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The mechanism of increased postprandial nonesterified fatty acid (NEFA) appearance in the circulation in impaired glucose tolerance (IGT) is due to increased adipose tissue lipolysis but could also be contributed to by reduced adipose tissue (AT) dietary fatty acid (DFA) trapping and increased "spillover" into the circulation. Thirty-one subjects with IGT (14 women, 17 men) and 29 with normal glucose tolerance (NGT, 15 women, 14 men) underwent a meal test with oral and intravenous palmitate tracers and the oral [ 18 F]-fluoro-thia-heptadecanoic acid positron emission tomography method. Postprandial palmitate appearance (Ra palmitate ) was higher in IGT versus NGT ( P < 0.001), driven exclusively by Ra palmitate from obesity-associated increase in intracellular lipolysis ( P = 0.01), as Ra palmitate from DFA spillover was not different between the groups ( P = 0.19) and visceral AT DFA trapping was even higher in IGT versus NGT ( P = 0.02). Plasma glycerol appearance was lower in IGT ( P = 0.01), driven down by insulin resistance and increased insulin secretion. Thus, we found higher AT DFA trapping, limiting spillover to lean organs and in part offsetting the increase in Ra palmitate from intracellular lipolysis. Whether similar findings occur in frank diabetes, a condition also characterized by insulin resistance but relative insulin deficiency, requires further investigation (Clinicaltrials.gov: NCT04088344, NCT02808182). NEW & NOTEWORTHY We found higher adipose tissue dietary fatty acid trapping, limiting spillover to lean organs, that in part offsets the increase in appearance rate of palmitate from intracellular lipolysis in prediabetes. These results point to the adaptive nature of adipose tissue trapping and dietary fatty acid spillover as a protective mechanism against excess obesity-related palmitate appearance rate from intracellular adipose tissue lipolysis.

To investigate the biodisponibility of placental transfer of fatty acids, rats pregnant for 20 days were given tracer amounts of [(14)C]palmitic (PA), oleic (OA), linoleic (LA), α-linolenic (LNA), or docosahexaenoic acid (DHA) orally and euthanized at 0.5, 1.0, 2.0, or 8.0 h thereafter. Maternal plasma radioactivity in lipids initially increased only to decline at later times. Most of the label appeared first as triacylglycerols (TAG); later, the proportion in phospholipids (PhL) increased. The percentage of label in placental lipids was also always highest shortly after administration and declined later; again, PhL increased with time. Fetal plasma radioactivity increased with time, with its highest value at 8.0 h after DHA or LNA administration. DHA initially appeared primarily in the nonesterified fatty acids (NEFA) and PA, OA, LA, and LNA as TAG followed by NEFA; in all cases, there was an increase in PhL at later times. Measurement of fatty acid concentrations allowed calculation of specific (radio)activities, and the ratio (fetal/maternal) of these in the plasmas gave an index of placental transfer activity, which was LNA > LA > DHA = OA > PA. It is proposed that a considerable proportion of most fatty acids transferred through the placenta are released into the fetal circulation in the form of TAG., (Copyright © 2016 the American Physiological Society.)

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

A noninvasive method to determine postprandial fatty acid tissue partition may elucidate the link between excess dietary fat and type 2 diabetes. We hypothesized that the positron-emitting fatty acid analog 14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid ((18)FTHA) administered orally during a meal would be incorporated into chylomicron triglycerides, allowing determination of interorgan dietary fatty acid uptake. We administered (18)FTHA orally at the beginning of a standard liquid meal ingested in nine healthy men. There was no significant (18)FTHA uptake in the portal vein and the liver during the 1st hour. Whole body PET/CT acquisition revealed early appearance of (18)FTHA in the distal thoracic duct, reaching a peak at time 240 min. (18)FTHA mean standard uptake value increased progressively in the liver, heart, quadriceps, and subcutaneous and visceral adipose tissues between time 60 and 240 min. Most circulating (18)F activity between time 0 and 360 min was recovered into chylomicron triglycerides. Using Triton WR-1339 treatment in rats that received (18)FTHA by gavage, we confirmed that >90% of this tracer reached the circulation as triglycerides. This novel noninvasive method to determine tissue dietary fatty acid distribution in humans should prove useful in the study of the mechanisms leading to lipotoxicity.

Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-l-thyronine (T(2)) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T(2) to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 h after its injection into hypothyroid rats. Administration of T(2) induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoylcarnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T(2) was able to 1) activate the AMPK-ACC-malonyl-CoA metabolic signaling pathway known to direct lipid partitioning toward oxidation and 2) increase the importing of fatty acids into the mitochondrion. These results suggest that T(2) stimulates mitochondrial fatty acid oxidation by activating several metabolic pathways, such as the fatty acid import/beta-oxidation cycle/FADH(2)-linked respiratory pathways, where fatty acids are imported. T(2) also enhanced skeletal muscle mitochondrial thermogenesis by activating pathways involved in the dissipation of the proton-motive force not associated with ATP synthesis ("proton leak"), the effect being dependent on the presence of free fatty acids inside mitochondria. We conclude that skeletal muscle is a target for T(2), and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis, T(2) could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.

Because excess triacylglycerol (TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of long-chain acyl-CoAs toward beta-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 microM oleate was present, both TAG mass and the incorporation of [14C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [14C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were approximately 40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtGPAT1. A 50-60% decrease was also observed in [14C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14C]TAG secretion increased appropriately in vector control-infected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [14C]TAG as the vector control cells. Thus, in isolated hepatocytes, mtGPAT1 may synthesize a cytosolic pool of TAG that cannot be secreted.

To determine the role of AMP-activated protein kinase (AMPK) activation on the regulation of fatty acid (FA) uptake and oxidation, we perfused rat hindquarters with 6 mM glucose, 10 microU/ml insulin, 550 microM palmitate, and [14C]palmitate during rest (R) or electrical stimulation (ES), inducing low-intensity (0.1 Hz) muscle contraction either with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). AICAR treatment significantly increased glucose and FA uptake during R (P < 0.05) but had no effect on either variable during ES (P > 0.05). AICAR treatment significantly increased total FA oxidation (P < 0.05) during both R (0.38 +/- 0.11 vs. 0.89 +/- 0.1 nmol x min(-1) x g(-1)) and ES (0.73 +/- 0.11 vs. 2.01 +/- 0.1 nmol x min(-1) x g(-1)), which was paralleled in both conditions by a significant increase and significant decrease in AMPK and acetyl-CoA carboxylase (ACC) activity, respectively (P < 0.05). Low-intensity muscle contraction increased glucose uptake, FA uptake, and total FA oxidation (P < 0.05) despite no change in AMPK (950.5 +/- 35.9 vs. 1,067.7 +/- 58.8 nmol x min(-1) x g(-1)) or ACC (51.2 +/- 6.7 vs. 55.7 +/- 2.0 nmol x min(-1) x g(-1)) activity from R to ES (P > 0.05). When contraction and AICAR treatment were combined, the AICAR-induced increase in AMPK activity (34%) did not account for the synergistic increase in FA oxidation (175%) observed under similar conditions. These results suggest that while AMPK-dependent mechanisms may regulate FA uptake and FA oxidation at rest, AMPK-independent mechanisms predominate during low-intensity muscle contraction.

Oxidation and adipose tissue uptake of dietary fat can be measured by adding fatty acid tracers to meals. These studies were conducted to measure between-study variability of these types of experiments and assess whether dietary fatty acids are handled differently in the follicular vs. luteal phase of the menstrual cycle. Healthy normal-weight men (n = 12) and women (n = 12) participated in these studies, which were block randomized to control for study order, isotope ([3H]triolein vs. [14C]triolein), and menstrual cycle. Energy expenditure (indirect calorimetry), meal fatty acid oxidation, and meal fatty acid uptake into upper body and lower body subcutaneous fat (biopsies) 24 h after the experimental meal were measured. A greater portion of meal fatty acids was stored in upper body subcutaneous adipose tissue (24 +/- 2 vs. 16 +/- 2%, P < 0.005) and lower body fat (12 +/- 1 vs. 7 +/- 1%, P < 0.005) in women than in men. Meal fatty acid oxidation (3H2O generation) was greater in men than in women (52 +/- 3 vs. 45 +/- 2%, P = 0.04). Leg adipose tissue uptake of meal fatty acids was 15 +/- 2% in the follicular phase of the menstrual cycle and 10 +/- 1% in the luteal phase (P = NS). Variance in meal fatty acid uptake was somewhat (P = NS) greater in women than in men, although menstrual cycle factors did not contribute significantly. We conclude that leg uptake of dietary fat is slightly more variable in women than in men, but that there are no major effects of menstrual cycle on meal fatty acid disposal.

