FFA Metabolism Research Articles

Multi-Omics Analysis of Genes Encoding Proteins Involved in Alpha-Linolenic Acid Metabolism in Chicken.

Alpha-linolenic acid (ALA, ω-3) is an antioxidant that reduces triglyceride (TG) levels in blood, a component of cell membranes and a precursor compound of eicosapentaenoic acid (EPA, ω-3) and eicosatrienoic acid (DHA, ω-3). Fatty acid content is a quantitative trait regulated by multiple genes, and the key genes regulating fatty acid metabolism have not been systematically identified. This study aims at investigating the protein-encoding genes regulating ω-3 polyunsaturated fatty acid (PUFA) content in chicken meat. We integrated genomics, transcriptomics and lipidomics data of Jingxing yellow chicken (JXY) to explore the interactions and associations among multiple genes involved in the regulation of fatty acid metabolism. Several key genes and pathways regulating ω-3 fatty acid metabolism in chickens were identified. The upregulation of GRB10 inhibited the mTOR signaling pathway, thereby improving the content of EPA and DHA. The downregulation of FGFR3 facilitated the conversion of ALA to EPA. Additionally, we analyzed the effects of ALA supplementation dose on glycerol esters (GLs), phospholipid (PL) and fatty acyl (FA) contents, as well as the regulatory mechanisms of nutritional responses in FFA metabolism. This study provides a basis for identifying genes and pathways that regulate the content of FFAs, and offers a reference for nutritional regulation systems in production.

FRI114 PUFAs Implicated In Platelet Activation Differ In Receptor Mediated Vs Post-receptor Insulin Resistance

Abstract Disclosure: R. Klein: None. S.T. Chung: None. R.J. Brown: None. Background: Severe insulin resistance (IR) can be classified as receptor mediated, in which all downstream signaling is blocked, or post receptor IR where some downstream signaling pathways are decreased and others are increased. Receptor-mediated IR occurs in rare mutations of the insulin receptor (INSR), whereas in most forms of IR, including type 2 diabetes (T2DM) and lipodystrophy (LD), IR is post-receptor. LD is a group of syndromes characterized by deficiency of fat depots leading to metabolic dysfunction similar to obesity. Patients with post-receptor IR have increased atherosclerotic cardiovascular disease (ASCVD) risk. Thrombosis underlies pathology of ASCVD and is a result of platelet (PLT) activation. Polyunsaturated fatty acids (PUFAs) are embedded in the PLT membrane and play a key role in thrombosis. During PLT activation, cytosolic phospholipase A2 (cPLA2) hydrolyzes membrane lipids, generating free PUFAs, leading to synthesis of mediators of blood vessel constriction, inflammation, and thrombosis. Methods: This was a cross-sectional study of subjects with INSR (N=30) and LD (N=30) matched for age, sex, HbA1c and pubertal stage. Untargeted metabolomic (848 compounds) and lipidomic (984 compounds) analysis was completed on serum after an 8-12 hour fast. Analyses were conducted by t-test and Mann-Whitney with adjustment for multiplicity. Bonferroni corrected P<0.05 was considered significant. Results: Of 19 long chain PUFAs (ω=3, ω=6) measured, 14 were significantly elevated in LD vs INSR, including arachidonate, docosahexaenoate, docosapentaenoate, stearidonate, and adrenate. There was no difference in linoleate, dihomo-linoleate, tetradecadienoate, linolenate, or docosadienoate levels between LD vs INSR. Among 8 long chain saturated FAs, only 3 were significantly higher in LD, 4 were comparable, and 1 was higher in INSR. All long and medium chain monounsaturated FAs were similar in LD vs INSR. cPLA2 was not different between LD and INSR. Conclusions: Our study shows that liberated PUFAs were higher in the patients with LD compared to INSR. A similar trend was not seen for other FFA such as saturated and monounsaturated FFA, suggesting that higher PUFAs in LD are unlikely to be caused by global differences in FFA metabolism, such as different peripheral uptake of FFA via lipoprotein lipase activity or diet. Instead, our data suggest an increase in PLT membrane ability to store and release PUFAs. These data suggest increased ASCVD risk in post-receptor insulin resistance may be partially mediated through differences in platelet activation. Presentation: Friday, June 16, 2023

In vitro evaluation of fenfluramine and norfenfluramine as victims of drug interactions.

Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood‐onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter−mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t1/2 > 120 min). Two metabolites (nFFA and an N‐oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine (CYP2D6, 48%), phencyclidine (CYP2B6, 42%), and furafylline (CYP1A2, 32%) and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%−20% metabolism and no clear inhibition of nFFA metabolism by any CYP‐selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 (10%–21% disappearance for 1 and 10 µM FFA, respectively), rCYP1A2 (22%−23%), rCYP2C19 (49%−50%), and rCYP2D6 (59%−97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism (e.g., stiripentol).

The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization, enhances hepatic β-oxidation, and induces ketosis

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-3H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and nonoxidative disposal (Rst). In the liver, clearance (Kβ-ox) and flux (Rβ-ox) of FFA into β-oxidation were estimated using [9,10-3H]-(R)-bromopalmitate/[U-14C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, Ra, Rox, and Rst with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased Kβ-ox, Rβ-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation.

Relationship between postabsorptive respiratory exchange ratio and plasma free fatty acid concentrations

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 <30 kg.m(-2)). Protocol 3 analyzed data from Nielsen et al. (Nielsen, S., Z. K. Guo, J. B. Albu, S. Klein, P. C. O'Brien, M. D. Jensen. 2003. Energy expenditure, sex and endogenous fuel availability in humans. J. Clin. Invest. 111: 981-988.) to understand how fasting RER and palmitate concentrations relate within individuals during four consecutive measurements. The results were as follows: 1) Fasting RER was correlated (r = 0.74, P < 0.001) with the previous day's average RER, and less so with RER variability. 2) Fasting RER was correlated (r = -0.39, P = 0.007) with fasting plasma palmitate concentrations. 3) The pattern of the RER/palmitate relationship was similar within individuals and between individuals; a negative slope was observed significantly more often than a positive slope (chi(2) test; P < 0.001). Our findings suggest that, despite a fixed food quotient, the slight departures from energy equilibrium in a controlled General Clinical Research Center environment can effect plasma FFA concentrations. We suggest that including indirect calorimetry as part of FFA metabolism studies may aid in data interpretation.

The use of energy by the splanchnic tissues and their metabolism of VFA, glucose and FFA in sheep fed high- and low-fibre diets*

The use of energy by the splanchnic tissues and their metabolism of VFA, glucose and FFA in sheep fed high- and low-fibre diets*

Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.

Abstract 5385: Transport of Free Fatty Acids in Cardiac Myocytes is Mediated by a Plasma Membrane Pump as Revealed by Monitoring Cytoplasmic Unbound Free Fatty Acids

The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA i ]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA u ) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA i ] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA u ([FFA o ]) using FFA-BSA complexes, which clamped [FFA o ] at fixed values. The time course of influx was monitored from the change in [FFA i ], which rose exponentially to a steady state level (k influx ~ 0.01 s −1 ). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA o ]. Efflux was monitored by the decrease in [FFA i ] which, like influx, revealed exponential behavior (k efflux ~ 0.02 s −1 ). At steady state [FFA i ] was greater than [FFA o ] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA i ] &gt; [FFA o ]) revealed saturation with increasing [FFA o ]. The initial rate of influx saturated at [FFA o ] &gt; 200 nM, while the [FFA i ] &gt; [FFA o ] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA o ] &gt; 200 nM. The efflux rate constant decreased for [FFA o ] &gt; zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA u . Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA i ] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada &amp; Utah).

Regional Fat Deposition as a Factor in FFA Metabolism

Humans have a large variability in body fat distribution, which has tremendous implications for metabolic health. Obese individuals with an upper-body-fat distribution have increased health complications such as dyslipidemia, hypertension, insulin resistance, and type 2 diabetes in comparison with lower-body-obese individuals. Additionally, females have more body fat, a greater proportion of fat in their lower body, and much less visceral fat than do lean males at the same body mass index. The reasons for these differences in body fat distribution have not been clearly identified but could be important. Herein we review what has been learned about regional differences in triglyceride storage capacity and lipolysis as they relate to the causes and consequences of regional fat accumulation. Both sex and site differences in regional fat storage have been described. In contrast, with the exception of variations between men and women in the contribution of visceral adipose tissue to hepatic FFA delivery, most studies have failed to show important sex differences in regional lipolysis in vivo.

