Leg free fatty acid kinetics during exercise in men and women

We previously reported that epinephrine stimulates leg free fatty acid (FFA) release in men but not in women. The present studies were conducted to determine whether the same is true during exercise. Six men and six women bicycled for 90 min at 45% of peak O(2) consumption, during which time systemic and leg FFA kinetics ([9, 10-(3)H]palmitate) were measured. The catecholamine and hormonal responses to exercise were not different in men and women. The baseline systemic and leg palmitate release was 94 +/- 15 vs. 114 +/- 5 micromol/min and 16 +/- 2 and 20 +/- 3 micromol/min, respectively, in men and women [P = nonsignificant (NS)]. Systemic and leg palmitate release increased (both P < 0.001) to 251 +/- 18 vs. 212 +/- 16 micromol/min and 73 +/- 19 vs. 80 +/- 12 micromol/min in men and women, respectively, during the last 30 min of exercise (P = NS, men vs. women). We conclude that the systemic and leg adipose tissue lipolytic response to exercise is not different in nonobese men and women.     

Modeling glucose and free fatty acid kinetics in glucose and meal tolerance test

Background

Quantitative evaluation of insulin regulation on plasma glucose and free fatty acid (FFA) in response to external glucose challenge is clinically important to assess the development of insulin resistance (World J Diabetes 1:36–47, 2010). Mathematical minimal models (MMs) based on insulin modified frequently-sampled intravenous glucose tolerance tests (IM-FSIGT) are widely applied to ascertain an insulin sensitivity index (IEEE Rev Biomed Eng 2:54–96, 2009). Furthermore, it is important to investigate insulin regulation on glucose and FFA in postprandial state as a normal physiological condition. A simple way to calculate the appearance rate (Ra) of glucose and FFA would be especially helpful to evaluate glucose and FFA kinetics for clinical applications.

Methods

A new MM is developed to simulate the insulin modulation of plasma glucose and FFA, combining IM-FSIGT with a mixed meal tolerance test (MT). A novel simple functional form for the appearance rate (Ra) of glucose or FFA in the MT is developed. Model results are compared with two other models for data obtained from 28 non-diabetic women (13 African American, 15 white).

Results

The new functional form for Ra of glucose is an acceptable empirical approximation to the experimental Ra for a subset of individuals. When both glucose and FFA are included in FSIGT and MT, the new model is preferred using the Bayes Information Criterion (BIC).

Conclusions

Model simulations show that the new MM allows consistent application to both IM-FSIGT and MT data, balancing model complexity and data fitting. While the appearance of glucose in the circulation has an important effect on FFA kinetics in MT, the rate of appearance of FFA can be neglected for the time-period modeled.

     

Splanchnic glucagon kinetics in exercising alloxan-diabetic dogs.

The purpose of this study was to define the relationship between arterial immunoreactive glucagon (IRG) and IRG that perfuses the liver via the portal vein during exercise in the diabetic state. Dogs underwent surgery >16 days before the experiment, at which time flow probes were implanted in the portal vein and the hepatic artery, and Silastic catheters were inserted in the carotid artery, portal vein, and hepatic vein for sampling. Dogs were made diabetic with alloxan injected intravenously approximately 3 wk before study (AD) or were studied in the nondiabetic state (ND). Each study consisted of a 30-min basal period and a 150-min moderate-exercise period on a treadmill. The findings from these studies indicate that the exercise-induced increment in portal vein IRG can be substantially greater in AD compared with ND, even when arterial and hepatic vein increments are not different. The larger IRG gradient from the portal vein to the systemic circulation in AD dogs is a function of a twofold greater increase in nonhepatic splanchnic IRG release and a fivefold greater hepatic fractional IRG extraction during exercise. In conclusion, during exercise, arterial IRG concentrations greatly underestimate the IRG levels to which the liver is exposed in ND, and this underestimation is considerably greater in dogs with poorly controlled diabetes.     

The plasma free fatty acid rebound induced by nicotinic acid

The time course of the nicotinic acid-induced changes in levels of plasma free fatty acids (FFA) was examined. The plasma FFA response of fasted dogs to graded doses of nicotinic acid was shown to be biphasic: an initial depression of the level of plasma FFA was followed by a rebound elevation to supernormal levels. FFA rebound was not seen after the administration of the nicotinic acid homologue, pyridylacetic acid, or a variety of nicotinic acid metabolities. A similar pattern of FFA response was observed in fasted, normal rats. Adrenalectomy did not abolish the secondary elevation of FFA but did cause a somewhat delayed response. Hypophysectomy modified the time course of the response-the initial FFA decrease was prolonged-and the intensity of the FFA rebound was diminished. No rebound was observed in hypophysectomized, adrenalectomized rats. In normal rats, nicotinic acid caused a significant rise in the level of plasma corticosterone. A normal rebound pattern was observed in thyroidectomized rats. Reserpine, administered on a schedule designed to deplete catecholamine stores, altered the time course of plasma FFA changes only slightly. The results indicate that both the pituitary and adrenal functions are required for the expression of the rebound phenomenon after nicotinic acid administration.     