Isotopic techniques were used to test the hypothesis that exercise and nitric oxide synthase (NOS) inhibition have distinct effects on tissue-specific fatty acid and glucose uptakes in a conscious, chronically catheterized mouse model. Uptakes were measured using the radioactive tracers (125)I-labeled beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) and deoxy-[2-(3)H]glucose (DG) during treadmill exercise with and without inhibition of NOS. [(125)I]BMIPP uptake at rest differed substantially among tissues with the highest levels in heart. With exercise, [(125)I]BMIPP uptake increased in both heart and skeletal muscles. In sedentary mice, NOS inhibition induced by nitro-L-arginine methyl ester (L-NAME) feeding increased heart and soleus [(125)I]BMIPP uptake. In contrast, exercise, but not L-NAME feeding, resulted in increased heart and skeletal muscle [2-(3)H]DG uptake. Significant interactions were not observed in the effects of combined exercise and L-NAME feeding on [(125)I]BMIPP and [2-(3)H]DG uptakes. In the conscious mouse, exercise and NOS inhibition produce distinct patterns of tissue-specific fatty acid and glucose uptake; NOS is not required for important components of exercise-associated metabolic signaling, or other mechanisms compensate for the absence of this regulatory mechanism.

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 microM palmitate and [1-(14)C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 microU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 +/- 0.6 vs. 11.0 +/- 0.5 micromol x g(-1) x h(-1); P > 0.05). Total palmitate uptake and oxidation were not significantly different (P > 0.05) between the G (10.1 +/- 1.0 and 0.8 +/- 0.1 nmol x min(-1) x g(-1)) and I (10.2 +/- 0.6 and 1.1 +/- 0.2 nmol. min(-1) x g(-1)) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion (P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups (P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.

The adipocyte-derived hormone leptin has been shown to acutely increase fatty acid (FA) oxidation and decrease esterification in resting rodent skeletal muscle. However, the effects of leptin on human skeletal muscle FA metabolism are completely unknown. In these studies, we have utilized an isolated human skeletal muscle preparation combined with the pulse-chase technique to measure FA metabolism with and without leptin in lean and obese human skeletal muscle. Under basal conditions (in the absence of leptin), muscle from the obese demonstrated significantly elevated levels of total FA uptake (+72%, P = 0.038) and enhanced rates of FA esterification into triacylglycerol (+102%, P = 0.042) compared with lean subjects. In the presence of leptin, lean muscle had elevated rates of endogenous (+103%, P = 0.01) and exogenous (+150%, P = 0.03) palmitate oxidation. When the ratio of esterification to exogenous oxidation was examined, leptin reduced this ratio (-47%, P = 0.032), demonstrating the increased partitioning of FA toward oxidation and away from storage. Contrary to these findings in lean muscle, leptin had no effect on FA metabolism in skeletal muscle of the obese. This study provides the first evidence that leptin increases FA oxidation in skeletal muscle of lean, but not obese humans, thus demonstrating the development of leptin resistance in obese human skeletal muscle.

Efficient uptake and channeling of long-chain fatty acids (LCFAs) are critical cellular functions. Although spontaneous flip-flop of nonionized LCFAs from one leaflet of a bilayer to the other is rapid, evidence is emerging that proteins are important mediators and/or regulators of trafficking of LCFAs into and within cells. Genetic screens have led to the identification of proteins that are required for fatty acid import and utilization in prokaryotic organisms. In addition, functional screens have elucidated proteins that facilitate fatty acid import into mammalian cells. Although the mechanisms by which these proteins mediate LCFA import are not well understood, studies in both prokaryotic and eukaryotic organisms provide compelling evidence that uptake of LCFAs across cellular membranes is coupled to esterification by acyl-CoA synthetases. This review will summarize results of studies of non-protein-mediated and protein-mediated LCFA transport and discuss how these different mechanisms may contribute to cellular metabolism.

We tested for gender differences in dietary fatty acid metabolism in 12 nonobese men and 12 nonobese women using the meal fatty acid tracer/adipose tissue biopsy study design. In addition to determining body composition, measurements of regional adipose tissue lipoprotein lipase activity, blood flow, and fat cell size were performed to place the meal fatty acid kinetic studies in perspective. Twenty-four hours after ingesting the test meal, the concentration of meal fatty acids was greater (P < 0.05) in abdominal subcutaneous than in thigh adipose tissue in both men (0. 61 +/- 0.12 vs. 0.45 +/- 0.09 mg/g) and women (0.59 +/- 0.10 vs. 0. 43 +/- 0.05) but was not different between men and women. A greater percentage of dietary fat was stored in subcutaneous adipose tissue in women than in men (38 +/- 3 vs. 24 +/- 3%, respectively, P < 0. 05), and a greater portion of meal fatty acid disposal was unaccounted for in men. Significant gender differences in regional adipose tissue blood flow after meal ingestion were noted; the differences were in the direction that could support greater nutrient storage in lower body fat in women.