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This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48–77) versus 38 (18–58) μmol/min; simple steatosis: 62 (46–78) versus 45 (25–65) μmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P

Most research into red blood cell (RBC) lipids focuses on membrane phospholipids and their relationships to metabolic conditions and diet. Triglycerides (TGs) exist in most cells; the TG-fatty acids serve as readily available fuel for oxidative phosphorylation. Because RBCs lack mitochondria, they would not be expected to store fatty acids in TG. We followed up on a previous in vitro study that found FFA can be incorporated into RBC-TG by testing whether intravenously infused [U-13C]palmitate could be detected in RBC-TG. We also quantified RBC-TG fatty acid concentrations and profiles as they relate to plasma FFA and lipid concentrations. We found that 1) RBC-TG concentrations measured by glycerol and LC/MS were correlated (r = 0.77; P < 0.001) and averaged

Diacylglycerols (DAG) are important lipid metabolites thought to induce muscle insulin resistance when present in excess; they can be synthesized de novo from plasma free fatty acids (FFA) or generated by hydrolysis of preexisting intracellular lipids. We present a new method to simultaneously measure intramyocellular concentrations of and the incorporation of [U-13C]palmitate from an intravenous infusion into individual DAG species. DAG were extracted from pulverized muscle samples using isopropanol:water:ethyl acetate (35:5:60; v:v:v). Chromatographic separation was conducted on reverse-phase column in binary gradient using 1.5 mM ammonium formate, 0.1% formic acid in water as solvent A, and 2 mM ammonium formate, 0.15% formic acid in methanol as solvent B. We used UPLC-ESI+-MS/MS in the multiple reaction monitoring (MRM) mode to separate the ions of interest from sample. Because DAG are a neutral lipid class, they were monitored as an ammonium adduct [M+NH4]+. To measure isotopic enrichment (for 13C16:0/16:0-DAG and 13C16:0/C18:1-DAG), we monitored the basic ions as [M+2+NH4]+ and the enriched compounds as [M+16+NH4]+. We were able to measure concentration and enrichment using 20 mg of skeletal muscle samples obtained from rats receiving a continuous infusion of [U-13C]palmitate. Applying this protocol to biological muscle samples proves that the method is sensitive, accurate, and efficient.

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-14C]palmitate versus [9,10-3H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (14C- or 3H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [3H]acetate recovery as 3H2O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-14C]acetate recovery as expired 14CO2 was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-14C]palmitate and [9,10-3H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [3H]palmitate. In conclusion, intravenous boluses of [9,10-3H]palmitate versus [1-14C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.

CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. Western blotting is the most widely used method to measure tissue CD36 protein content, but it is time consuming, technically demanding, and semiquantitative. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. By using serial dilutions of each sample and including a calibrator sample and quality control sample on each plate, we could achieve inter- and intra-assay variability of ∼ 10%. We found that CD36 content in omental and abdominal subcutaneous adipose tissue varied over a 2–5-fold range depending upon the means of data expression (per units of tissue protein, weight, or lipid). Omental CD36 content in women decreased markedly (P = 0.01) as a function of fat cell size. For the most part, tissue CD36 content was not correlated with CD36 mRNA. This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake.

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of 13C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of 13C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.

The relationship between overnight postabsorptive (fasting) respiratory exchange ratio (RER) and plasma FFA concentrations was addressed using data from three separate protocols, each of which involved careful control of the antecedent diet. Protocol 1 examined the relationship between fasting RER and the previous daytime RER. In Protocol 2 fasting, RER and plasma palmitate concentrations were measured in 29 women and 31 men (body mass index

