Mechanisms underlying skeletal muscle insulin resistance induced by fatty acids: importance of the mitochondrial function

Insulin resistance condition is associated to the development of several syndromes, such as obesity, type 2 diabetes mellitus and metabolic syndrome. Although the factors linking insulin resistance to these syndromes are not precisely defined yet, evidence suggests that the elevated plasma free fatty acid (FFA) level plays an important role in the development of skeletal muscle insulin resistance. Accordantly, in vivo and in vitro exposure of skeletal muscle and myocytes to physiological concentrations of saturated fatty acids is associated with insulin resistance condition. Several mechanisms have been postulated to account for fatty acids-induced muscle insulin resistance, including Randle cycle, oxidative stress, inflammation and mitochondrial dysfunction. Here we reviewed experimental evidence supporting the involvement of each of these propositions in the development of skeletal muscle insulin resistance induced by saturated fatty acids and propose an integrative model placing mitochondrial dysfunction as an important and common factor to the other mechanisms.

Palmitoleic acid (n-7) increases white adipocyte lipolysis and lipase content in a PPARα-dependent manner

We investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from wild-type or PPARα-deficient mice treated with 16:1n7 (300 mg•kg(-1)•day(-1)) or oleic acid (18:1n9, 300 mg•kg(-1)•day(-1)) by gavage for 10 days were evaluated for lipolysis, TAG, and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) and protein content of ATGL and pSer(660)-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild-type but not PPARα-deficient mice with 16:1n7 increased primary adipocyte basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild-type and PPARα-deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARα

N-3 fatty acids and cardiovascular prevention

In this study we elucidate the role of polyunsaturated fatty acids (PUFAs) in the prevention of cardiovascular diseases, focusing the attention on their role in the modulation of acyl composition of cell lipids and of gene expression. Regarding this latter mechanism, the effectiveness of PUFAs as activators of two transcriptional factors, SREBPs and PPARs, have been considered. Two different model system have been used: primary cultures of neonatal rat cardiomyocytes and an human hepatoma cell line (HepG2). Cells have been supplemented with different PUFAs at physiological concentration, and special attention has been devoted to the main n-3 PUFAs, EPA and DHA. PUFAs influence on global gene expression in cardiomyocytes has been evaluated using microarray technique. Furthermore, since it is not fully elucidated which transcription factors are involved in this modulation in the heart, expression and activation of the three different PPAR isoforms have been investigated. Hepatocytes have been used as experimental model system in the evaluation of PUFAs effect on SREBP activity. SREBPs are considered the main regulator of cholesterol and triglyceride synthesis, which occur mainly in the liver. In both experimental models the modification of cell lipid fatty acid composition subsequent to PUFAs supplementation has been evaluated, and related to the effects observed at molecular level. The global vision given by the obtained results may be important for addressing new researches and be useful to educators and policy makers in setting recommendations for reaching optimal health through good nutrition.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

A key role for lipoic acid synthesis during Plasmodium liver stage development.

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

The maize lipoxygenase, ZmLOX10 , mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, majorregulatory genes and the signals that modulate these defense metabolites are vastly understudied, especiallyin important agro-economic monocot species. Here we show that products and signals derived from a singleZea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbiv-ory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substratefor the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the spe-cialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indi-cating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression ofJA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling isrequired for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to par-tially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produceGLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness toparasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic linkto the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compro-mised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence thatLOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene toagro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Quantitative analysis of arachidonic acid, endocannabinoids, N-acylethanolamines and steroids in biological samples by LCMS/MS: Fit to purpose.

Arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid; C20:4) (arachidonate, AA) is a vital polyunsaturated omega-6 fatty acid (PUFA) without its presence the mammalian brain, muscles, and possibly other organs cannot develop or function [1] and [2]. AA fulfils numerous known and possibly yet unknown functions as integral part of mammalian phospholipid membranes and as free AA which also acts as a precursor of a variety of biologically active lipid mediators generally referred to as eicosanoids (e.g., prostaglandins, leukotrienes). A more recent class of eicosanoids is composed of the endogenous cannabinoids (endocannabinoids) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA), which act on cannabinoid CB1 and CB2 receptors but also modulate ion channels and nuclear receptors [3] and [4]. In recent years, the role of endocannabinoids as prominent anti-inflammatory and neuromodulatory eicosanoids has been shown by numerous studies [5].