Basal and insulin-regulated free fatty acid and glucose metabolism in humans

These studies were done to examine the effects of body composition, resting energy expenditure (REE), sex, and fitness on basal and insulin-regulated FFA and glucose metabolism. We performed 137 experiments in 101 nondiabetic, premenopausal women and men, ranging from low normal weight to class III obese (BMI 18.0-40.5 kg/m2). Glucose flux was measured using [6-(2)H2]glucose and FFA kinetics with [9,10-(3)H]oleate under either basal (74 experiments) or euglycemic hyperinsulinemic (1.0 mU.kg FFM(-1).min(-1)) clamp conditions (63 experiments). Consistent with our previous findings, REE and sex independently predicted basal FFA flux, whereas fat-free mass was the best predictor of basal glucose flux; in addition, percent body fat was independently and positively associated with basal glucose flux (total r2 = 0.52, P < 0.0001). Insulin-suppressed lipolysis remained significantly associated with REE (r = 0.25, P < 0.05), but percent body fat also contributed (total adjusted r2 = 0.36, P < 0.0001), whereas sex was not significantly related to insulin-suppressed FFA flux. Glucose disposal during hyperinsulinemia was independently associated with peak VO2, percent body fat, and FFA concentrations (total r2 = 0.63, P < 0.0001) but not with sex. We conclude that basal glucose production is independently related to both FFM and body fatness. In addition, hyperinsulinemia obscures the sex differences in FFA release relative to REE, but brings out the effects of fatness on lipolysis.

The effect of high-dose simvastatin on free fatty acid metabolism in patients with type 2 diabetes mellitus

Statins improve all major lipid fractions, reduce coronary heart disease risk, and may have a minor effect on glucose tolerance. A reduction in free fatty acid flux and concentrations could be partly responsible for these effects. We measured nocturnal and postprandial plasma palmitate concentrations and rate of appearance ( R a) on 2 occasions in 12 obese dyslipidemic subjects with type 2 diabetes mellitus, using a single-blind, crossover format (placebo followed by simvastatin, 80 mg/d), and also on 1 occasion in 6 untreated control subjects. The diabetic subjects had increased average nocturnal (127 ± 13 vs 80 ± 10 μmol/L, P < .05) and 2-hour postprandial (49 ± 6 vs 17 ± 2 μmol/L, P < .001) palmitate concentrations, as well as increased nocturnal (31.6 ± 3.7 vs 19.5 ± 3.7 mmol/m 2 over 9 hours, P < .05) and postprandial (11.5 ± 3.7 vs 5.5 ± 3.7 mmol/m 2 every 4 hours, P < .005) integrated palmitate R a compared to normal controls. High-dose simvastatin reduced serum triglycerides by 35% but had no effect on plasma palmitate concentrations or R a. These results suggest that the triglyceride-lowering effect of statins is not mediated through an effect on FFA metabolism.

Physiologic Cardiac Hypertrophy During Pregnancy Involves a Change in Gene Expression Regulating Cardiac Metabolism

Pregnancy is a chronic volume-overload state that necessitates maternal cardiovascular adaptations in order to support fetal development. Conscious dogs were studied using echocardiography before (Control), 60 days (60D) of pregnancy, and ~14 days post partum (PP). The Left Ventricular (LV) end-diastolic diameter, LV end-diastolic volume, heart rate, stroke volume, and cardiac output were significantly elevated at 60D vs. control (9%, 11%, 27%, 12% and 40% respectively, all p<0.05), and returned to normal levels PP. LV free wall mass was significantly larger in hearts at 60D (21% increase, p<0.05), indicating ventricular hypertrophy. We examined the changes in gene expression at those time points via the Affymetrix GeneChip® Canine Genome Array. We found elevated mRNA levels of genes that were indicative of cardiac hypertrophy, i.e.: MHC class II DLA beta chain; Microtubule-associated protein 1B; cardiac ankyrin repeat protein, metalloproteinase 9; myomegalin (80%, 61%, 60%, 52%, and 50% respectively, all p<0.05). Genes responsible for myocyte calcium signaling; sarcoplasmic reticulum Ca2+-transport ATPase; and cardiac ryanodine receptor 2 were significantly elevated (100%, and 60% respectively, p<0.05). Genes associated with glucose and glycogen metabolism such as; glycogen phosphorylase; pyruvate dehydrogenase; 6-phosphofructokinase were down regulated (−1.7, −1.6, −1.6 Fold change respectively, all p<0.05). Genes involved in free fatty acid oxidation such as isocitrate dehydrogenase; 3-ketoacyl-CoA thiolase; acetyl-coenzyme A synthetase (1.6, 1.6, 1.5 Fold change respectively, all p<0.05) among others were elevated. This indicates that during normal pregnancy the heart adapts by hypertrophy, increasing calcium signaling (inotropic state) and emphasizing FFA metabolism over glucose metabolism.

