Regulation Of Fatty Acid Uptake Research Articles

Dynamic palmitoylation of STX11 controls injury-induced fatty acid uptake to promote muscle regeneration

Different types of cells uptake fatty acids in response to different stimuli or physiological conditions; however, little is known about context-specific regulation of fatty acid uptake. Here, we show that muscle injury induces fatty acid uptake in muscle stem cells (MuSCs) to promote their proliferation and muscle regeneration. In humans and mice, fatty acids are mobilized after muscle injury. Through CD36, fatty acids function as both fuels and growth signals to promote MuSC proliferation. Mechanistically, injury triggers the translocation of CD36 in MuSCs, which relies on dynamic palmitoylation of STX11. Palmitoylation facilitates the formation of STX11/SNAP23/VAMP4 SANRE complex, which stimulates the fusion of CD36- and STX11-containing vesicles. Restricting fatty acid supply, blocking fatty acid uptake, or inhibiting STX11 palmitoylation attenuates muscle regeneration in mice. Our studies have identified a critical role of fatty acids in muscle regeneration and shed light on context-specific regulation of fatty acid sensing and uptake.

Vitamin D improves hepatic steatosis in NAFLD via regulation of fatty acid uptake and β-oxidation.

The study aimed to explore the association of serum 25(OH)D3 and hepatic steatosis in non-alcoholic fatty liver disease (NAFLD) patients and to determine whether the effect of vitamin D (VD) is mediated by activation of the peroxisome proliferator-activated receptor α (PPARα) pathway. The study contained a case-control study, in vivo and in vitro experiments. A case-control study was conducted to compare serum parameters between NAFLD patients and controls and to evaluate the association of 25(OH)D3 and NAFLD. In vivo study, male Wistar rats were randomly divided into control and model groups, fed a standard chow diet and a high-fat diet (HFD), respectively, for 7 weeks to generate an NAFLD model. Then, the rats were treated with VD and a PPARα antagonist (MK886) for 7 weeks. Tissue and serum were collected and assessed by biochemical assays, morphological analysis, histological analysis, and western blot analysis. In vitro, HepG2 cells were incubated with oleic acid (OA) to induce steatosis, which was evaluated by staining. HepG2 cells were pretreated with MK886 followed by calcitriol treatment, and differences in lipid metabolism-related proteins were detected by western blot. NAFLD patients were characterized by impaired liver function, dyslipidemia, and insulin resistance. Serum 25(OH)D3 was negatively associated with alanine aminotransferase (ALT) in NAFLD. VD deficiency was a risk factor for patients with no advanced fibrosis. Adequate VD status (25(OH)D3 >20 ng/mL) had a protective effect in patients after adjustment for confounding variables. NAFLD rats showed hyperlipidemia with severe hepatic steatosis, systematic inflammation, and lower serum 25(OH)D3. VD treatment ameliorated hepatic steatosis both in NAFLD rats and OA-induced HepG2 cells. Further, MK886 inhibited the anti-steatosis effect of VD. The study revealed that an adequate VD level may act as a protective factor in NAFLD and that VD may alleviate hepatic steatosis via the PPARα signaling pathway.

Biological targets of berberine hydrochloride and the mechanisms by which it alleviates metabolic-associated fatty liver disease