Hyperinsulinemia is common in obesity, but whether it plays a role in intramyocellular triglyceride (imcTG) buildup is unknown. In this study, hyperinsulinemic-euglycemic clamp experiments were performed in overnight-fasted lean and high-fat-fed obese rats, awake, to determine the effect of insulin on imcTG synthesis (incorporation of [14C]glycerol, [14C]glucose, and [3H]oleate). Insulin infusion at 25 (low insulin) and 100 (high insulin) pmol/kg/min increased plasma insulin by 5- and 16-fold, respectively, whereas plasma and intramyocellular glycerol, FFAs, triglycerides, and glucose levels were maintained at their basal levels by co-infusion of exogenous glycerol, FFAs, and triglycerides at fixed rates and glucose at varying rates. In obese rats, insulin suppressed incorporation of glycerol into the imcTG-glycerol moiety dose dependently (P < 0.01–P < 0.001) in gastrocnemius and tibialis anterior, but only the high insulin suppressed it in soleus (P < 0.05). The low insulin suppressed glucose incorporation into imcTG-glycerol in all three muscles (P = 0.01–P < 0.01). However, the low insulin did not affect (P > 0.05) and the high insulin suppressed (P < 0.05–P < 0.01) fatty acid incorporation into imcTG in all three muscles. Insulin also suppressed glycerol incorporation in lean rats (P < 0.01–P < 0.04). On the other hand, imcTG pool size was not affected by insulin (P > 0.05). These observations suggest that acute hyperinsulinemia inhibits imcTG synthesis and thus does not appear to promote imcTG accumulation via the synthetic pathway, at least in the short term.

Hypertriglyceridemia is considered a cardiovascular risk factor in diabetic and nondiabetic subjects. In this study, we aimed to determine potential regulators of very low density lipoprotein-triglyceride (TG) production. VLDL-TG kinetics were measured in 13 men and 12 women {body mass index [mean (range)]: 24.8 (20.2–35.6) kg/m2}. VLDL-TG production was assessed from the plasma decay of a bolus injection of ex vivo labeled VLDL particles ([1-14C]triolein-VLDL-TG). Similar VLDL-TG production (μmol/min) was found in men and women. VLDL-TG production was not significantly correlated with palmitate flux ([9,10-3H]palmitate) (r = 0.09, P = 0.67) or palmitate concentration (r = −0.29, P = 0.2) but was correlated significantly with fasting insulin concentration (r = 0.46, P < 0.05) and resting energy expenditure (REE) (r = 0.45, P < 0.05). The latter correlation improved when adjusted for sex. The best multivariate model with VLDL-TG production as the dependent variable and REE, body composition, hormones, and substrate levels as independent variables included fasting insulin (P = 0.02) and REE (P = 0.02) (r2 = 0.32, P < 0.001). We conclude that VLDL kinetics are similar in men and women and that REE and plasma insulin are significant independent predictors of VLDL-TG production. FFA availability and body fat distribution are unrelated to VLDL production. We suggest that REE plays a greater role in VLDL-TG production than previously anticipated. REE and insulin should be taken into account when VLDL-TG production comparisons between groups are made.

Adipose tissue lipolysis provides circulating FFAs to meet the body’s lipid fuel demands. FFA release is well regulated in normal-weight individuals; however, in upper-body obesity, excess lipolysis is commonly seen. This abnormality is considered a cause for at least some of the metabolic defects (dyslipidemia, insulin resistance) associated with upper-body obesity. “Normal” lipolysis is sex-specific and largely determined by the individual’s resting metabolic rate. Women have greater FFA release rates than men without higher FFA concentrations or greater fatty acid oxidation, indicating that they have greater nonoxidative FFA disposal, although the processes and tissues involved in this phenomenon are unknown. Therefore, women have the advantage of having greater FFA availability without exposing their tissues to higher and potentially harmful FFA concentrations. Upper-body fat is more lipolytically active than lower-body fat in both women and men. FFA released by the visceral fat depot contributes only a small percentage of systemic FFA delivery. Upper-body subcutaneous fat is the dominant contributor to circulating FFAs and the source of the excess FFA release in upper-body obesity. We believe that abnormalities in subcutaneous lipolysis could be more important than those in visceral lipolysis as a cause of peripheral insulin resistance. Understanding the regulation of FFA availability will help to discover new approaches to treat FFA-induced abnormalities in obesity.

