Regulation of direct adipose tissue free fatty acid storage during mixed meal ingestion and high free fatty acid concentration conditions.

We found that direct free fatty acid (FFA) storage (fatty acid cycling back into adipose tissue) in leg vs. abdominal subcutaneous fat is related to regional differences in adipose tissue diacylglycerol acyltransferase (DGAT) activity under high-FFA conditions and to differences in adipose tissue acyl-CoA synthetase (ACS)activity under meal ingestion conditions. We also found that direct FFA storage rates in leg fat were significantly less in physically active than sedentary adults. Direct FFA storage into adipocytes relates to body fat distribution. Adipose tissue CD36, ACS, and DGAT may account for some of the between-depot and interindividual variability in FFA storage. These studies were to test whether CD36, ACS, or DGAT might be important for direct palmitate storage under meal ingestion or high-FFA conditions. We measured upper (UBSQ) and lower body subcutaneous (LBSQ) adipose tissue FFA storage rates by infusing palmitate tracers intravenously and performing adipose biopsies under hypoinsulinemic (high-FFA) and mixed-meal conditions. We recruited five postmenopausal women, physically active males (5) and females (5), and sedentary males (5) and females (5). We found that 1) the ratio of UBSQ to LBSQ DGAT activity predicted the ratio of palmitate storage [adjusted R = 0.25, F = 8.0, P = 0.01, 95% CI (0.07, 0.48)] under high-FFA conditions; 2) the ratio of UBSQ to LBSQ ACS activity predicted the ratio of palmitate storage under meal conditions [adjusted R = 0.18, F = 6.3, P = 0.02, 95% CI (0.12, 1.28)]; 3) LBSQ direct palmitate storage rates were significantly less in physically active than sedentary and 4) adipose tissue CD36 protein content, ACS, or DGAT activities did not independently predict palmitate storage rates. We conclude that physically active adults have lesser fatty acid cycling back into adipose tissue and that adipose ACS and DGAT may affect competition between UBSQ and LBSQ adipose for direct palmitate storage.

Pyrroloquinoline Quinone Modifies Lipid Profile, but Not Insulin Sensitivity, of Palmitic Acid-Treated L6 Myotubes.

Pyrroloquinoline quinone (PQQ) is a novel stimulator of mitochondrial biogenesis and cellular energy metabolism. This is the first study investigating regulatory mechanisms and metabolic responses underlying PQQ's action in palmitate-exposed L6 myotubes. Particularly, we assessed alterations in lipid content and composition, expression of metabolic enzymes, and changes in glucose transport. The experiments were conducted using muscle cells subjected to short (2 h) and prolonged (24 h) incubation with PQQ in a sequence of pre- and post-palmitic acid (PA) exposure. We demonstrated the opposite effects of 2 and 24 h treatments with PQQ on lipid content, i.e., a decline in the level of free fatty acids and triacylglycerols in response to short-time PQQ incubation as compared to increases in diacylglycerol and triacylglycerol levels observed after 24 h. We did not demonstrate a significant impact of PQQ on fatty acid transport. The analysis of metabolic enzyme expression showed that the vast majority of PQQ-dependent alterations cumulated in the PA/PQQ 24 h group, including elevated protein amount of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), sirtuin-1 (SIRT1), phosphorylated 5'AMP-activated protein kinase (pAMPK), carnitine palmitoyltransferase I (CPT1), citrate synthase (CS), fatty acid synthase (FAS), and serine palmitoyltransferase, long chain base subunit 1 (SPT1). In conclusion, the results mentioned above indicate PQQ-dependent activation of both fatty acid oxidation and lipid synthesis in order to adapt cells to palmitic acid-rich medium, although PQQ did not attenuate insulin resistance in muscle cells.

Palmitic Acid-Rich High-Fat Diet Exacerbates Experimental Pulmonary Fibrosis by Modulating Endoplasmic Reticulum Stress.