<p indent="0mm">Metabolic-associated fatty liver disease (MAFLD) is a common disease worldwide and is closely related to cardiovascular disease, obesity, diabetes and other metabolic diseases. However, there is no efficient medication available yet. Researches on potential drugs, including antioxidants, insulin sensitizers and lipid-lowering drugs, have indicated that these drugs are effective in reducing the liver fat content and some histological lesions but have limited effects in alleviating liver fibrosis. Berberine hydrochloride (BBR) is an isoquinoline alkaloid derived from many medicinal plants. It was used as an anti-diarrhoea drug in the early stage. However, a large number of studies have shown that it has rich pharmacological effects in the protection against cardiovascular diseases, cancers and other aspects. Increasing evidence has indicated that BBR can regulate metabolism and has beneficial effects on MAFLD. In the target organ of the liver, BBR can activate AMPK by regulating the phosphorylation of AMPK. This phosphorylation inhibits the expression of downstream genes related to lipid synthesis and metabolism, and thus improves hepatic steatosis. The mitochondrial membrane protein UCP2 is considered to be one of the key proteins in reducing fatty liver that acts by targeting liver mitochondria to improve cellular oxidative stress. Improving insulin resistance may also be one of the important mechanisms by which BBR alleviates MAFLD. This mechanism is mainly related to the glucose transporter and insulin resistance-related protein IRS. However, BBR is absorbed and first eliminated in the small intestine. More than 99% of BBR does not enter the bloodstream. It is speculated that BBR mainly plays a biological role in alleviating hepatic steatosis by acting through the intestinal tract, but the specific mechanism and target of BBR remain unclear. Some studies have indicated that the effect of BBR on the improvement of the intestinal microenvironment and the inhibition of intestinal lipid uptake may be the key mechanism. BBR can alleviate MAFLD by improving intestinal dysregulation, reducing blood lipids and inflammatory cytokines, and restoring bile acids. In addition, BBR, absorbed by intestinal epithelial cells, can regulate intestinal lipid uptake. Soat2 may be an important regulatory target of BBR in the intestine. By regulating Soat2, BBR can inhibit the expression of proteins related to lipid uptake in the intestinal epithelium and inhibit the uptake of free fatty acids and cholesterol in the intestinal tract. Recent studies have found that Soat2 is a target gene of hepatic X receptor (LXRα) transcriptional regulation. If the LXR inverse activator acts only in the gut, it can effectively inhibit Soat2 expression, reduce cholesterol reabsorption, and lower plasma LDL levels. Therefore, the LXR-SOAT2 regulatory mechanism may also be involved in the regulation of fatty acid uptake in the gut by BBR. In this review, we briefly summarize the mechanism by which BBR acts in the direct regulation of liver lipid metabolism, including activating the AMPK pathway and alleviating liver inflammation and oxidative stress. A potential biological target of BBR in the intestinal tract that could ameliorate MAFLD by improving the intestinal microenvironment and inhibiting intestinal lipid uptake was also reviewed.

Stomatin modulates adipogenesis through the ERK pathway and regulates fatty acid uptake and lipid droplet growth

Regulation of fatty acid uptake, lipid production and storage, and metabolism of lipid droplets (LDs), is closely related to lipid homeostasis, adipocyte hypertrophy and obesity. We report here that stomatin, a major constituent of lipid raft, participates in adipogenesis and adipocyte maturation by modulating related signaling pathways. In adipocyte-like cells, increased stomatin promotes LD growth or enlargements by facilitating LD-LD fusion. It also promotes fatty acid uptake from extracellular environment by recruiting effector molecules, such as FAT/CD36 translocase, to lipid rafts to promote internalization of fatty acids. Stomatin transgenic mice fed with high-fat diet exhibit obesity, insulin resistance and hepatic impairments; however, such phenotypes are not seen in transgenic animals fed with regular diet. Inhibitions of stomatin by gene knockdown or OB-1 inhibit adipogenic differentiation and LD growth through downregulation of PPARγ pathway. Effects of stomatin on PPARγ involves ERK signaling; however, an alternate pathway may also exist.

A Dichotomous Role for FABP7 in Sleep and Alzheimer's Disease Pathogenesis: A Hypothesis.

Fatty acid binding proteins (FABPs) are a family of intracellular lipid chaperone proteins known to play critical roles in the regulation of fatty acid uptake and transport as well as gene expression. Brain-type fatty acid binding protein (FABP7) is enriched in astrocytes and has been implicated in sleep/wake regulation and neurodegenerative diseases; however, the precise mechanisms underlying the role of FABP7 in these biological processes remain unclear. FABP7 binds to both arachidonic acid (AA) and docosahexaenoic acid (DHA), resulting in discrete physiological responses. Here, we propose a dichotomous role for FABP7 in which ligand type determines the subcellular translocation of fatty acids, either promoting wakefulness aligned with Alzheimer’s pathogenesis or promoting sleep with concomitant activation of anti-inflammatory pathways and neuroprotection. We hypothesize that FABP7-mediated translocation of AA to the endoplasmic reticulum of astrocytes increases astrogliosis, impedes glutamatergic uptake, and enhances wakefulness and inflammatory pathways via COX-2 dependent generation of pro-inflammatory prostaglandins. Conversely, we propose that FABP7-mediated translocation of DHA to the nucleus stabilizes astrocyte-neuron lactate shuttle dynamics, preserves glutamatergic uptake, and promotes sleep by activating anti-inflammatory pathways through the peroxisome proliferator-activated receptor-γ transcriptional cascade. Importantly, this model generates several testable hypotheses applicable to other neurodegenerative diseases, including amyotrophic lateral sclerosis and Parkinson’s disease.

