lipases, which, in turn, released oxylipids from triglycerides and phospholipids in plasma lipoproteins. Some of that lipolysis may have occurred during sample storage. Measurements of nonesterified lipids in human plasma may be distorted if heparin is administered to subjects before blood is drawn and if lipase inhibitors are omitted from stored samples.