The impact of lipotoxicity on the development of lung fibrosis is unclear. Saturated fatty acids, such as palmitic acid (PA), activate endoplasmic reticulum (ER) stress, a cellular stress response associated with the development of idiopathic pulmonary fibrosis (IPF). We tested the hypothesis that PA increases susceptibility to lung epithelial cell death and experimental fibrosis by modulating ER stress. Total liquid chromatography and mass spectrometry were used to measure fatty acid content in IPF lungs. Wild-type mice were fed a high-fat diet (HFD) rich in PA or a standard diet and subjected to bleomycin-induced lung injury. Lung fibrosis was determined by hydroxyproline content. Mouse lung epithelial cells were treated with PA. ER stress and cell death were assessed by Western blotting, TUNEL staining, and cell viability assays. IPF lungs had a higher level of PA compared with controls. Bleomycin-exposed mice fed an HFD had significantly increased pulmonary fibrosis associated with increased cell death and ER stress compared with those fed a standard diet. PA increased apoptosis and activation of the unfolded protein response in lung epithelial cells. This was attenuated by genetic deletion and chemical inhibition of CD36, a fatty acid transporter. In conclusion, consumption of an HFD rich in saturated fat increases susceptibility to lung fibrosis and ER stress, and PA mediates lung epithelial cell death and ER stress via CD36. These findings demonstrate that lipotoxicity may have a significant impact on the development of lung injury and fibrosis by enhancing pro-death ER stress pathways.

Subcutaneous adipose tissue free fatty acid uptake measured using positron emission tomography and adipose biopsies in humans.

Positron emission tomography (PET) radiopharmaceuticals can noninvasively measure free fatty acid (FFA) uptake into adipose tissue. We studied 29 volunteers to test whether abdominal and femoral subcutaneous adipose tissue FFA uptake measured using [1-11C]palmitate PET agrees with FFA storage rates measured using an intravenous bolus of [1-14C]palmitate and adipose biopsies. The dynamic left ventricular cavity PET images combined with blood sample radioactivity corrected for the 11CO2 content were used to create the blood time activity curve (TAC), and the constant (Ki) was determined using Patlak analysis of the TACs generated for regions of interest in abdominal subcutaneous fat. These data were used to calculate palmitate uptake rates in abdominal subcutaneous adipose tissue (µmol·kg-1·min-1). Immediately after the dynamic imaging, a static image of the thigh was taken to measure the standardized uptake value (SUV) in thigh adipose tissue, which was scaled to each participant's abdominal adipose tissue SUV to calculate thigh adipose palmitate uptake rates. Abdominal adipose palmitate uptake using PET [1-11C]palmitate was correlated with, but significantly (P < 0.001) greater than, FFA storage measured using [1-14C]palmitate and adipose biopsy. Thigh adipose palmitate measured using PET calculation was positively correlated (R2 = 0.44, P < 0.0001) with and not different from the biopsy approach. The relative differences between PET measured abdominal subcutaneous adipose tissue palmitate uptake and biopsy-measured palmitate storage were positively correlated (P = 0.03) with abdominal subcutaneous fat. We conclude that abdominal adipose tissue FFA uptake measured using PET does not equate to adipose FFA storage measured using biopsy techniques.

A Peptide-Based HIV-1 Fusion Inhibitor with Two Tail-Anchors and Palmitic Acid Exhibits Substantially Improved In Vitro and Ex Vivo Anti-HIV-1 Activity and Prolonged In Vivo Half-Life.

Enfuvirtide (T20) is the first U.S. FDA-approved HIV fusion inhibitor-based anti-HIV drug. Its clinical application is limited because of its low potency and short half-life. We previously reported that peptide HP23-E6-IDL, containing both N- and C-terminal anchor-tails, exhibited stronger potency and a better resistance profile than T20. Here we designed an analogous peptide, YIK, by introducing a mutation, T639I, and then a lipopeptide, YIK-C16, by adding palmitic acid (C16) at the C-terminus of YIK. We found that YIK-C16 was 4.4- and 3.6-fold more potent than HP23-E6-IDL and YIK against HIV-1IIIB infection and 13.3- and 10.5-fold more effective than HP23-E6-IDL and YIK against HIV-1Bal infection, respectively. Consistently, the ex vivo anti-HIV-1IIIB activity, as determined by the highest dilution-fold of the serum causing 50% inhibition of HIV-1 infection, of YIK-C16 in the sera of pretreated mice was remarkably higher than that of YIK or HP23-E6-IDL. The serum half-life (t1/2 = 5.9 h) of YIK-C16 was also significantly longer than that of YIK (t1/2 = 1.3 h) and HP23-E6-IDL (t1/2 = 1.0 h). These results suggest that the lipopeptide YIK-C16 shows promise for further development as a new anti-HIV drug with improved anti-HIV-1 activity and a prolonged half-life.