Abstract 169: miR-21-5p Regulation of Lipid Content and Peroxidation in H9C2 Cells

Cardiovascular pathologies are the leading single cause of death in chronic kidney disease (CKD) patients. We have found that the 5/6 nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle 7 weeks after surgery, targeting peroxisome proliferator-activated receptor alpha (PPARα). PPARα is a regulator of fatty acid uptake and metabolism. In our model we find that suppression of miR-21-5p alters the expression of numerous genes involved with fatty acid oxidation and glycolysis, presumably through its regulatory action on PPARα and/or additional targets. We also find that 5/6Nx rats exhibit dyslipidemia and increased left ventricular lipid content at this time. In this study we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content and peroxidation in H9C2 cells. Cells were transfected with anti-miR-21-5p (40nM), pre-miR-21-5p (20nM) or appropriate scrambled oligonucleotide controls. After 24 hours medium was changed and half of the cells from each transfection group were treated with lipid (0.66 mM oleic acid and 0.33 mM palmitic acid) for 48 hours (n=6/treatment group for each set of experiments). Lipid content, measured by AdipoRed assay (Lonza) was significantly increased with lipid treatment (nearly two-fold). Overexpression of miR-21-5p significantly attenuated this increase (228.0 ± 9.7 vs. 198.2 ± 8.9% of untreated control), while suppression of miR-21-5p augmented lipid content (235.8 ± 11.2 vs. 328.1 ± 12.3% of untreated control). These results were supported by imaging of Oil Red O stained cells. We found that the abundance of malondialdehyde (MDA), a product of lipid peroxidation, was significantly increased in response to lipid treatment. Overexpression of miR-21-5p reduced MDA content in untreated and lipid treated cells, suggesting that miR-21-5p reduces oxidative stress. Suppression of miR-21-5p had no effect on MDA levels. These results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. Ongoing studies aimed at evaluation of alterations in fatty acid oxidation and oxidative stress will further aid in determining the functional impact of miR-21-5p on associated pathways in cardiac tissue.

Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT pathway in a diet-induced mouse model of nonalcoholic fatty liver disease.

Flavonoids and related compounds seem to have favorable effects on nonalcoholic fatty liver disease (NAFLD) progression, although the exact mechanisms implicated are poorly understood. In this study, we aimed to investigate the effect of the flanovol quercetin on gene expression deregulation involved in the development of NAFLD, as well as the possible implication of phosphatidylinositol 3-kinase (PI3K)/AKT pathway modulation. We used an in vivo model based on methionine- and choline-deficient (MCD) diet-fed mice and an in vitro model consisting of Huh7 cells incubated with MCD medium. MCD-fed mice showed classical pathophysiological characteristics of nonalcoholic steatohepatitis, associated with altered transcriptional regulation of fatty acid uptake- and trafficking-related gene expression, with increased lipoperoxidation. PI3K/AKT pathway was activated by MCD and triggered gene deregulation causing either activation or inhibition of all studied genes as demonstrated through cell incubation with the PI3K-inhibitor LY294002. Treatment with quercetin reduced AKT phosphorylation, and oxidative/nitrosative stress, inflammation and lipid metabolism-related genes displayed a tendency to normalize in both in vivo and in vitro models. These results place quercetin as a potential therapeutic strategy for preventing NAFLD progression by attenuating gene expression deregulation, at least in part through PI3K/AKT pathway inactivation.