CD36 deficiency in mice impairs lipoprotein lipase-mediated triglyceride clearance

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36(-)(/)(-)) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36(-)(/)(-) mice. cd36(-)(/)(-) mice showed no differences in hepatic VLDL-TG production or intestinal [(3)H]TG uptake compared with wild-type littermates. cd36(-)(/)(-) mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36(-/-) mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36(-)(/)(-) mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36(-)(/)(-) mice by infusion prolonged the plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05). We conclude that the increased plasma TG levels observed in cd36(-)(/)(-) mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.

The adrenergic control of hepatic glucose and FFA metabolism in rainbow trout ( Oncorhynchus mykiss): Increased sensitivity to adrenergic stimulation with fasting

The adrenergic control of glucose and FFA release was studied in hepatocytes of rainbow trout ( Oncorhynchus mykiss), which were either normally fed or fasted for 3 weeks. Isolated hepatocytes were incubated with adrenaline, noradrenaline, or isoprenaline (nonselective β-agonist). Identification of the hepatic β-adrenoceptor was combined with quantification of the difference in its affinity for adrenaline and noradrenaline. To identify the β-adrenoceptor subtype, isoprenaline incubations were combined with atenolol (selective β 1-antagonist) or ICI 118,551 (selective β 2-antagonist). Stimulation of the β-adrenoceptor resulted in mobilisation of glucose, which was inhibited by ICI 118,551 thus pointing to a β 2-subtype. The affinity of the β 2-adrenoceptor for isoprenaline and adrenaline (p D 2-values of 8.3 and 7.9) was clearly higher than for noradrenaline (p D 2-value of 6.5). This indicates that at physiological concentrations β 2-adrenoceptors in trout are mainly stimulated by adrenaline and not by noradrenaline. A significant effect of β-adrenoceptor stimulation on the FFA release was also found, although only at high concentrations (i.e., 10 −6 and 10 −5 M). Again the β 2-adrenoceptor appeared to mediate the stimulation of hepatic FFA release. Upon fasting, both the basal glucose and FFA release were strongly decreased. The ratio between glucose and FFA release decreased from 15.4 to 4.3 upon fasting and at this ratio the energy output for both metabolites became equal. The mobilisation of FFA upon adrenergic stimulation was relatively conserved, namely −35% upon fasting, as opposed to −89% in mobilisation of glucose. This indicates that upon fasting FFA gain importance in hepatic metabolism. The hepatic sensitivity to adrenergic stimulation is enhanced upon fasting, as indicated by an increased p D 2-value from 8.3 to 8.9.

Effects of atorvastatin on fasting and postprandial complement component 3 response in familial combined hyperlipidemia

VLDL overproduction by enhanced hepatic FFA flux is a major characteristic of familial combined hyperlipidemia (FCHL). The postprandial complement component 3 (C3) response has been associated with impaired postprandial FFA metabolism in FCHL. We investigated the effects of 16 weeks of treatment with atorvastatin on postprandial C3 and lipid changes in 12 FCHL patients. Atorvastatin significantly lowered fasting plasma C3 and triglyceride (TG) in FCHL. Fasting TG and insulin sensitivity were the best predictors of fasting and postprandial C3. Postprandial triglyceridemia and C3 response, estimated as area under the curve (AUC), were significantly lowered by atorvastatin by 19% and 12%, respectively, albeit still elevated, compared with 10 matched controls. Postprandial FFA-AUC and postheparin plasma lipolytic activities remained unchanged after atorvastatin, suggesting no major effect on lipolysis. After atorvastatin, postprandial hydroxybutyric acid-AUC, which was elevated in untreated FCHL patients, was decreased, reaching values similar to those in controls. The present data show reduction of postprandial hepatic FFA flux in FCHL by atorvastatin, providing an additional mechanistic explanation for the reduction of VLDL secretion reported previously for atorvastatin. This was accompanied by a decrease in fasting plasma C3 concentrations and a blunted postprandial C3 response to an acute oral fat load.

