Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.     

Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite''s carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.     

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.     

p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene

p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73–ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.     

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A₁ or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.     

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD

1. Download high-res image (315KB)
2. Download full-size image

     

Mefluidide对玉米叶片线粒体膜脂组成的影响及其与抗冷力的关系

Mcfluidide叶面喷洒处理玉米幼苗,常温下可提高叶片线粒体膜脂中磷脂酰胆碱含量和脂肪酸中亚油酸含量,降低棕榈酸含量。当处理和未处理幼苗在5℃低温下生长6d期间,两者的磷脂酰胆碱含量均随低温时间的延长而递减,但处理苗的含量一直高于对照苗的含量。对照苗的亚油酸含量亦随低温时间的延长而递减,处理苗的亚油酸含量则在6d低温期间基本保持稳定,且一直高于对照。对照苗的棕榈酸含量随低温时间延长而递增,处理苗表现为短期低温棕榈酸含量降低,长期低温含量增加,但仍较对照为低,因而处理苗的脂肪酸不饱和度和亚油酸/棕榈酸比值始终高于对照苗。     

Lipid Class and Fatty Acid Composition of the Protozoan Parasite of Oysters, Perkinsus marinus Cultivated in Two Different Media

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20: (n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2delta7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.     

Features of the Uncoupling Effect of Fatty Acids in Liver Mitochondria of Mammals with Different Body Weight

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.     

Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.     

Phosphatidylethanolamine Synthesis in the Parasite Mitochondrion Is Required for Efficient Growth but Dispensable for Survival of Toxoplasma gondii

Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (∼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.     

Astragaloside IV ameliorates fat metabolism in the liver of ageing mice through targeting mitochondrial activity

Astragaloside IV (AST) is a major bioactive compound of Radix Astragali with medical and health benefits. Previous studies have found that AST can reduce the body weights of high-fat diet fed mice. However, the effect of AST on fat metabolism of ageing mice is unclear. In this study, naturally ageing mice were administered intragastrically with AST at 30 mg/kg/day (ageing + AST-L group) and 90 mg/kg/day (ageing + AST-H group) for 16–20 months. Adult (4 months old) and ageing mice were given 1% sodium carboxyl methylcellulose as vehicle. Energy metabolism-related biological parameters of living mice were examined. Moreover, mRNA and protein levels of key enzymes/proteins involved in triglyceride (TG) lipolysis, fatty acid β-oxidation (FAO), ketone body (KB) production and mitochondrial respiratory chain were also examined after sacrifice. Results demonstrated that treatment with AST significantly reduced body weight, white fat and liver/body weight ratio of ageing mice, significantly reduced serum/hepatic TG levels, respiratory quotient, promoted fatty acid mobilization in white adipose tissue, mitochondrial FAO and KB production and mitochondrial biosynthesis/functions in the liver of ageing mice. AST also up-regulated the expression of phosphorylated AMP-activated protein kinase, acetyl-CoA carboxylase, acetyl-coenzyme A synthetase, carnitine palmitoyltransferase 1a/1b, enoyl coenzyme A hydratase-short chain, acyl-CoA dehydrogenase medium chain and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase-2 involved in fat metabolism. These results indicated that mitochondrial activity could be the target of AST to treat abnormal fat metabolism during ageing.     

Evolution of Plant-Like Crystalline Storage Polysaccharide in the Protozoan Parasite Toxoplasma gondii Argues for a Red Alga Ancestry

Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates. Toxoplasma gondii displays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of the Toxoplasma gondii, green alga Chlamydomonas reinhardtii, and dinoflagellate Crypthecodinium cohnii storage polysaccharides. We show Toxoplasma gondii amylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that the Toxoplasma gondii amylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red alga Cyanidioschyzon merolae and, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan–water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes.This article contains online-only supplementary material.Reviewing Editor:Dr. Patrick Keeling     

Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii

In the process of autophagy, the Atg8 protein is conjugated, through a ubiquitin-like system, to the lipid phosphatidylethanolamine (PE) to associate with the membrane of forming autophagosomes. There, it plays a crucial role in the genesis of these organelles and in autophagy in general. In most eukaryotes, the cysteine peptidase Atg4 processes the C terminus of cytosolic Atg8 to regulate its association with autophagosomal membranes and also delipidates Atg8 to release this protein from membranes. The parasitic protist Toxoplasma gondii contains a functional, yet apparently reduced, autophagic machinery. T. gondii Atg8 homolog, in addition to a cytosolic and occasionally autophagosomal localization, also localizes to the apicoplast, a nonphotosynthetic plastid bounded by four membranes. Our attempts to interfere with TgATG8 function showed that it appears to be essential for parasite multiplication inside its host cell. This protein also displays a peculiar C terminus that does not seem to necessitate processing prior to membrane association and yet an unusually large Toxoplasma homolog of ATG4 is predicted in the parasite genome. A TgATG4 conditional expression mutant that we have generated is severely affected in growth, and displays significant alterations at the organellar level, noticeably with a fragmentation of the mitochondrial network and a loss of the apicoplast. TgATG4-depleted parasites appear to be defective in the recycling of membrane-bound TgATG8. Overall, our data highlight a role for the TgATG8 conjugation pathway in maintaining the homeostasis of the parasite’s organelles and suggest that Toxoplasma has evolved a specialized autophagic machinery with original regulation.     

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons'' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.     