Docosahexaenoic acid attenuates the detrimental effect of palmitic acid on human endothelial cells by modulating genes from the atherosclerosis signaling pathway.

The formation of atherosclerotic changes leads to dysfunction in numerous cell types, especially endothelial cells. In the current experiment, we aimed to show the therapeutic effect of Docosahexaenoic acid on palmitic-induced atherosclerotic changes in the human endothelial lineage. Human Umbilical Vein Endothelial cells were incubated with 1 mM palmitic acid for 48 hours and then exposed to 40 µM docosahexaenoic acid for next 24 hours. Cellular atherosclerosis and lipid removal were confirmed by the application of Oil red O solution. The cell survival rate was studied by using MTT assay and flow cytometry analysis of Annexin V. We also measured the protein level of tumor necrosis factor-α and granulocyte-macrophage colony-stimulating factor by immunofluorescence imaging. The transcription level of genes participating in the atherosclerosis signaling pathway was monitored in atherosclerotic endothelial cells before and after treatment with docosahexaenoic acid. The viability of the cells was reduced after 48 hours incubation with palmitic acid. It is noteworthy that the number of viable endothelial cells was increased after exposure to docosahexaenoic acid. Compared with the cells that received palmitic acid, Oil red O staining showed a decrease in the cellular content of fatty acid after incubation with docosahexaenoic acid (P < 0.05). PCR array indicated that the modulation of key genes played a role in atherosclerosis and reached near-control levels. These data support the notion that incubation of atherosclerotic human endothelial cells with docosahexaenoic acid could return the detrimental effects of palmitic acid by modulation of the atherosclerosis signaling pathway.

Fate of orally administered radioactive fatty acids in the late-pregnant rat.

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.

Effect of palmitic acid on the characteristics and release profiles of rotigotine-loaded microspheres.

BACKGROUND: The initial burst release is a major obstacle to the development of microsphere-formulated drug products. PURPOSE: To investigate the influence of palmitic acid on the characteristics and release profiles of rotigotine-loaded poly(d,l-lactide-co-glycolide) microspheres. MATERIALS AND METHODS: Rotigotine-loaded microspheres (RMS) were prepared using the oil-in-water emulsion solvent evaporation technique. The in vitro characteristics of the RMS were evaluated with scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and a particle size analyzer. The in vitro drug release and in vivo pharmacokinetics of the RMS were investigated. RESULTS AND DISCUSSION: The SEM results showed that the addition of palmitic acid changed the surface morphology of the microspheres from smooth to dimpled and then to non-smooth as the palmitic acid content increased. DSC revealed the existence of molecularly dispersed forms of palmitic acid in the microspheres. The in vitro and in vivo release profiles indicated that the addition of 5% and 8% palmitic acid significantly decreased the burst release of rotigotine from the microspheres, and the late-stage release was delayed as the palmitic acid content increased across the investigated range (5-15%). CONCLUSION: The addition of palmitic acid to the microspheres significantly affects the release profile of rotigotine from RMS.

High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry.

The constitutive activity of the ghrelin receptor is of high physiological and pathophysiological relevance. In-depth structure-activity relationship studies revealed a palmitoylated ghrelin receptor ligand that displays an in vitro binding affinity in the low nanomolar range. Activity studies revealed inverse agonistic as well as antagonistic properties and in vitro metabolic analysis indicated a high stability in blood serum and liver homogenate. For metabolic testing in vivo, a combined approach of stable isotopic labeling and mass spectrometry-based analysis was established. Therefore, a heavy isotopic version of the peptide containing a (13)C-labeled palmitic acid was synthesized and a 1:1 ratio of a (12)C/(13)C-peptide mixture was injected into rats. Biological samples were analyzed by multiple reaction monitoring allowing simultaneous peptide detection and quantification. Measurements revealed a suitable bioavailability over 24h in rat serum and subsequent high-resolution mass spectrometry investigations showed only negligible degradation and slow body clearance. Hence, this method combination allowed the identification and evaluation of a highly potent and metabolically stable ghrelin receptor ligand in vivo.