Selective parasympathetic innervation of subcutaneous and intra-abdominal fat — functional implications

The wealth of clinical epidemiological data on the association between intra-abdominal fat accumulation and morbidity sharply contrasts with the paucity of knowledge about the determinants of fat distribution, which cannot be explained merely in terms of humoral factors. If it comes to neuronal control, until now, adipose tissue was reported to be innervated by the sympathetic nervous system only, known for its catabolic effect. We hypothesized the presence of a parasympathetic input stimulating anabolic processes in adipose tissue. Intra-abdominal fat pads in rats were first sympathetically denervated and then injected with the retrograde transneuronal tracer pseudorabies virus (PRV). The resulting labeling of PRV in the vagal motor nuclei of the brain stem reveals that adipose tissue receives vagal input. Next, we assessed the physiological impact of these findings by combining a fat pad–specific vagotomy with a hyperinsulinemic euglycemic clamp and RT-PCR analysis. Insulin-mediated glucose and FFA uptake were reduced by 33% and 36%, respectively, whereas the activity of the catabolic enzyme hormone-sensitive lipase increased by 51%. Moreover, expression of resistin and leptin mRNA decreased, whereas adiponectin mRNA did not change. All these data indicate an anabolic role for the vagal input to adipose tissue. Finally, we demonstrate somatotopy within the central part of the autonomic nervous system, as intra-abdominal and subcutaneous fat pads appeared to be innervated by separate sympathetic and parasympathetic motor neurons. In conclusion, parasympathetic input to adipose tissue clearly modulates its insulin sensitivity and glucose and FFA metabolism in an anabolic way.

Selective parasympathetic innervation of subcutaneous and intra-abdominal fat--functional implications.

The wealth of clinical epidemiological data on the association between intra-abdominal fat accumulation and morbidity sharply contrasts with the paucity of knowledge about the determinants of fat distribution, which cannot be explained merely in terms of humoral factors. If it comes to neuronal control, until now, adipose tissue was reported to be innervated by the sympathetic nervous system only, known for its catabolic effect. We hypothesized the presence of a parasympathetic input stimulating anabolic processes in adipose tissue. Intra-abdominal fat pads in rats were first sympathetically denervated and then injected with the retrograde transneuronal tracer pseudorabies virus (PRV). The resulting labeling of PRV in the vagal motor nuclei of the brain stem reveals that adipose tissue receives vagal input. Next, we assessed the physiological impact of these findings by combining a fat pad-specific vagotomy with a hyperinsulinemic euglycemic clamp and RT-PCR analysis. Insulin-mediated glucose and FFA uptake were reduced by 33% and 36%, respectively, whereas the activity of the catabolic enzyme hormone-sensitive lipase increased by 51%. Moreover, expression of resistin and leptin mRNA decreased, whereas adiponectin mRNA did not change. All these data indicate an anabolic role for the vagal input to adipose tissue. Finally, we demonstrate somatotopy within the central part of the autonomic nervous system, as intra-abdominal and subcutaneous fat pads appeared to be innervated by separate sympathetic and parasympathetic motor neurons. In conclusion, parasympathetic input to adipose tissue clearly modulates its insulin sensitivity and glucose and FFA metabolism in an anabolic way. The implications of these findings for the (patho)physiology of fat distribution are discussed.

ApoA-II Versus ApoA-I

Apolipoprotein A-I (apoA-I), the major protein of HDL, is probably among the most intensively studied of all proteins. It functions in HDL assembly, in the removal of excess cholesterol from cells, and in the transport of cholesterol from peripheral tissues to the liver, a process known as “reverse cholesterol transport.” It is a cofactor for lecithin:cholesterol acyl transferase (LCAT), the enzyme responsible for cholesterol esterification in HDL, and it stabilizes certain antioxidant enzymes, such as serum paraoxonase, that are carried on HDL.1,2 Rare null mutations of apoA-I in human populations are associated with early coronary heart disease,3 and overexpression of apoA-I in transgenic mice protects against atherogenesis.4 An exciting recent development was the 4-angstrom-resolution x-ray structure for the helical part of apoA-I, encompassing residues 44 to 243 (reviewed by Segrest et al5). See page 1977 In contrast, apolipoprotein A-II (apoA-II), the second most abundant protein of HDL, has no known function, and it has been the subject of few detailed studies. Its presence in HDL has been reported to decrease,6 or have no influence on, cholesterol efflux from cells,7,8 nor is it a cofactor for any known enzyme. Rare individuals lacking apoA-II appear to have normal plasma lipids.9 In fact, all of the known effects of apoA-II are deleterious. In studies with mice and humans, apoA-II levels have been associated with increased susceptibility to atherosclerosis,10–13 increased free fatty acid levels,14,15 increased body fat,15,16 and increased insulin resistance.15,17 Undoubtedly, apoA-II has some unknown beneficial functions, possibly related to fatty acid metabolism or host defense. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , …

AGING AND SKELETAL MUSCLE FREE FATTY ACID (FFA) METABOLISM DURING EXERCISE

We have recently reported that muscle FFA utilization is lower in older vs. young individuals when exercising at the same relative intensity (i.e., 50% of VO2peak). The purpose of this study was to determine whether this is also true during exercise at the same absolute intensity, when metabolic rate and the demand for substrate is similar. Healthy young (27 ± 1 y; n = 5) and older (65 ± 2 y; n = 6) subjects were studied during 1 h of cycling at a power output of ∼35 W (VO2 = ∼0.8 L/min in both groups). Leg FFA metabolism was quantified via infusion of [1-13C]oleate and sampling of femoral arterial and venous blood, while leg blood flow was measured using indocyanine green dye. Despite a lower plasma flow in the older compared to the younger subjects (1231 ± 89 vs. 1688 ± 187 mL/min; P < 0.05), fractional extraction (11 ± 1 vs. 18 ± 1%) and clearance (141 ± 16 vs. 311 ± 38 mL/min) of FFA across the leg were significantly lower (P < 0.01) in the older group. The rate of leg FFA uptake did not differ significantly between groups, however, because arterial FFA concentration was ∼50% higher in the older subjects (0.50 ± 0.09 vs. 0.33 ± 0.03 μmol/mL; P = 0.13). No difference existed between groups in the percentage of FFA uptake by the leg that was oxidized, which averaged 82 ± 4%. We conclude that aging appears to impair the intrinsic capacity of muscle to utilize FFA during mild to moderate exercise. This does not appear to be due to any change in the metabolic fate of the FFA that are taken up. Supported by NIH AG14769

ROLE OF FATS IN EXERCISE: Types and Quality

Plasma TGs, FFAs, and muscle TG are oxidizable lipid fuel sources for skeletal muscle metabolism during prolonged exercise. Plasma FFAs are a major fuel oxidized by skeletal muscle, and their rate of use by muscle depends on several factors, including plasma FFA availability, transport from plasma to the mitochondria, and intracellular metabolism. Mobilization of FFAs from adipose tissue is the first committed step in FFA metabolism, and it depends on the rate of adipose tissue lipolysis. Adipose tissue lipolysis increases with exercise duration and exercise intensity up to intensities of approximately 60% to 65%. Evidence suggests that FFAs are transported from plasma to the mitochondria by FFA transporter proteins that include the plasma membrane and cytosolic FABPPM and FABPC. Plasma FFA use can also be regulated at the mitochondrial transport step by changing the activity of carnitine palmitoyltransferase (CPT-1). Although results from biopsy and tracer studies indicate that muscle TG contribute to skeletal muscle oxidative metabolism during exercise, their exact contribution is difficult to ascertain. Evidence shows that muscle TG use depends on exercise intensity, duration, and mode. The contribution of plasma TG to skeletal muscle metabolism is small. The rate of use of plasma TG is dependent on lipoprotein lipase activity, which is correlated with the oxidative capacity of the muscle fibers. Dietary manipulations can modulate substrate use during exercise and can potentially affect exercise performance. High carbohydrate availability before exercise is associated with an increase in blood glucose and plasma insulin concentrations, which can ultimately decrease the rate of adipose tissue lipolysis and the availability of plasma FFAs. Increased glucose flux has also been shown to decrease lipid oxidation by directly inhibiting the transport of FFAs across the mitochondrial membranes. High lipid availability can be changed by short-term or long-term exposure to high-fat diets. Because carbohydrate reserves are diminished with exposure to high-fat diets, improvements in exercise performance have been difficult to measure under these conditions.