高果糖引起的脂肪肝大鼠肾脏脂质合成相关基因和蛋白的表达

大量研究表明,高果糖可引起脂肪肝,但对肾脏脂质代谢的影响尚不清楚。该实验研究给予10％果糖水5周后诱导的脂肪肝大鼠肾脏的脂质代谢情况,并探讨其可能机制。将16只雄性SD大鼠随机分为正常组（con）和果糖组（fru）,果糖组给予10％（W／V）果糖水,第5N末称体重、取血、处死,检测血浆GLU、TG、TC和INSULIN含量。取肾脏、肝脏和白色脂肪称重,采用形态学方法观察肝脏和肾脏脂质沉积情况,酶法测其TG、TC含量,以Real time—PCR检测肾脏、肝脏中脂质合成和脂质氧化相关基因水平,以Westemblot检测肾、肝细胞核脂质合成转录因子的蛋白表达。结果显示,果糖组大鼠血浆TG、INSULIN明显升高,并出现肥胖体征,肝脏脂质沉积严重,其调控脂质合成的两个关键的转录因子ChREBP和SREBPlcmRNA和核蛋白表达都明显升高,并且它们靶向的脂质合成相关酶FAS、ACCl、SCDlmRNA表达也显著增加。但是,在肾脏中,高果糖没有引起TG含量的变化,调控脂质重新合成的基因和蛋白的表达也未发生变化。因此,与果糖致脂肪肝不同,高果糖饮食并没有造成肾脏的脂质沉积和脂质合成相关基因、蛋白的变化。     

The NTPase activity of the double FYVE domain–containing protein 1 regulates lipid droplet metabolism

Lipid droplets (LDs) are transient lipid storage organelles that can be readily tapped to resupply cells with energy or lipid building blocks and therefore play a central role in cellular metabolism. However, the molecular factors and underlying mechanisms that regulate the growth and degradation of LDs are poorly understood. It has emerged that proteins that establish contacts between LDs and the endoplasmic reticulum play a critical role in regulating LD metabolism. Recently, the autophagy-related protein, double FYVE domain–containing protein 1 (DFCP1/ZFYVE1) was shown to reside at the interface of the endoplasmic reticulum and LDs, however, little is known about the involvement of DFCP1 in autophagy and LD metabolism. Here, we show that DFCP1 is a novel NTPase that regulates free fatty acid metabolism. Specifically, we show that DFPC1-knockdown, particularly during starvation, increases cellular free fatty acids and decreases the levels of cellular TAGs, resulting in accumulated small LDs. Using selective truncations, we demonstrate that DFCP1 accumulation on LDs in cells and in vitro is regulated by a previously unknown NTPase domain. Using spectroscopic approaches, we show that this NTPase domain can dimerize and can hydrolyze both ATP and GTP. Furthermore, mutations in DFCP1 that either impact nucleotide hydrolysis or dimerization result in changes in the accumulation of DFCP1 on LDs, changes in LD density and size, and colocalization of LDs to autophagosomes. Collectively, our findings suggest that DFCP1 is an NTPase that modulates the metabolism of LDs in cells.     

Saccharomyces cerevisiae和Yarrowia lipolytica对自由饱和脂肪酸的选择性吸收及胞内积累特性研究

利用产油微生物生产特殊功能、高附加值的脂肪酸,具有良好的开发利用前景。以酿酒酵母(S.cerevisiae)和解脂耶氏酵母(Y.lipolytica)为出发菌株,以链长C4-C18的单一自由饱和脂肪酸作为唯一碳源,探究了两类酵母吸收利用、积累脂肪酸情况及胞内脂肪酸组成情况。结果表明:当碳链长C≤10时不能被利用,而且抑制细胞的生长,特别是当碳链长C≤8时,细胞很快被杀死;当碳链长C=11时,对细胞的生长有一定的抑制作用,菌体长势缓慢;碳链长C≥12时,对细胞生长没有影响;脂肪酸利用速度,偶数C脂肪酸奇数C脂肪酸;Nile red全细胞脂类染色显示,S.cerevisiae胞内脂质主要集中于胞内周边膜部位,Y.lipolytica主要以脂质体形式存在胞内,及少部分在胞内周边膜部位;GC/Mass脂类成分分析表明,菌株S.cerevisiae S228C BY4741-pox1和S.cerevisiae S228C BY4741-pox1,3可以积累培养基添加的相应脂肪酸,而其他供试菌积累的脂肪酸链长C≥16,没有检测到培养基含有相应的脂肪酸。这些结果为利用酵母生产特殊功能脂肪酸,及开发特色高附加值油脂提供了有意义的参考。     

Fatty Acids Induce Chloride Permeation in Rat Liver Mitochondria by Activation of the Inner Membrane Anion Channel (IMAC)

The inner membrane of freshly isolated mammalian mitochondria is poorly permeable to Cl(-). Low, nonlytic concentrations (< or =30 microM) of long-chain fatty acids or their branched-chain derivatives increase permeation of Cl(-) as indicated from rapid large-scale swelling of mitochondria suspended in slightly alkaline KCl medium (supplemented with valinomycin). Myristic, palmitic, or 5-doxylstearic acid are powerful inducers of Cl(-) permeation, whereas lauric, phytanic, stearic, or 16-doxylstearic acid stimulate Cl(-) permeation in a lesser extent. Fatty acid-induced Cl(-) permeation across the inner membrane correlates well with the property of nonesterified fatty acids to release endogenous Mg(2+) from mitochondria. Myristic acid stimulates anion permeation in a selective manner, similar as was described for A23187, an activator of the inner membrane anion channel (IMAC). Myristic acid-induced Cl(-) permeation is blocked by low concentrations of tributyltin chloride (IC(50) approximately 1.5 nmol/mg protein). Moreover, myristic acid activates a transmembrane ion current in patch-clamped mitoplasts (mitochondria with the outer membrane removed) exposed to alkaline KCl medium. This current is best ascribed to the opening of an ion channel with a single-channel conductance of 108 pS. We propose that long-chain fatty acids can activate IMAC by withdrawal of Mg(2+) from intrinsic binding sites.