Abstract 1870: Omental adipocytes promote overexpression of CD36 in ovarian cancer cells and enhance tumorigenicity.

Abstract OBJECTIVES: Epithelial ovarian cancers frequently metastasize and grow to a considerable size in the omentum, a peritoneal fold largely comprised of adipocytes. We have previously shown ovarian cancer cells induce lipolysis in adipocytes and utilize adipocyte-derived fatty acids as an energy source. However, the mechanism of adipocyte-induced fatty acid uptake in ovarian cancer has not yet been established. The goal of this study was to determine whether adipocytes alter the expression of fatty acid transport receptors in ovarian cancer cells and whether inhibition of these receptors impedes tumor progression. METHODS: Immunofluorescence microscopy and western blot analysis were used to study the expression profile of fatty acid transport receptors in a panel of ovarian cancer cell lines cultured in the presence and absence of omental adipocytes. To determine the impact of the identified fatty acid receptor on tumor progression, its expression was silenced in SKOV3ip1 cells using a short hairpin RNA (shRNA) lentiviral vector. Stably-transfected SKOV3ip1-shRNA cells (1x10ˆ6) were injected intraperitoneally into nude mice. Fatty acid transporter mRNA levels of laser-microdissected human high-grade serous ovarian cancer cells and their corresponding omental metastases were measured using real-time quantitative PCR analysis. RESULTS: Among the fatty acid transport receptors evaluated, CD36 was found to be selectively upregulated in ovarian cancer cells in the presence of omental adipocytes. CD36 knockdown in SKOV3ip1 cells reduced lipid accumulation and fatty acid uptake (81% and 35%, respectively, p&amp;lt;0.01) and prevented adhesion to extracellular matrix proteins (collagen I and laminin). CD36-shRNA cells exhibited reduced clone formation (5-fold, p&amp;lt;0.01) in a soft agar clonogenicity assay, compared to scrambled shRNA controls. Metastatic tumor burden was significantly reduced in CD36-shRNA injected mice compared to scrambled shRNA controls, as assessed by tumor weight and number of metastatic foci (1.19g vs. 0.20g, p&amp;lt;0.01 and 188 vs. 18, p&amp;lt;0.01, respectively). In human omental metastases samples (n=10), CD36 mRNA levels were elevated 6-fold as compared to their primary tumor counterparts. CONCLUSIONS: In the presence of omental adipocytes, CD36 is an important regulator of fatty acid uptake in ovarian cancer cells. Disruption of fatty acid trafficking may offer a novel treatment approach in reducing tumor dissemination within the abdominal cavity. Citation Format: Andras Ladanyi, Kristin Nieman, Carla Penicka, Anirban Mitra, Hilary Kenny, Katja Gwin, Rebecca Wolsky, S Diane Yamada, Ernst Lengyel. Omental adipocytes promote overexpression of CD36 in ovarian cancer cells and enhance tumorigenicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1870. doi:10.1158/1538-7445.AM2013-1870

Low RIP140 expression rescues insulin‐mediated FA oxidation via a rise in MCAD gene expression in skeletal muscle cells exposed to high palmitate availability

We have shown that in L6 cells incubated with high palmitate, low Receptor Interacting Protein 140 (RIP140) expression alters the regulation of muscle fatty acid (FA) uptake and FA oxidation under insulin‐mediated conditions. To provide mechanistic insights for these metabolic changes, we measured mRNA and/or protein expression of several genes involved in the regulation of FA uptake and oxidation. Cells were incubated with insulin (1000 nM) ± palmitate (400 μM) and with siRNA sequences for either RIP140 or a negative control. Under insulin‐mediated conditions, short‐term incubation with palmitate decreased (P&lt;0.05) FA uptake (24%) and oxidation (42%). Under these conditions, low RIP140 expression reduced (P&lt;0.05) FA uptake (35%) and restored FA oxidation so that it was not different (P&gt;;0.05) from the control rate. Incubation with palmitate increased (P&lt;0.05) CD36 (70%) and FATP1 (224%) mRNA, decreased (P&lt;0.05) CPT1 (45%) mRNA and did not change MCAD (P&gt;;0.05) mRNA. High palmitate was accompanied by reductions (P&lt;0.05) in protein content for FATP1 (68%) and total (59%) and plasma membrane (31%) CD36 but it did not affect (P&gt;;0.05) CPT1. Under those same conditions, low RIP140 expression was accompanied by a rise in the mRNA expression of MCAD (83%; P&lt;0.05) and FATP1 (38%; P=0.08) but no change (P&gt;;0.05) in mRNA expression of CPT1 and CD36. Protein content of CPT1, FATP1, CD36 (total and plasma membrane) were not affected (P&gt;;0.05) by low RIP140 expression. Our results indicate that low RIP140 expression in the context of high palmitate availability increases gene expression of MCAD and that this may in part explain the restoration of insulin‐mediated FA oxidation.USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund

Effects of adipocyte lipoprotein lipase on de novo lipogenesis and white adipose tissue browning

Efficient storage of dietary and endogenous fatty acids is a prerequisite for a healthy adipose tissue function. Lipoprotein lipase (LPL) is the master regulator of fatty acid uptake from triglyceride-rich lipoproteins. In addition to LPL-mediated fatty acid uptake, adipocytes are able to synthesize fatty acids from non-lipid precursor, a process called de novo lipogenesis (DNL). As the physiological relevance of fatty acid uptake versus DNL for brown and white adipocyte function remains unclear, we studied the role of adipocyte LPL using adipocyte-specific LPL knockout animals (aLKO). ALKO mice displayed a profound increase in DNL-fatty acids, especially palmitoleate and myristoleate in brown adipose tissue (BAT) and white adipose tissue (WAT) depots while essential dietary fatty acids were markedly decreased. Consequently, we found increased expression in adipose tissues of genes encoding DNL enzymes (Fasn, Scd1, and Elovl6) as well as the lipogenic transcription factor carbohydrate response element binding protein-β. In a high-fat diet (HFD) study aLKO mice were characterized by reduced adiposity and improved plasma insulin and adipokines. However, neither glucose tolerance nor inflammatory markers were ameliorated in aLKO mice compared to controls. No signs of increased BAT activation or WAT browning were detected in aLKO mice either on HFD or after 1week of β3-adrenergic stimulation using CL316,243. We conclude that despite a profound increase in DNL-derived fatty acids, proposed to be metabolically favorable, aLKO mice are not protected from metabolic disease per se. In addition, induction of DNL alone is not sufficient to promote browning of WAT. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

Contraction-induced signaling: evidence of convergent cascades in the regulation of muscle fatty acid metabolism

The regulation of fatty acid utilization during muscle contraction and exercise remains to be fully elucidated. Evidence suggests that the metabolic responses of skeletal muscle induced by the contraction-induced changes in energy demand are mediated by the activation of a multitude of intracellular signaling cascades. This review addresses the roles played by 3 intracellular signaling cascades of interest in the regulation of fatty acid uptake and oxidation in contracting skeletal muscle; namely, the AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinases (CaMKs), and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascades. Data delineating the potential role of AMPK in cross-talk with CaMKII, CaMK kinase (CaMKK), and ERK1/2 are presented. Collectively, data show that in perfused rodent muscle, regulation of fatty acid uptake and oxidation occurs via (i) CaMKII signaling via both AMPK-dependent and -independent cascades, (ii) CaMKK signaling via both AMPK-dependent and -independent cascades, (iii) AMPK signaling in a time- and intensity-dependent manner, and (iv) ERK1/2 signaling in an intensity-dependent manner.

High levels of arachidonic acid and peroxisome proliferator-activated receptor-alpha in breast cancer tissues are associated with promoting cancer cell proliferation

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER–), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.

