Control of fatty acid and glycerol release in adipose tissue lipolysis

Adipose tissue lipolysis is the catabolic process leading to the breakdown of triglycerides stored in fat cells and the release of fatty acids and glycerol. Recent work has revealed that lipolysis is not a simple metabolic pathway stimulated by catecholamines and inhibited by insulin. New discoveries on the regulation of lipolysis by endocrine and paracrine factors and on the proteins involved in triglyceride hydrolysis have led to a reappraisal of the complexity of the various signal transduction pathways. The steps involved in the dysregulation of lipolysis observed in obesity have partly been identified.     

FGF1 and insulin control lipolysis by convergent pathways

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Analysis of lipolysis in adipocytes using a fluorescent fatty acid derivative

For facilitation of the experimental analysis of the mechanism and regulation of mobilization of fatty acids from adipose triacylglycerol (TAG) stores, which also represents important targets for pharmacological intervention with the pathogenesis of diabetes and obesity, we developed a convenient and reliable non-radioactive cell-based assay. Isolated rat adipocytes are incubated with the fluorescent fatty acid derivative, 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid (NBD-FA), in the presence of insulin. The resulting NBD-FA-labeled TAG is efficiently cleaved by hormone-sensitive lipase (HSL) in vitro. After removal of insulin and excess of free NBD-FA, lipolysis is initiated by addition of isoproterenol and/or adenosine deaminase. The amount of NBD-FA generated in total or released into the incubation medium in the presence of modulatory hormones or compounds is then monitored by thin layer chromatography and fluorescence imaging. Release of NBD-FA, glycerol and [3H]oleic acid from TAG follows similar kinetics and concentration dependence in response to various lipolytic and anti-lipolytic stimuli as well as inhibitors of HSL. Release of NBD-FA from adipocytes correlates well to translocation of HSL from the cytosol to TAG droplets. In addition, we found that a cell-free system consisting of NBD-FA-labeled TAG droplets with endogenous associated HSL closely reflects the lipolytic state of the adipocytes used for its preparation. In conclusion, release of NBD-FA from TAG in vivo and in vitro can be used as accurate index for (regulation of) lipolysis in primary and cultured adipocytes.     

Increased lipolysis in transgenic animals overexpressing the epithelial fatty acid binding protein in adipose cells

Fatty acid binding proteins (FABPs) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967-972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered. These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type.     

Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats

We have reported previously that randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) resulted in significantly lower body fat accumulation and higher hepatic fatty acid oxidation than from long-chain triacylglycerol (LCT) in rats. To understand the metabolic changes occurring in white adipose tissue, the fatty acid oxidation and synthesis, and the adipocytokine level were measured in rats fed with MLCT or LCT for 2 weeks. In comparison with LCT, MLCT lowered not only the fatty acid synthase and glycerol-3-phosphate dehydrogenase activities in perirenal adipose tissue, but also the serum insulin and leptin levels, in addition to significantly reducing the body fat accumulation. In contrast, fatty acid oxidation measured as the carnitine palmitoyltransferase activity in the tissue was significantly higher in the MLCT-fed rats than in the LCT-fed rats. It seems that the altered fatty acid metabolism in adipose tissue per se was also responsible for the lower adiposity by dietary MLCT.     

Cell-associated nonesterified fatty acid levels and their alteration during lipolysis in the isolated mouse adipose cell

A rapid and flexible method has been developed for measuring cell-associated, probably intracellular, nonesterified fatty acids (CAFA) in isolated mouse adipose cells. A variety of lipolytic agents as well as various concentrations of epinephrine elevate CAFA levels in rough proportion to their stimulation of glycerol and fatty acid release. Insulin reduces epinephrine-elevated CAFA levels. A detailed, quantitative study of the relationship among lipolytic activity, CAFA levels, and the extracellular molar ratio of fatty acids to albumin has been carried out. Epinephrine-elevated CAFA levels rise linearly with, while epinephrine-stimulated lipolytic activity is independent of, fatty acid to albumin ratios below 2-3. As the ratio increases from 3 to 5, CAFA levels continue to increase, whereas lipolytic activity decreases. Above ratios of 5, fatty acid release almost completely ceases; CAFA levels increase dramatically with residual glycerol release. A temperature-dependent efflux of epinephrine-elevated CAFA can be elicited through blockade of stimulated lipolysis with propranolol, but only in the presence of extracellular fatty acid to albumin ratios below 3. These observations suggest that during stimulated lipolysis, a fatty acid gradient exists between the cell and extracellular serum albumin and that CAFA represent the intracellular component of this gradient. In addition, these observations support the concept that intracellular fatty acids play a role in the feedback regulation of adipose cell function as extracellular fatty acids accumulate during the lipolytic response.     

Uptake of long-chain fatty acid methyl esters by mammalian cells

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.     