Intramyocellular diacylglycerol concentrations and [U-¹³C]palmitate isotopic enrichment measured by LC/MS/MS.

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.

Gastric bypass surgery is associated with near-normal insulin suppression of lipolysis in nondiabetic individuals.

We hypothesized that individuals who have undergone gastric bypass have greater insulin sensitivity that obese subjects but less compared with lean. We measured free fatty acid (FFA) and glucose kinetics during a two-step, hyperinsulinemic euglycemic clamp in nondiabetic subjects who were 38 ± 5 mo post-gastric bypass surgery (GB; n = 15), in lean subjects (L; n = 15), and in obese subjects (O; n = 16). Fasting FFAa were not significantly different between the three study groups but during both doses of insulin were significantly higher in O than in either GB or L. The effective insulin concentration resulting in half-maximal suppression of FFA was similar in L and GB and significantly less in both groups compared with O. Glucose infusion rates during low-dose insulin were not significantly different in GB compared with either L or O. During high-dose insulin, glucose infusion rates were significantly greater in GB than in O but less than in L. Endogenous glucose production in GB was significantly lower than O only during low dose of insulin. We conclude that gastric bypass is associated with improvements in adipose tissue insulin sensitivity to levels similar to lean, healthy persons and also with improvements in the response of glucose metabolism to insulin. These changes may be due to preferential reduction in visceral fat and decreased FFA availability. However, some differences in insulin sensitivity in GB remain compared with L. Residual insulin resistance may be related to excess total body fat or abnormal lipolysis and requires further study.

Assessment of myocardial triglyceride oxidation with PET and 11C-palmitate.

BACKGROUND: The goal of this study was to test whether myocardial triglyceride (TG) turnover including oxidation of TG-derived fatty acids (FA) could be assessed with PET and (11)C-palmitate. METHODS AND RESULTS: A total of 26 dogs were studied fasted (FAST), during Intralipid infusion (IL), during a hyperinsulinemic-euglycemic clamp without (HIEG), or with Intralipid infusion (HIEG + IL). (11)C-palmitate was injected, and 45 minutes were allowed for labeling of myocardial TG pool. 3D PET data were then acquired for 60 minutes, with first 15 minutes at baseline followed by 45 minutes during cardiac work stimulated with constant infusion of either phenylephrine (FAST, n = 6; IL, n = 6; HIEG + IL, n = 6) or dobutamine (FAST, n = 4; HIEG, n = 4). Myocardial (11)C washout during adrenergic stimulation (AS) was fitted to a mono-exponential function (Km(PET)). To determine the source of this (11)C clearance, Km(PET) was compared to direct coronary sinus-arterial measurements of total (11)C activity, (11)C-palmitate, and (11)CO(2). Before AS, PET curves in all groups were flat indicating absence of net clearance of (11)C activity from heart. In both FAST groups, AS resulted in negligible net (11)C activity and (11)CO(2) production higher than net (11)C-palmitate uptake. AS with phenylephrine resulted in net myocardial uptake of total (11)C activity and (11)C-palmitate in IL and HIEG + IL, and (11)CO(2) production lower than (11)C-palmitate uptake. In contrast, AS with dobutamine in HIEG resulted in net clearance of all (11)C metabolites (total (11)C activity, (11)C-palmitate and (11)CO(2)) with (11)CO(2) contributing 66% to endogenous FA oxidation. The AS resulted in significant Km(PET) in all the groups, except HIEG + IL. However, positive correlation between Km(PET) and (11)CO(2) was observed only in HIEG (R (2) = 0.83, P = .09). CONCLUSIONS: This is the first study to demonstrate that using PET and pre-labeling of intracardiac TG pool with (11)C-palmitate, noninvasive assessment of myocardial TG use is feasible under metabolic conditions that favor endogenous TG use such as increased metabolic demand (beta-adrenergic stimulation of cardiac work) with limited availability of exogenous substrate (HIEG).

