Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.     

A defect in the activity of Delta6 and Delta5 desaturases may be a factor in the initiation and progression of atherosclerosis

Atherosclerosis is a dynamic process. Dyslipidemia, diabetes mellitus, hypertension, obesity, and shear stress of blood flow, the risk factors for the development of atherosclerosis, are characterized by abnormalities in the metabolism of essential fatty acids (EFAs). Gene expression profiling studies revealed that at the sites of atheroslcerosis-prone regions, endothelial cells showed upregulation of pro-inflammatory genes as well as antioxidant genes, and endothelial cells themselves showed changes in cell shape and proliferation. Uncoupled respiration (UCP-1) precedes atherosclerosis at lesion-prone sites but not at the sites that are resistant to atherosclerosis. UCP-1 expression in aortic smooth muscle cells causes hypertension, enhanced superoxide anion production and decreased the availability of NO, suggesting that inefficient metabolism in blood vessels causes atherosclerosis without affecting cholesterol levels. Thus, mitochondrial dysfunction triggers atherosclerosis. Atherosclerosis-free aortae have abundant concentrations of the EFA-linoleate, whereas fatty streaks (an early stage of atherosclerosis) are deficient in EFAs. EFA deficiency promotes respiratory uncoupling and atherosclerosis. I propose that a defect in the activity of Delta6 and Delta5 desaturases decreases the formation of gamma-linolenic acid (GLA), dihomo-DGLA (DGLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) from dietary linoleic acid (LA) and alpha-linolenic acid (ALA). This, in turn, leads to inadequate formation of prostaglandin E1 (PGE1), prostacyclin (PGI2), PGI3, lipoxins (LXs), resolvins, neuroprotectin D1 (NPD1), NO, and nitrolipids that have anti-inflammatory and platelet anti-aggregatory actions, inhibit leukocyte activation and augment wound healing and resolve inflammation and thus, lead to the initiation and progression atheroslcerosis. In view of this, it is suggested that Delta6 and Delta5 desaturases could serve as biological target(s) for the discovery and development of pharmaceuticals to treat atherosclerosis.     

A defect in the activity of Delta6 and Delta5 desaturases may be a factor predisposing to the development of insulin resistance syndrome

GLUT-4 (glucose transporter) receptor, tumor necrosis factor-alpha (TNF-alpha), interleukins-6 (IL-6), daf-genes and PPARs (peroxisomal proliferation activator receptors) play a role in the development of insulin resistance syndrome and associated conditions. But, the exact interaction between these molecules/factors and the mechanism(s) by which they produce insulin resistance syndrome is not clear. I propose that a defect in the activity of the enzymes Delta6 and Delta5 desaturases that are essential for the formation of long chain metabolites of essential fatty acids, linoleic acid and alpha-linolenic acid, is a factor in the development of insulin resistance syndrome. Long chain polyunsaturated fatty acids (LCPUFAs) increase cell membrane fluidity and enhance the number of insulin receptors and the affinity of insulin to its receptors; suppress TNF-alpha, IL-6, macrophage migration inhibitory factor (MIF) and leptin synthesis; increase the number of GLUT-4 receptors, serve as endogenous ligands of PPARs, modify lipolysis, and regulate the balance between pro- and anti-oxidants, and thus, play a critical role in the pathogenesis of insulin resistance. In the nematode, Caenorhabditis elegans, the protein encoded by daf-2 is 35% identical to the human insulin receptor; daf-7 codes a transforming growth factor-beta (TGF-beta) type signal and daf-16 enhances superoxide dismutase (SOD) expression. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Calorie restriction enhances the activity of Delta6 and Delta5 desaturases, melatonin production, decreases daf-2 signaling, free radical generation, and augments anti-oxidant defenses that may explain the beneficial effect of diet control in the management of obesity, insulin resistance, and type II diabetes mellitus. These evidences suggest that the activities of Delta6 and Delta5 enzymes play a critical role in the expression and regulation of GLUT-4, TNF-alpha, IL-6, MIF, daf-genes, melatonin, and leptin by modulating the synthesis and tissue concentrations of LCPUFAs. Caloric restriction delays ageing by activating Sir 2 deacetylase in yeast, and expression of Sir 2 (SIRT1) in human cells. Both insulin and insulin-like growth factor-1 (IGF-1) attenuated this response. SIRT1 sequesters the proapoptotic factor Bax, prevents stress-induced apoptosis of cells, and thus, prolongs survival. In addition, SIRT1 repressed PPAR-gamma, and overexpression of SIRT1 attenuated adipogenesis, and upregulation of SIRT in differentiated fat cells triggered lipolysis and loss of fat, events that are known to attenuate insulin resistance and prolong life span. It remains to be seen whether LCPUFAs have a regulatory role in SIRT1 expression and control Sir 2 deacetylase activity. Thus, calorie restriction or reduced food intake has a role not only in the pathobiology of insulin resistance, but also in other associated conditions such as obesity, type II diabetes mellitus, ageing, and longevity.     

Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate

Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet beta-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet beta-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 mum) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary beta-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca(2+)] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A(2) (iPLA(2)beta) hydrolyzes beta-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA(2)beta prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in beta-cells from iPLA(2)beta(-/-) mice. Stably transfected INS-1 cells that overexpress iPLA(2)beta hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet beta-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca(2+) entry and insulin secretion.     

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E β-cells alongside altered metabolism-secretion coupling

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1–0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.     

Cloning and functional expression of the first plant fatty acid elongase specific for Delta(6)-polyunsaturated fatty acids

In order to elucidate the biosynthesis of long-chain polyunsaturated fatty acids (PUFAs) in plants we searched for a cDNA encoding a Delta(6)-specific PUFA elongase from Physcomitrella patens, which is known to contain high proportions of arachidonic acid (20:4 Delta(5,8,11,14)). An EST clone from P. patens was identified by its low homology to the yeast gene ELO1, which is required for the elongation of medium-chain fatty acids. We functionally characterized this cDNA by heterologous expression in Saccharomyces cerevisiae grown in the presence of several fatty acids. Analysis of the fatty acid profile of the transgenic yeast revealed that the cDNA encodes a protein that leads to the elongation of the C(18) Delta(6)-polyunsaturated fatty acids gamma-linolenic acid (18:3 Delta(6,9,12)) and stearidonic acid (18:4 Delta(6,9,12,15)), which were recovered to 45-51% as their elongation products. In contrast, linoleic and alpha-linolenic acids were hardly elongated and we could not measure any elongation of saturated and mono-unsaturated fatty acids (including 18:1 Delta(6)), indicating that the elongase is highly specific for the polyunsaturated nature of the fatty acid acting as substrate.     

Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a Delta 6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (Delta 5 and Delta 6) occur predominantly at the sn-2 position of phosphatidylcholine when using Delta 5- and Delta 6-desaturases from lower plants, fungi, worms, and algae. The specificity of these Delta 6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of gamma-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human Delta 6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that Delta 12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Liver triacylglycerols and free fatty acids in streptozotocin-induced diabetic rats have atypical n-6 and n-3 pattern