Niacin mediates lipolysis in adipose tissue through its G-protein coupled receptor HM74A

A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue.     

Separation of long-chain fatty acid esters of retinol by high-performance liquid chromatography

Individual long-chain fatty acid esters of retinol can be resolved by high-performance liquid chromatography using an octyl- or phenyl-substituted reverse-phase column and mixtures of acetonitrile with water as mobile phase. This simple procedure provides good resolution of biologically important retinyl esters including retinyl palmitate and retinyl oleate. Using an isocratic elution system, it is shown that nine synthetic esters of retinol, ranging in fatty acyl chain length from 12 to 20 carbons, each elute with a unique elution volume and produce an absorbance signal at 340 nm proportional to molar concentration. The method is suitable for analysis of various esters of retinol in biological samples including lymph chylomicrons and blood plasma. The octyl-substituted reverse-phase column can also be used to separate more polar neutral retinoids including retinol and retinaldehyde.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

CTRP5 ameliorates palmitate-induced apoptosis and insulin resistance through activation of AMPK and fatty acid oxidation

Lipotoxicity resulting from a high concentration of saturated fatty acids is closely linked to development of insulin resistance, as well as apoptosis in skeletal muscle. CTRP5, an adiponectin paralog, is known to activate AMPK and fatty acid oxidation; however, the effects of CTRP5 on palmitate-induced lipotoxicity in myocytes have not been investigated. We found that globular domain of CTRP5 (gCTRP5) prevented palmitate-induced apoptosis and insulin resistance in myocytes by inhibiting the activation of caspase-3, reactive oxygen species accumulation, and IRS-1 reduction. These beneficial effects of gCTRP5 are mainly attributed to an increase in fatty acid oxidation through phosphorylation of AMPK. These results provide a novel function of CTRP5, which may have preventive and therapeutic potential in management of obesity, insulin resistance, and type 2 diabetes mellitus.     

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal?/?) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal?/? mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal?/? mice.     

AMPK-mediated increase in myocardial long-chain fatty acid uptake critically depends on sarcolemmal CD36

CD36, also named fatty acid translocase, has been identified as a putative membrane transporter for long-chain fatty acids (LCFA). In the heart, contraction-induced 5′ AMP-activated protein kinase (AMPK) signaling regulates cellular LCFA uptake through translocation of CD36 and possibly of other LCFA transporters from intracellular storage compartments to the sarcolemma. In this study, isolated cardiomyocytes from CD36^+/+- and CD36^−/− mice were used to investigate to what extent basal and AMPK-mediated LCFA uptake are CD36-dependent. Basal LCFA uptake was not altered in CD36^−/− cardiomyocytes, most likely resulting from a (1.8-fold) compensatory upregulation of fatty acid-transport protein-1. The stimulatory effect of contraction-mimetic stimuli, oligomycin (2.5-fold) and dipyridamole (1.6-fold), on LCFA uptake into CD36^+/+ cardiomyocytes was almost completely lost in CD36^−/− cardiomyocytes, despite that AMPK signaling was fully intact. CD36 is almost entirely responsible for AMPK-mediated stimulation of LCFA uptake in cardiomyocytes, indicating a pivotal role for CD36 in mediating changes in cardiac LCFA fluxes.     

Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Long-chain fatty acids can transfer passively across mammalian cell membranes. However, under physiological conditions of low fatty acid to albumin ratios in the circulation, the major fraction of uptake appears to be mediated by a saturable, protein-facilitated component. A simple diffusion process becomes significant at high molar ratios of fatty acid to albumin as the concentration of free fatty acid in solution is increased. Identification of the mammalian membrane fatty acid transporter(s) has been the focus of active investigation by several research groups. In this review we discuss three candidate proteins: FABPm, FAT/CD36 and FATP which have been cloned and are currently being characterized. Recent evidence arguing for an important role of the fatty acid transport step in general metabolism and linking these proteins to physiologic or metabolic abnormalities is described.     

New insights into the regulation of cAMP synthesis beyond GPCR/G protein activation: implications in cardiovascular regulation

Regulation of intracellular concentrations of cyclic AMP is one of the most ubiquitous mechanisms for regulating cellular functions. Further, the manner in which cAMP production is regulated via G proteins at the level of adenylyl cyclase activation has been studied extensively. This review focuses instead on the recently identified mechanisms and roles for regulation of adenylyl cyclase functions beyond G protein activation. These mechanisms include: a) the coupling of particular isoforms of adenylyl cyclase to function within a single cell type b) regulation of membrane trafficking of higher order enzyme aggregates and c) raf kinase-dependent phosphorylation and sensitization of adenylyl cyclases--an important pathway for crosstalk between tyrosine kinase signaling cascades with regulation of cAMP-mediated responses.