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.     

Accelerated esterification of free fatty acid using pulsed microwaves

It was demonstrated that pulsed microwave irradiation is a more effective method to accelerate the esterification of free fatty acid with a heterogeneous catalyst than continuous microwave irradiation. A square-pulsed microwave with a 400 Hz repetition rate and a 10-20% duty cycle with the same energy as the continuous microwave were used in this study. The pulsed microwaves improved the esterification conversion from 39.9% to 66.1% after 15 min in comparison with the continuous microwave under the same reaction conditions. These results indicated that pulsed microwaves with repetitive strong power could enhance the efficiency of biodiesel production relative to the use of continuous microwave with mild power.     

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of plasma free fatty acid concentration on the content and composition of the free fatty acid fraction in rat skeletal muscles.

Skeletal muscles contain a fraction of free (unesterified) fatty acids. This fraction is very small, but important since it contributes to the creation of the plasma-myocyte free fatty acid concentration gradient. Maintenance of this gradient is necessary for blood-borne fatty acids to be transported into the cell. There are no data on the regulation of the content and composition of the free fatty acid fraction in the cell. The aim of the present study was to examine the effect of an elevation and a reduction in the plasma-borne free fatty acid concentration on the content and composition of the free fatty acid fraction in different skeletal muscle types. The experiments were carried out on male Wistar rats with 280 - 310 g body weight. They were divided into four groups - 1, control; 2, exercised 3 h on a treadmill moving with a speed of 1,200 m/h and set at + 10 degrees incline; 3, treated with heparin; and 4, treated with nicotinic acid. Samples of the soleus as well as the red and white sections of the gastrocnemius muscles were taken. These muscles are composed mostly of slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibres, respectively. Lipids were extracted from the muscle samples and from the blood; the free fatty acid fraction was isolated by means of thin-layer chromatography. The individual free fatty acids were identified and quantified using gas-liquid chromatography. The plasma concentration of free fatty acids was as follows: control group, 236.1 +/- 32.9; after exercise, 407.4 +/- 117.5; after heparin, 400.8 +/- 36.8; and after nicotinic acid, 102.5 +/- 26.1 micromol/l (p < 0.01 vs. control values in each case). The total content of the free fatty acid fraction in the control group was as follows: white gastrocnemius, 27.6 +/- 7.3; red gastrocnemius, 52.2 +/- 13.9; soleus, 72.3 +/- 10.2 nmol/g. Elevation in plasma free acid concentration during exercise increased the total content of free fatty acids in the white gastrocnemius (38.7 +/- 13.9) and in the soleus (103.4 +/- 15.9 nmol/g; rest-exercise: p < 0.05 and p < 0.01, respectively), but had no effect in the red gastrocnemius. Neither elevation in the plasma free fatty acid concentration with heparin nor reduction with nicotinic acid affected the total content of the free fatty acid fraction in the muscles examined. The ratio of plasma concentration of individual acid to muscle concentration for the same acid varied greatly, depending on acid, muscle type and experimental group. The ratio was positive (above unity) for each acid almost in all cases with the exception of certain acids in the nicotinic acid-treated group where it was below unity. We conclude that the skeletal myocytes maintain a stable level of free fatty acid fraction in the wide range of plasma free fatty acid concentrations.     

Plasma free fatty acid composition in Ainu and Japanese men

The ratio of oleic acid in the plasma FFA fraction was higher in the Japanese than in the Ainu. When glucose was administered, the proportion of oleic acid was reduced, while that of stearic acid increased in both groups. In the Japanese, moreover, a decrease in linoleic acid and increases in palmitic and myristic acids were also observed. As the plasma FFA level rose after injection of norepinephrine, the proportion of oleic acid increased and that of palmitic, stearic, myristic and lauric acids decreased, and the composition approached the pattern of depot fat. It was inferred that the difference observed in the plasma FFA composition between the Ainu and Japanese was attributable to the difference in the plasma levels of FFA which was significantly lower in the Ainu.

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Zusammenfassung Das Verhältnis von Oelsäure in der freien Fettsäurenfraktion (FFA) im Plasma war bei Japanern (n=9) höher als bei Ainus (n=5). Nach Glukose war das Oelsäure-Verhältnis vermindert, während in beiden Gruppen Stearinsäure anstieg. Bei den Japanern ergaben sich ausserdem ein Abfall der Linolensäure und Austiege der Palmitin- und Myristinsäure. Wenn der Plasma FFA-Spiegel nach Noradrenalin-Injektion anstieg, stieg der Oelsäureanteil und fielen die Palmitin-, Myristin- und Laurinsäureanteile und die Zusammensetzung war wie die von Depotfett. Es wird angenommen, dass die Unterschiede in der Zusammensetzung der Plasma FFA bei Japanern und Ainus Folge der unterschiedlichen FFA-Spiegel waren, die bei den Ainu signifikant tiefer lagen.