Probing Mechanism for the Enhancement of Uptake of Fatty Acid into Cells by the Membrane Protein CD36

CD36 is a membrane protein found in various cell types including adipocytes and endothelial cells. Physiological studies have suggested that CD36 is associated with insulin-resistant diabetes, however, the mechanism remains poorly understood. It is well recognized as a receptor for multiple ligands such as collagen and thrombospondin 1, and possibly acts as a catalyst to enhance the rate of transmembrane movement of fatty acid. To separate the two independent events transport across the plasma membrane and the subsequent intracellular metabolism, we have applied biophysical approaches and metabolic analyses to cells in vitro. Our results using cultured cells (HEK293 cells overexpressing CD36 and 3T3-L1 adipocyte cells) as well as mice adipocyte cells isolated from a CD36 null mouse showed that fatty acids diffuse through the plasma membrane rapidly with or without CD36. In HEK 298 cells, which normally synthesize triglycerides very slowly and to a limited extent, expression of CD36 enhanced the rate and extent of synthesis. Even in the presence of CD36, incorporation into triglycerides is a much slower process (min) relative to the transmembrane movement (sec), indicating that the rate-limiting step of the regulation of fatty acid uptake by CD36 is intracellular metabolism. Lastly, by PCR array analysis of 84 proteins, we have identified several enzymes involved in human fatty acid metabolism with gene expression levels altered by overexpression of CD36. Taken together, our results showed that CD36 increases fatty acid uptake by enhancing triglycerides synthesis rather than acting as a membrane transporter, but as yet by unidentified molecular mechanisms.

Low RIP140 Expression Partially Restores Metabolic Flexibility in L6 Muscle Cells Treated with High FA

To elucidate the role of RIP140 in the regulation of fatty acid uptake (FAU) and oxidation (FAO) and glucose uptake (GU) in insulin‐resistant muscle cells, L6 myotubes were transfected with RNAi to genetically silence a control sequence (CS) or RIP140 (RIP). Cells were then incubated ± palmitic acid (FA: 400μM, 12h) to induce insulin resistance followed by incubation ± insulin (I: 1000 nM) before measurement of glucose and FA kinetics. In line with the presence of insulin resistance, FA treatment raised basal GU (122.2±5.2% vs 230.0±18.8%; P&lt;0.05) and obliterated the I‐induced increase in GU (P&lt;0.05). FA treatment decreased basal FAU (107.1±3.7% vs 45.4±10.5%; P&lt;0.05) and FAO (176.8±19.6 vs 82.7±13.9%; P&lt;0.05). I‐induced FAU and FAO was reduced by 24% (P&lt;0.05) and 44% (P&lt;0.05) with FA treatment. Silencing of RIP140 in FA‐treated cells did not affect basal GU or FAO but it was associated with higher FAU (45.4±10.5% vs 75.0±12.1%; P&lt;0.05). With I stimulation, silencing of RIP140 in FA‐treated cells was associated with higher GU (29%; P&lt;0.05), lower FAU (35%; P&lt;0.05) and no change in FAO (P&lt;0.05). Our data show that reduced RIP140 expression in FA‐treated muscle cells rescues insulin‐mediated GU and basal FAU but does not affect basal or I‐mediated FAO. Together, our results indicate that low RIP140 expression may partially restore insulin sensitivity when insulin resistance is induced by high FA treatment.USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund

Targeting of acyl‐CoA synthetase 3 to lipid droplets

Lipid droplets emerge as dynamic organelles highly relevant for lipid homeostasis and the pathophysiology of metabolic diseases. We are using fatty acyl‐CoA synthetases (ACS), especially ACSL3 as a paradigm to investigate the formation and growth of lipid droplets. Out of eleven mammalian long chain ACS, ACSL3 is the only family member localizing specifically to lipid droplets and the ER. The distribution of ACSL3 between these two organelles depends on the cell type and whether exogenous fatty acids are being supplied. The N‐terminal region of ACSL3 is sufficient to direct reporter proteins to lipid droplets which we analysed by confocal microscopy. Mutational analysis demonstrated that only amino acids 12 to 51 are essential for targeting. ACSL3 is tightly membrane associated by an amphipathic helix contained within the N‐terminal sorting signal. During lipid droplet biogenesis, ACSL3 moves from the bilayer membrane of the ER to the phospholipid monolayer of nascent lipid droplets as we demonstrated by live microscopy. Overexpression of ACSL3 results not only in increased cellular enzyme activity but also in the enhanced uptake of exogenous fatty acids. This suggests that ACSL3 is functionally relevant for the regulation of fatty acid uptake. Moreover, we speculate that the specific localization of ACSL3 is important for the local biosynthesis of phospholipids at the surface of the lipid droplets.