In diabetes there is a decrease in membrane arachidonic (AA) and docosahexaenoic (DHA) acids and a concomitant increase in linoleic (LA) and alpha-linolenic (ALA) acids. This metabolic perturbation is thought to be due to impaired activity of Delta(6)- and Delta(5)-desaturases. Triacylglycerols are the major lipid pool in plasma and liver tissue and have a significant influence on fatty acid composition of membrane and circulating phospholipids. Data on the distribution of n-6 and n-3 polyunsaturated fatty acids of triacylglycerols in diabetes are sparse. We investigated whether streptozotocin-induced diabetes in Sprague-Dawley rats alters fatty acid composition of triacylglycerols and free fatty acids of liver tissue. The animals were fed a breeding diet prior to mating, during pregnancy and lactation. On days 1-2 of pregnancy, diabetes was induced in 10 of the 25 rats. Liver was obtained at post partum day 16 for analysis. Relative levels of LA (P=0.03), dihomo-gamma-linolenic acid (DHGLA) (P=0.02), AA (P=0.049), total n-6 (P=0.02), ALA (P=0.013), eicosapentaenoic acid (EPA) (P=0.004), docosapentaenoic acid (22:5n-3, DPA) (P=0.013), DHA (P=0.033), n-3 metabolites (P=0.015) and total n-3 (P=0.011) were significantly higher in the triacylglycerols of the diabetics compared with the controls. Similarly, liver free fatty acids of the diabetics had higher levels of LA (P=0.0001), DHGLA (P=0.001), AA (P=0.001), n-6 metabolites (P=0.002), total n-6 (P=0.0001), ALA (P=0.003), EPA (P=0.015), docosapentaenoic (22:5n-3, P=0.003), DHA (P=0.002), n-3 metabolites (P=0.005) and total n-3 (P=0.001). We conclude that impaired activity of desaturases and/or long chain acyl-CoA synthetase could not explain the higher levels of AA, DHA and n-6 and n-3 metabolites in the diabetics. This seems to be consistent with an alteration in the regulatory mechanism, which directs incorporation of polyunsaturated fatty acids either into triacylglycerols or phospholipids.     

Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.     

Effects of stable suppression of Group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells

Studies involving pharmacologic inhibition or transient reduction of Group VIA phospholipase A2 (iPLA2beta) expression have suggested that it is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels, rates of arachidonate incorporation into phospholipids, and degradation of excess phosphatidylcholine (PC). In insulin-secreting islet beta-cells and some other cells, in contrast, iPLA2beta signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 beta-cell lines in which iPLA2beta expression is stably suppressed by small interfering RNA. Two such iPLA2beta knockdown (iPLA2beta-KD) cell lines express less than 20% of the iPLA2beta of control INS-1 cell lines. The iPLA2beta-KD INS-1 cells exhibit impaired insulin secretory responses and reduced proliferation rates. Electrospray ionization mass spectrometric analyses of PC and LPC species that accumulate in INS-1 cells cultured with arachidonic acid suggest that 18:0/20:4-glycerophosphocholine (GPC) synthesis involves sn-2 remodeling to yield 16:0/20:4-GPC and then sn-1 remodeling via a 1-lyso/20:4-GPC intermediate. Electrospray ionization mass spectrometric analyses also indicate that the PC and LPC content and composition of iPLA2beta-KD and control INS-1 cells are nearly identical, as are the rates of arachidonate incorporation into PC and the composition and remodeling of other phospholipid classes. These findings indicate that iPLA2beta plays signaling or effector roles in beta-cell secretion and proliferation but that stable suppression of its expression does not affect beta-cell GPC lipid content or composition even under conditions in which LPC is being actively consumed by conversion to PC. This calls into question the generality of proposed housekeeping functions for iPLA2beta in PC homeostasis and remodeling.     

A novel Delta12-fatty acid desaturase gene from methylotrophic yeast Pichia pastoris GS115

The methylotrophic yeast Pichia pastoris GS115, a widely used strain in production of various heterologous proteins, especially membrane-bound enzymes, can also produce linoleic and linolenic acids, which indicates the existence of membrane-bound Delta12 and Delta15-fatty acid desaturases. This paper describes the cloning and functional characterization of a novel Delta12-fatty acid desaturase gene from this methylotrophic yeast. The open reading frame of the gene (named Pp-FAD12) is 1263 bp in size and encodes a 420-amino-acid peptide. The deduced Pp-FAD12 protein shows high identity (50-67%) with Delta12-fatty acid desaturases from other fungi. It also shows a high identity (57%) with Delta15-fatty acid desaturase (named Sk-FAD15) from Saccharomyces kluyveri. Expression of Pp-FAD12 in polyunsaturated fatty acids non-producing yeast Saccharomyces cerevisiae demonstrated that its product converted oleic acid (18 : 1) to linoleic acid (18 : 2). This result suggests that Pp-FAD12 encodes a novel Delta12-fatty acid desaturase in P. pastoris GS115. This is the first report about the cloning and functional characterization of Delta12-fatty acid desaturase gene in methylotrophic yeast.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Cellular distribution and contribution of cyclooxygenase COX-2 to diabetogenesis in NOD mouse