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Resume La proportion d'acide oleique dans l'ensemble des acides gras libres (FFA) du plasma sanguin est plus marquée chez les Japonais (n=9) que chez les Aïnous (n=5). La proportion d'acide oleique a diminuê après l'administration de glucose, car, dans les deux groupes, la teneur en acide stéarique s'est renforcée. On constante en outre une diminution de l'acide linoleique et une augmentation des acides palmitique et myristique. Lorsque, après injection de noradrénaline, le taux de FFA du plasma se relève, on constate une augmentation des quantités relatives de l'acide oleique au détriment de la part prise par les acides palmitique, myristique et laurique et leur composition est semblable aux dépots adipeux. On peut ainsi admettre que les différences de composition du FFA plasmatique découlent du taux dissemblable du FFA entre Aïnous et Japonais. Ce taux est significativement inférieur chez les premiers.

     

Unbound free fatty acid concentrations are increased in cardiac ischemia

Monitoring increased plasma unbound free fatty acid (FFA_u) concentrations has been proposed as a biomarker for myocardial ischemia. In the current study, 30 acute coronary syndrome (ACS) patients presenting in the emergency department, with chest pain within 12 h of onset, were clinically evaluated along with serial cardiac troponin I (cTnI) and FFA_u measurements. Increased FFA_u were found in 28 of 30 (93%) of ACS patients, ranging from 2.0 to 430 nM. For the nine ACS patients with myocardial infarction (MI), FFA_u levels were increased at presentation for all (100%). In contrast, cTnI was increased in only 9 of 30 (30%) patients, mean 0.7 μg/L, and in only 2 of 9 (22%) MI patients, mean 1.3 μg/L. During the 24 h following admission, cTnI increased in all 9 MI patients. FFA_u concentrations increased in every sample in which cTnI increased. Our findings suggest that FFA_u is increased in ischemia regardless of the presence or absence of myocardial necrosis, as reflected by increased or normal cTnI, respectively.     

Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membrane's role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFA(i)]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFA(i)] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFA(i)] in response to changing the extracellular unbound FFA concentration ([FFA(o)]). [FFA(o)] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant approximately 0.02 s(-1)) and saturable (K(o) approximately 200 nm). Efflux is twice as fast as influx, for zero [FFA(o)], but decreases with increasing [FFA(o)]. Surprisingly, at steady state [FFA(i)] is 2-5-fold (average 2-fold) greater than [FFA(o)] and this [FFA(i)]/[FFA(o)] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFA(o)]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membrane's lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.     

Comparison of the effect of semisynthetic human insulin and porcine insulin on glucose kinetics, plasma free fatty acid and amino acid levels in man

The effect of semisynthetic human insulin on hepatic glucose output, peripheral glucose clearance, plasma levels of C-Peptide, free fatty acids and amino acids was compared with purified pork insulin using the glucose clamp technique. 8 normal overnight-fasted subjects received intravenous infusions of either human or porcine insulin at 20 mU/m2.min(-1) during 120 min achieving plasma insulin levels of approximately equal to 50 mU/l. Plasma glucose levels were maintained at euglycaemia by variable rates of glucose infusion. Hepatic glucose production measured by continuous infusion of 3-(3) H-glucose was similarly suppressed by both insulins to rates near zero. The metabolic clearance rate of glucose increased during infusion of human insulin by 120%, C-peptide levels decreased by 41% and plasma FFA concentrations fell by 74%. The respective changes during infusion of pork insulin were similar, 118%, 48% and 72%. Both insulins decreased the plasma levels of branched-chain amino acids, tyrosine, phenylalanine, methionine, serine and histidine similarly. Thus, the results demonstrate that semisynthetic human and porcine insulin are aequipotent with respect to suppression of hepatic glucose output, stimulation of glucose clearance, inhibition of insulin secretion, lipolysis and proteolysis.     

Multi-exponential analysis of plasma free amino acid kinetics in the rat

A multi-exponential analysis has been made of the curve of decreasing specific radioactivity with time after intravenous injection of a mixture of (U-^{1 4}-C)-labelled amino acids in rats. The data were shown to accord well with a linear model which was tested using an approximate linear regression method. This confirmed the satisfactory fit of the data to the model. Values for total entry into and of irreversible disposal from plasma were calculated and the extent of external recycling of amino acids obtained by difference.     

Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism

Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.