Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats

Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague-Dawley rats were adapted to a 3h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil (n-3) or triolein (n-9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD+triolein, and the FFD+PUFA by 80%, 75%, and 50%, respectively, when assessed 2h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism and gene expression.     

Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A-I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A-I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism.     

Dietary fatty acids and arthritis

Musculoskeletal complaints are the second most frequent reason for medical treatments. Within these diseases rheumatoid arthritis (RA) and, especially, osteoarthritis (OA) are common. Although the causes of arthritis are multifactorial and not fully understood, clinical trials have generally shown benefit from dietary n-3 polyunsaturated fatty acids. This has usually been attributed to their anti-inflammatory properties. Recently we have used in vitro model systems to study the molecular mechanism(s) by which n-3 PUFAs may act to alleviate the symptoms of arthritis. These experiments showed that n-3 PUFAs reduce expression of cartilage-degrading proteinases, cyclooxygenase-2 and inflammatory cytokines. Eicosapentaenoic acid (EPA) was more effective than docosahexaenoic acid (DHA) or alpha-linolenic acid. The data provide a scientific rationale for the consumption of n-3 fatty acids as part of a healthy diet and perhaps in treating arthritis.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Effect of pitavastatin on apolipoprotein A-I production in HepG2 cell

There are few reports describing the mechanism of HDL-elevating action of HMG-CoA reductase inhibitors (statins). As it is considered that the key step of HDL production is the secretion of apolipoprotein A-I (apoA-I), we investigated the effect of statins on apoA-I synthesis and secretion by HepG2 cell to elucidate the mechanism of the action. Each statin induced apoA-I expression (mRNA and protein) dose-dependently: the rank order of the apoA-I induction pitavastatin (3 μM) > simvastatin (10 μM) > atorvastatin (50 μM). The induction of apoA-I by statins disappeared with addition of mevalonate, which indicates that the effect is HMG-CoA reductase inhibition-dependent. Based on HMG-CoA reductase inhibition, pitavastatin-induced apoA-I more efficiently than simvastatin and atorvastatin. Further study revealed that pitavastatin increased ABCA1 mRNA in HMG-CoA reductase-dependent manner and that Rho and Rho kinase inhibitor (C3T and Y27632) increased apoA-I production in the HepG2 cells. These results suggest that pitavastatin efficiently increases apoA-I in the culture medium of HepG2 cells by promoting apoA-I production through inhibition of HMG-CoA reductase and suppression of Rho activity and by protecting apoA-I from catabolism through ABCA1 induction and lipidation of apoA-I.     

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.     

Phosphate limitation as crucial factor to enhance yeast lipid production from short-chain fatty acids

Microbial lipids for chemical synthesis are commonly obtained from sugar-based substrates which in most cases is not economically viable. As a low-cost carbon source, short-chain fatty acids (SCFAs) that can be obtained from food wastes offer an interesting alternative for achieving an affordable lipid production process. In this study, SCFAs were employed to accumulate lipids using Yarrowia lipolytica ACA DC 50109. For this purpose, different amounts of SCFAs, sulfate, phosphate and carbon: phosphate ratios were used in both synthetic and real SCFAs-rich media. Although sulfate limitation did not increase lipid accumulation, phosphate limitation was proved to be an optimal strategy for increasing lipid content and lipid yields in both synthetic and real media, reaching a lipid productivity up to 8.95 g/L h. Remarkably, the highest lipid yield (0.30 g/g) was achieved under phosphate absence condition (0 g/L). This fact demonstrated the suitability of using low-phosphate concentrations to boost lipid production from SCFAs.     

Determination of apolipoprotein A-I synthesis in intestinal explants from fetal and neonatal rats

The ability of rat intestine and liver to synthesize the main constitutive apoproteins of HDL (apolipoproteins (apo) A-I, A-IV and E) was studied by incorporation of [3H]leucine in vitro at different stages of perinatal life. In both organs, apoprotein synthesis was barely detectable at day 18 of gestation; it was initiated 2 days before the end of gestation. Apo A-I synthesis leveled off at birth in the intestine but kept increasing in the liver during suckling. Intestinal apo A-IV and hepatic apo E synthesis became stable 5 days after birth. Hormonal determination of apo A-I synthesis was examined at different ages in jejunum cultured for 48 h in vitro in the presence of effectors. The addition of dexamethasone to the culture medium was without effect on intestine explanted either at day 18 of gestation or at different postnatal ages (0, 2 and 5 days), but induced the specific stimulation of apo A-I synthesis at day 20 of gestation. At this stage, triiodothyronine alone was ineffective, whereas it enhanced the dexamethasone-induced stimulation. Apo A-I synthesis remained unaffected by insulin alone or combined with the glucocorticoid. Administration of cortisone acetate to pregnant rats from day 14 of gestation onwards resulted in a stimulation of apo A-I synthesis only when it was prolonged after the 20th day of gestation. No effect of dietary substrates could be obtained in vitro. It is concluded that glucocorticoids specifically potentiate prenatal apo A-I synthesis in the rat intestine but that their action is limited to the days immediately preceding birth. They cannot induce early maturation nor stimulate existing synthesis.     