Preferential uptake of dietary Fatty acids in adipose tissue and muscle in the postprandial period.

Despite consistent evidence that abnormalities of fatty acid delivery and storage underlie the metabolic defects of insulin resistance, physiological pathways by which fat is stored in adipose tissue and skeletal muscle are not clear. We used a combination of stable isotope labeling and arteriovenous difference measurements to elucidate pathways of postprandial fat deposition in adipose tissue and skeletal muscle in healthy humans. A test meal containing [U-(13)C]palmitate was combined with intravenous infusion of [(2)H(2)]palmitate to label plasma fatty acids and VLDL-triglyceride. Both dietary (chylomicron) and VLDL-triglyceride were cleared across adipose tissue and muscle, though with greater fractional extraction of the chylomicron-triglyceride. In adipose tissue there was significant uptake of plasma nonesterified fatty acids (NEFAs) in the postprandial but not the fasting state. However, this was minor in comparison with chylomicron-triglyceride fatty acids. We modeled the fate of fatty acids released by lipoprotein lipase (LPL). There was clear preferential uptake of these fatty acids compared with plasma NEFAs. In muscle, there was unexpected evidence for release of LPL-derived fatty acids into the plasma. With this integrative physiological approach, we have revealed hidden complexities in pathways of fatty acid uptake in adipose tissue and skeletal muscle.

Alterations in branched-chain amino acid kinetics in nonobese but insulin-resistant Asian men.

Background: Branched-chain amino acids (BCAAs) are elevated in the insulin-resistant (IR) state. The reasons for this increase remain unclear, but it may be related to abnormalities in BCAA metabolism and free fatty acid (FFA) metabolism. Objective: In this study, we quantified BCAA and FFA kinetics of IR and insulin-sensitive (IS) nonobese Asian men with the use of stable-isotope tracers. We hypothesized that in addition to greater substrate flux, the BCAA oxidative pathway is also impaired to account for the higher plasma BCAA concentration in the IR state. Design: We recruited 12 IR and 14 IS nonobese and healthy Asian men. Oral-glucose-tolerance tests (OGTTs) were performed to quantify insulin sensitivity, and subjects underwent 2 stable-isotope infusion studies. [U-13C6]Leucine was infused to measure leucine flux and oxidation as indexes of BCAA metabolism, whereas [U-13C16]palmitate was infused to measure palmitate flux and oxidation to represent FFA metabolism, The 2H2O dilution method was used to estimate body composition. Results: IR subjects had greater adiposity and significantly higher fasting and post-OGTT glucose and insulin concentrations compared with the IS group. However, none of the subjects were diabetic. Despite similar dietary protein intake, IR subjects had a significantly higher plasma BCAA concentration and greater leucine flux. Leucine oxidation was also greater in the IR group, but the relation between leucine oxidation and flux was significantly weaker in the IR group than in the IS group (r = 0.530 compared with 0.695, P < 0.0388 for differences between slope). FFA oxidation was, however, unaffected despite higher FFA flux in the IR group. Conclusion: The higher plasma BCAA concentration in healthy nonobese individuals with IR is associated with a weaker relation between BCAA oxidation and BCAA flux and this occurs in the presence of accelerated FFA flux and oxidation.

Fatty acid induced glioma cell growth is mediated by the acyl-CoA synthetase 5 gene located on chromosome 10q25.1-q25.2, a region frequently deleted in malignant gliomas.