Unlike most other mammalian cells, beta-cells of Langerhans constitutively express cyclooxygenase (COX)-2 rather than COX-1. COX-2 is also constitutively expressed in type 1 diabetes (T1D) patients' periphery blood monocytes and macrophage. To understand the role of COX-2 in the beta-cell, we investigated COX-2 expression in beta-cells and islet infiltrates of NOD and BALB/c mice using fluorescence immunohistochemistry and cytochemical confocal microscopy and Western blotting. Immunostaining showed that COX-2 is expressed in islet-infiltrating macrophages, and that the expression of insulin and COX-2 disappeared concomitantly from the beta-cells when NOD mice progressed toward overt diabetes. Also cultured INS-1E cells coexpressed insulin and COX-2 but clearly in different subcellular compartments. Treatment with celecoxib increased insulin release from these cells in a dose-dependent manner in glucose concentrations ranging from 5 to 17 mM. Excessive COX-2 expression by the islet-infiltrating macrophages may contribute to the beta-cell death during insulitis. The effects of celecoxib on INS-1E cells suggest that PGE(2) and other downstream products of COX-2 may contribute to the regulation of insulin release from the beta-cells.     

Hormonal modulation of delta6 and delta5 desaturases: case of diabetes

Animal biosynthesis of high polyunsaturated fatty acids from linoleic, alpha-linolenic and oleic acids is mainly modulated by the delta6 and delta5 desaturases through dietary and hormonal stimulated mechanisms. From hormones, only insulin activates both enzymes. In experimental diabetes mellitus type-1, the depressed delta6 desaturase is restored by insulin stimulation of the gene expression of its mRNA. However, cAMP or cycloheximide injection prevents this effect. The depression of delta6 and delta5 desaturases in diabetes is rapidly correlated by lower contents of arachidonic acid and higher contents of linoleic in almost all the tissues except brain. However, docosahexaenoic n-3 acid enhancement, mainly in liver phospholipids, is not explained yet. In experimental non-insulin dependent diabetes, the effect upon the delta6 and delta5 desaturases is not clear. From all other hormones glucagon, adrenaline, glucocorticoids, mineralocorticoids, oestriol, oestradiol, testosterone and ACTH depress both desaturases, and a few hormones: progesterone, cortexolone and pregnanediol are inactive.     

Fatty acid desaturation and the regulation of adiposity in Caenorhabditis elegans

Monounsaturated fatty acids are essential components of membrane and storage lipids. Their synthesis depends on the conversion of saturated fatty acids to unsaturated fatty acids by Delta9 desaturases. Caenorhabditis elegans has three Delta9 desaturases encoded by the genes fat-5, fat-6, and fat-7. We generated nematodes that display a range of altered fatty acid compositions by constructing double-mutant strains that combine mutations in fat-5, fat-6, and fat-7. All three double-mutant combinations have reduced survival at low temperatures. The fat-5;fat-6 double mutants display relatively subtle fatty acid composition alterations under standard conditions, but extreme fatty acid composition changes and reduced survival in the absence of food. The strain with the most severe defect in the production of unsaturated fatty acids, fat-6;fat-7, exhibits slow growth and reduced fertility. Strikingly, the fat-6;fat-7 double-mutant animals have decreased fat stores and increased expression of genes involved in fatty acid oxidation. We conclude that the Delta9 desaturases, in addition to synthesizing unsaturated fatty acids for properly functioning membranes, play key roles in lipid partitioning and in the regulation of fat storage.     

Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia.

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.     

Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.

Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.