Dietary fatty acids and membrane protein function

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.     

Functional lipidomics of oxidized products from polyunsaturated fatty acids

Because of their high degree of unsaturation, polyunsaturated fatty acids (PUFA) in mammals, with mainly 18, 20 and 22 carbons, can easily be autooxidized, and converted into many oxidized derivatives and degradation products. This short review reports on some of those relevant to the evaluation of oxidative stress in situ. In addition, the enzyme-dependent oxygenation by both dioxygenases and monooxygenases is briefly reviewed by functional and/or metabolic categories, pointing out the structure variety and the analytical approaches.     

Dietary fatty acids modulate adipocyte TNFa production via regulation of its DNA promoter methylation levels

The factors regulating TNF alpha (TNFa) levels could be considered therapeutic targets against metabolic syndrome development. DNA methylation is a potent regulator of gene expression and may be associated with protein levels. In this study we investigate whether the effect of dietary fatty acids on TNFa released from adipocytes might be associated with modifications of the TNFa promoter DNA methylation status. A group of rats was assigned to three diets with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of visceral adipose tissues were taken for adipocyte isolation, in which released TNFa levels were measured, and for methylation and expression studies. In addition, 3 T3-L1 cells were treated with palmitic, oleic and linoleic acids, with and without 5-Azacitydine (5-AZA). After treatments, cells and supernatants were included in the same analyses as rat samples. TNFa promoter methylation levels, gene expression and secretion were different according to the diets and fatty acid treatments associated with them. Cells treated with 5-AZA displayed higher TNFa levels than in the absence of 5-AZA, without differences between fatty acids. According to our results, dietary fatty acid regulation of adipocyte TNFa levels may be mediated by epigenetic modifications of the TNFa promoter DNA methylation levels.     

Properties of discoidal complexes of human apolipoprotein A-I with phosphatidylcholines containing various fatty acid chains

In this study we demonstrate that apolipoprotein A-I determined the common size classes of discoidal particles formed with numerous phosphatidylcholines, and with ether analogs of phosphatidylcholines. We show furthermore, that the nature of the lipids dictates the distribution of particles among the different size classes. These experiments were performed with discoidal complexes containing various phospholipids (phosphatidylcholines with saturated and unsaturated fatty acid chains of different lengths and the ether analog of 1-palmitoyl-2-oleoylphosphatidylcholine), cholesterol, and human apolipoprotein A-I, prepared by the sodium cholate dialysis method, and fractionated by Bio-Gel A-5m gel-filtration chromatography. The complex preparations were analyzed in terms of their average composition, spectral properties of the apolipoprotein, and the dynamic behavior of the lipid domains. Nondenaturing gradient gel electrophoresis was used to analyze the size classes of particles present in the complex preparations. Starting with reaction mixtures containing around 100:1, phospholipid/apolipoprotein A-I molar ratios, complexes were isolated with molar ratios from 40:1 to 100:1. In most complexes apolipoprotein A-I had high levels of alpha-helical structure (65-77% alpha-helix), and tryptophan residues in a nonpolar environment. The lipid domains of complexes exhibited the dynamic behavior expected of the main phospholipid components. In the average size range from 90 to 100 A diameters, discrete particle classes with 80, 87, 102, 108, or 112 A Stokes diameters were observed for all the complexes containing different phospholipids. These discrete, recurring particle sizes are attributed to distinct apolipoprotein A-I conformations and variable lipid content.     

Microbial production of odd-chain fatty acids

Odd-chain fatty acids (OcFAs) and their derivatives have attracted much attention due to their beneficial physiological effects and their potential to be alternatives to advanced fuels. However, cells naturally produce even-chain fatty acids (EcFAs) with negligible OcFAs. In the process of biosynthesis of fatty acids (FAs), the acetyl-CoA serves as the starter unit for EcFAs, and propionyl-CoA works as the starter unit for OcFAs. The lack of sufficient propionyl-CoA, the precursor, is usually regarded as the main restriction for large-scale bioproduction of OcFAs. In recent years, synthetic biology strategies have been used to modify several microorganisms to produce more propionyl-CoA that would enable an efficient biosynthesis of OcFAs. This review discusses several reported and potential metabolic pathways for propionyl-CoA biosynthesis, followed by advances in engineering several cell factories for OcFAs production. Finally, trends and challenges of synthetic biology driven OcFAs production are discussed.     

Hepatic expression of apolipoprotein A-I gene in rats is upregulated by monounsaturated fatty acid diet

The effect of the degree of dietary fat saturation on the hepatic expression of apolipoprotein A-I mRNA was studied in male rats. Animals were maintained for two months on a high fat diet (40% w/w) containing 0.1% cholesterol. Two groups of control animals received either chow diet or chow plus 0.1% cholesterol, while experimental groups received their fat supplement as coconut, corn or olive oil respectively. Dietary cholesterol did not affect apolipoprotein A-I mRNA levels as compared to control animals. Corn oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA than those receiving cholesterol, or coconut oil plus cholesterol. Olive oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA when compared to all other dietary groups. Our data indicate that monounsaturated fatty acids supplied as olive oil play a major role in regulating the hepatic expression of apolipoprotein A-I in male rats.