Acyl-CoA synthetase (ACS) ligates fatty acid and CoA to produce acyl-CoA, an essential molecule in fatty acid metabolism and cell proliferation. ACS5 is a recently characterized ACS isozyme highly expressed in proliferating 3T3-L1 cells. Molecular characterization of the human ACS5 gene revealed that the gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683 amino acid protein. Two major ACS5 transcripts of 2.5- and 3.7-kb are distributed in a wide range of tissues with the highest expression in uterus and spleen. Markedly increased levels of ACS5 transcripts were detected in a glioma line, A172 cells, and primary gliomas of grade IV malignancy, while ACS5 expression was found to be low in normal brain. Immunohistochemical analysis also revealed strong immunostaining with an anti-ACS5 antibody in glioblastomas. U87MG glioma cells infected with an adenovirus encoding ACS5 displayed induced cell growth on exposure to palmitate. Consistent with the induction of cell growth, the virus infected cells displayed induced uptake of palmitate. These results demonstrate a novel fatty acid-induced glioma cell growth mediated by ACS5.

Absorption and transport of dietary long-chain fatty acids in cirrhosis: a stable-isotope-tracing study.

BACKGROUND: In rats, 30-70% of dietary fatty acids (FAs) are absorbed through the portal vein. Whether this occurs in humans is unknown, but it may occur in persons with cirrhosis, who show a blunted chylomicronemic response to dietary fat without significant steatorrhea. OBJECTIVE: The objective was to investigate whether portal FA absorption occurs in humans with cirrhosis. DESIGN: Six control subjects and 10 patients with (n = 5) and without (n = 5) cirrhotic ascites were fed [1-(13)C]palmitic and oleic acids in a test meal. Samples were drawn before and 30, 60, 90, 120, 240, 360, 480, and 720 min afterward for plasma [1-(13)C]-labeled FAs and breath (13)CO(2) assay. Fecal [1-(13)C]-labeled FAs were also measured. RESULTS: [1-(13)C]-Labeled FAs increased in chylomicrons in all groups, but less in ascitic cirrhotic patients, because their median area under the curve from 120 to 720 min was significantly lower than in the control subjects for labeled palmitate [520 (interquartile range: 192-1137) compared with 2862 (2674-4175) micromol . min/L] and oleate [829 (781-1263) compared with 3119 (2939-4986) micromol . min/L]. [1-(13)C]-Labeled FA enrichment of VLDL was also lower in cirrhotic patients. [1-(13)C]-Labeled FA in free FAs peaked earlier in ascitic than in nonascitic patients and control subjects, mainly for [1-(13)C]oleate, and the median area under the curve from 0 to 120 min was significantly higher in ascitic patients than in control subjects [301 (255-400) compared with 48 (34-185) micromol . min/L]. Fecal excretion of [1-(13)C]-labeled FA was negligible and not significantly different between groups. CONCLUSIONS: The low [1-(13)C]-labeled FA concentrations in chylomicrons and VLDL, without increased fecal losses, confirm previous data in cirrhotic patients with the use of an unlabeled fat load. The earlier [1-(13)C]-labeled FA appearance in free FAs supports the portal absorption of dietary fat in patients with advanced cirrhosis with spontaneous portal-systemic shunting.

Behaviour of dioxin in pig adipocytes.

Due to their lipophilic properties, dioxins can be integrated into the lipidic vacuole of adipocytes (fat cells). The aim of this study was to determine the kinetics of incorporation and release of 3H-labelled palmitic acid and 14C-labelled 2,3,7,8-TCDD in isolated adipocytes from pigs. The incorporation of 2,3,7,8-TCDD and palmitic acid was found to be concomitant under conditions of lipogenesis, under the effect of increasing quantities of insulin and in the presence of glucose. Release of these two compounds was found to be dependant on a lipolytic agent (epinephrine). These results suggest the risk of a strong increase of 2,3,7,8-TCDD, induced by lipolysis, in the blood of animals or humans previously exposed to this dioxin.

Nutrient modulation of palmitoylated 24-kilodalton protein in rat pancreatic islets.