There has been more interest in VLDL-triglyceride (TG) kinetics during the last decade. Unfortunately, robust measurement methods are elaborate and not readily available. Here, we describe a method using unique, ex vivo labeling of the fatty acid moiety of VLDL-TG followed by intravenous bolus infusion in the same person. We found that plasma disappearance of ex vivo-labeled VLDL-TG was comparable to that of in vivo-labeled VLDL-TG and that turnover rates can be safely estimated from the log linear decay of VLDL-TG specific activity. We found minor labeling of the plasma FFA (oleate) pool, which was largely attributable to coinfusion of free [14C]triolein; VLDL-TG did not contribute substantially to the plasma FFA pool. The plasma decay curve of VLDL-TG was not affected by the presence of tracer in the FFA pool, provided that the data from 2 h after the VLDL tracer bolus infusion was used. The FFA contamination problem was circumvented by minor modification of the VLDL-TG tracer preparation. The approach we describe should expand the opportunity to study processes that cannot be assessed if the FFA precursor pool is labeled. This method for VLDL-TG tracer preparation can allow measurement of VLDL turnover, tissue uptake of VLDL-TG, and oxidation of VLDL-TG.

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. There are more than 19 different isoforms of PLA2 in the mammalian system, but recent studies have focused on three major groups, namely, the group IV cytosolic PLA2, the group II secretory PLA2 (sPLA2), and the group VI Ca2+-independent PLA2. These PLA2s are involved in a complex network of signaling pathways that link receptor agonists, oxidative agents, and proinflammatory cytokines to the release of arachidonic acid (AA) and the synthesis of eicosanoids. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes. All major groups of PLA2 are present in the central nervous system (CNS). Therefore, this review is focused on PLA2 and AA release in neural cells, especially in astrocytes and neurons. In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies on PLA2 in cerebral ischemia, Alzheimer's disease, and neuronal injury due to excitotoxic agents.Information from these studies has provided clear evidence for the important role of PLA2 in regulating physiological and pathological functions in the CNS.

Mean diameters of fat cells from abdominal tissues from 31 volunteers were determined by three methods based on fat cell isolation after collagenase digestion and methylene blue staining. The three methods were direct microscopy (Micro), manual measurement of diameters from digital images by using the public domain NIH Image program (Scion), and automated measurement of diameters from digital images using a customized program developed by Biomedical Imaging Resource at Mayo Clinic (AdCount). There was excellent agreement between the methods' measurement of mean abdominal fat cell diameter (concordance correlation coefficient >0.84). The Scion method gave slightly but systematically lower mean abdominal fat cell diameters than did either AdCount or Micro. The AdCount approach produced results that are comparable to those from Micro. Comparison of AdCount and Micro in measuring diameters of fat cells from thigh confirmed the good comparability between the two methods independent of fat depot.AdCount is very reliable, and the quickest and most objective of the three methods in measuring fat cell diameters from various depots.

Previous methods for measuring plasma glycerol specific activity (SA) are suboptimal, making the determination of glycerol kinetics in vivo with radiotracers difficult. A new high performance liquid chromatography (HPLC) method is described that permits the accurate and specific measurement of glycerol SA. The method involves isolation of glycerol from plasma and the formation of a tribenzoyl derivative. Glycerol rate of appearance was measured in five human volunteers using both [2-3H]glycerol and [2H5] glycerol. There was close agreement between the glycerol appearance rates measured using the two approaches (1.66 ± 0.14 vs. 1.70 ± 0.10 μmol·kg-1· min-1, respectively, P = NS). This HPLC method offers improved specificity over existing methods of measuring glycerol turnover using radiotracers.—Judd, R. L., R. Nelson, S. Klein, M. D. Jensen, and J. M. Miles. Measurement of plasma glycerol specific activity by high performance liquid chromatography to determine glycerol flux. J. Lipid Res. 1998. 39: 1106–1110.