Regulation of fatty acid uptake into tissues: lipoprotein lipase- and CD36-mediated pathways

Cells obtain FAs either from LPL-catalyzed hydrolysis of lipoprotein triglyceride or from unesterified FFAs associated with albumin. LPL also influences uptake of esterified lipids such as cholesteryl and retinyl esters that are not hydrolyzed in the plasma. This process might not involve LPL enzymatic activity. LPL is regulated by feeding/fasting, insulin, and exercise. Although a number of molecules may affect cellular uptake of FFAs, the best characterized is CD36. Genetic deletion of this multiligand receptor reduces FFA uptake into skeletal muscle, heart, and adipose tissue, and impairs intestinal chylomicron production and clearance of lipoproteins from the blood. CD36 is regulated by some of the same factors that regulate LPL, including insulin, muscle contraction, and fasting, in part, via ubiquitination. LPL and CD36 actions in various tissues coordinate biodistribution of fat-derived calories.

The multigene family of fatty acid-binding proteins (FABPs): Function, structure and polymorphism

Fatty acid-binding proteins (FABPs) are members of the superfamily of lipid-binding proteins (LBP). So far 9 different FABPs, with tissue-specific distribution, have been identified: L (liver), I (intestinal), H (muscle and heart), A (adipocyte), E (epidermal), Il (ileal), B (brain), M (myelin) and T (testis). The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. The structure of all FABPs is similar - the basic motif characterizing these proteins is beta-barrel, and a single ligand (e.g. a fatty acid, cholesterol, or retinoid) is bound in its internal water-filled cavity. Despite the wide variance in the protein sequence, the gene structure is identical. The FABP genes consist of 4 exons and 3 introns and a few of them are located in the same chromosomal region. For example, A-FABP, E-FABP and M-FABP create a gene cluster. Because of their physiological properties some FABP genes were tested in order to identify mutations altering lipid metabolism. Furthermore, the porcine A-FABP and H-FABP were studied as candidate genes with major effect on fatness traits.

Regulation of fatty acid uptake and metabolism in L6 skeletal muscle cells by resistin

Resistin has been proposed as a potential link between obesity and insulin resistance. It is also well established that altered metabolism of fatty acids by skeletal muscle can lead to insulin resistance and lipotoxicity. However, little is known about the effect of resistin on long chain fatty acid uptake and metabolism in skeletal muscle. Here we show that treating rat skeletal muscle cells with recombinant resistin (50 nM, 24 h) decreased uptake of palmitate. This correlated with reduced cell surface CD36 content and lower expression of FATP1, but no change in FATP4 or CD36 expression. We also found that resistin decreased fatty acid oxidation by measuring 14CO 2 production from [1- 14C] oleate and an increase in intracellular lipid accumulation was detected in response to resistin. Decreased AMPK and ACC phosphorylation were observed in response to resistin while expression of ACC and AMPK isoforms was unaltered. Resistin mediated these effects without altering cell viability. In summary, our results demonstrate that chronic incubation of skeletal muscle cells with resistin decreased fatty acid uptake and metabolism via a mechanism involving decreased cell surface CD36 content, FATP1 expression and a decrease in phosphorylation of AMPK and ACC.

Energy translocation across cell membranes and membrane models.

Fatty acid transport is an important process in cellular energy distribution and storage in both normal and pathological states, especially obesity-linked type 2 diabetes mellitus. Fatty acid transport has been studied by the complementary approaches of cell biology and biophysics. According to the latter approach, specific proteins that enhance the uptake and storage of fatty acids are posited as fatty acid translocases, which facilitate fatty acid movement from the outer to inner leaflets of the plasma membrane. According to biophysical studies conducted in vitro, fatty acid translocation occurs by a rapid diffusive process that does not require a protein. Herein, we critically review these two mechanisms and their importance in the regulation of fatty acid uptake in vivo.

Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase (FAT/CD36)

Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase (FAT/CD36)