Protein acylation in glucose stimulation of insulin secretion in the beta-cells has been implicated. Accordingly, we attempted to identify the target(s) of acylation in the pancreatic islets. Rat pancreatic islets were labeled with [3H]palmitic acid for 1 h at 37 C, and the whole cell lysate was analyzed by SDS-PAGE and two-dimensional gel electrophoresis. The labeling of the proteins by [3H]palmitic acid was shown to be palmitoylation by chemical analyses. Palmitoylation of four distinct bands was recognized, and the palmitoylation was significantly reduced in all of them when the labeling was performed with high glucose. Quite interestingly, the degree of attenuation was particularly dominant for a 24-kDa doublet. Palmitoylation of the 24-kDa doublet was preferentially attenuated also by the mitochondrial fuels and an acylation inhibitor, cerulenin. The half-life of the labeling of the doublet was apparently shorter (approximately 45 min) than that of other bands on pulse chasing of the islets, irrespective of the presence or absence of high glucose. High glucose attenuation of the palmitoylation of the 24-kDa doublet was partially blocked by 20 mm mannoheptulose, a glucokinase inhibitor. Two-dimensional gel electrophoresis revealed that the doublet was composed of acidic peptides, and, by immunoprecipitation, it was shown not to be synaptosome-associated protein of 25 kDa. We identified rapidly turning over palmitoylated 24-kDa acidic proteins distinct from synaptosome-associated protein of 25 kDa in the pancreatic islets, which are preferentially modulated by fuel secretagogues. The data suggested a functional role of the palmitoylated 24-kDa doublet in nutrient stimulation of insulin secretion.

Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption.

BACKGROUND: Classic fat balance studies detect fat malabsorption but do not discriminate between the potential causes of malabsorption, such as impaired intestinal lipolysis or reduced uptake of fatty acids. OBJECTIVE: We aimed to validate a novel test for the specific, sensitive detection of impaired intestinal uptake of long-chain unesterified fatty acids in an appropriate rat model of fat malabsorption. DESIGN: The absorption and appearance in plasma of [(13)C]palmitic acid were determined in control rats and in rats with fat malabsorption due either to chronic bile deficiency (permanent bile diversion) or to oral administration of the lipase inhibitor orlistat (200 mg/kg diet). [(13)C]Palmitic acid results were compared with the percentage absorption of ingested dietary fat determined by fat balance. RESULTS: Between 1 and 6 h after intraduodenal administration, plasma [(13)C]palmitate concentrations in control rats were 4-10-fold higher than in bile-deficient rats (P < 0.05) but were not significantly different between orlistat-supplemented rats and their controls. In control and bile-deficient rats, plasma [(13)C]palmitate concentrations allowed complete discrimination between normal (>92%) and reduced (<92%) fat absorption, whereas the percentage absorption of [(13)C]palmitate over 48 h appeared to be highly correlated with the percentage absorption of ingested dietary fat (r = 0.89, P < 0.001). CONCLUSIONS: The [(13)C]palmitic acid absorption test detects impaired intestinal absorption of long-chain fatty acids selectively and sensitively in a rat model of fat malabsorption due to bile deficiency. Our data strongly support the use of the [(13)C]palmitic acid absorption test for the diagnosis of clinical fat malabsorption syndromes.

Effect of binding protein surface charge on palmitate uptake by hepatocyte suspensions.

1. Studies were directed at determining whether hepatocytes, isolated from female Sprague-Dawley rats, facilitate the uptake of protein-bound long-chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein-ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake. 2. The clearance rate of [3H]-palmitate in the presence of alpha 1-acid-glycoprotein (pI = 2.7), albumin (pI = 4.9) and lysozyme (pI = 11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (= 0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion-reaction model. 3. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein-palmitate complexes, the diffusion-reaction model predicted clearance rates were 4.9 microliters s-1/10(6) cells, 4.8 microliters s-1/10(6) cells and 5.5 microliters s-1/10(6) cells for alpha 1-acid-glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2 +/- 0.1 microliters s-1/10(6) cells, 2.3 +/- 0.3 microliters s-1/10(6) cells and 7.1 +/- 0.7 microliters s-1/10(6), respectively. 4. Hepatocyte palmitate clearance was significantly faster (P < 0.01) in the presence of lysozyme than albumin which was significantly faster than alpha 1-acid-glycoprotein (P < 0.01). The marked difference in clearance rates could not be explained by considering differences in solution viscosity. 5. Our results are consistent with the notion that ionic interactions between protein-ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diffusional distance of the unbound ligand and/or by facilitating the protein-ligand dissociation rate.