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

n-fatty acids of shale partings in the Franciscan bedded cherts

Normal saturated fatty acid (n-fatty acid) in marine sediments from coastal and pelagic environments were analyzed. The coastal sediments contain both short-chained n-fatty acids with carbon numbers from 12 to 18 and long-chained acids from 22 to 32, whereas the pelagic sediments contain predominantly short-chained acids. The relative abundance of short-chained to long-chained n-fatty acids, expressed by the molar ratio C16/C26, can be an indicator to assess the depositional environment of sedimentary rocks. The ratio of long-chained n-fatty acids (C22-C32) to the total n-fatty acids also has the potential to discriminate sedimentary environments. The indicators based on the n-fatty acids were applied to the Franciscan bedded cherts. The result shows that the bedded cherts had deposited in continuous environments from the pelagic to the coastal. This is in harmony with the same inference based on major, trace and rare earth elements and normal paraffins.

Fatty acids and intact polar lipids of laminated microbial sediments from St. Peter Ording, German Wadden Sea

Hidden for the untrained eye through a thin layer of sand, laminated microbial sediments occur in supratidal beaches along the North Sea coast. The inhabiting microbial communities organize themselves in response to vertical gradients of light, oxygen or sulfur compounds. We performed a fine-scale investigation on the vertical zonation of the microbial communities using a lipid biomarker approach, and assessed the biogeochemical processes using a combination of microsensor measurements and a 13C-labeling experiment. Lipid biomarker fingerprinting showed the overarching importance of cyanobacteria and diatoms in these systems, and heterocyst glycolipids revealed the presence of diazotrophic cyanobacteria even in 9 to 20 mm depth. High abundance of ornithine lipids (OL) throughout the system may derive from sulfate reducing bacteria, while a characteristic OL profile between 5 and 8 mm may indicate presence of purple non-sulfur bacteria. The fate of 13C-labeled bicarbonate was followed by experimentally investigating the uptake into microbial lipids, revealing an overarching importance of cyanobacteria for carbon fixation. However, in deeper layers, uptake into purple sulfur bacteria was evident, and a close microbial coupling could be shown by uptake of label into lipids of sulfate reducing bacteria in the deepest layer. Microsensor measurements in sediment cores collected at a later time point revealed the same general pattern as the biomarker analysis and the labeling experiments. Oxygen and pH-microsensor profiles showed active photosynthesis in the top layer. The sulfide that diffuses from deeper down and decreases just below the layer of active oxygenic photosynthesis indicates the presence of sulfur bacteria, like anoxygenic phototrophs that use sulfide instead of water for photosynthesis.

Carbon isotopic compositions and concentrations of major fatty acids in the Beggiatoa covered sediment core at the crest of southern Hydrate Ridge

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as -103 per mill. Specific fatty acids released from bacterial membranes include C 16:1 omega 5c , C 17:1 omega 6c , and cyC 17:0 omega 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest d13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 omega 5c and cyC 17:0 omega 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.

Fatty acids, alcohols and hydrocarbons of microbial mats from Lake 2A on Kiritimati Island

Modern microbial mats are widely recognized as useful analogs for the study of biogeochemical processes relevant to paleoenvironmental reconstruction in the Precambrian. We combined microscopic observations and investigations of biomarker composition to investigate community structure and function in the upper layers of a thick phototrophic microbial mat system from a hypersaline lake on Kiritimati (Christmas Island) in the Northern Line Islands, Republic of Kiribati. In particular, an exploratory incubation experiment with 13C-labeled bicarbonate was conducted to pinpoint biomarkers from organisms actively fixing carbon. A high relative abundance of the cyanobacterial taxa Aphanocapsa and Aphanothece was revealed by microscopic observation, and cyanobacterial fatty acids and hydrocarbons showed 13C-uptake in the labeling experiment. Microscopic observations also revealed purple sulfur bacteria (PSB) in the deeper layers. A cyclic C19:0 fatty acid and farnesol were attributed to this group that was also actively fixing carbon. Background isotopic values indicate Calvin-Benson cycle-based autotrophy for cycC19:0 and farnesol-producing PSBs. Biomarkers from sulfate-reducing bacteria (SRB) in the top layer of the mat and their 13C-uptake patterns indicated a close coupling between SRBs and cyanobacteria. Archaeol, possibly from methanogens, was detected in all layers and was especially abundant near the surface where it contained substantial amounts of 13C-label. Intact glycosidic tetraether lipids detected in the deepest layer indicated other archaea. Large amounts of ornithine and betaine bearing intact polar lipids could be an indicator of a phosphate-limited ecosystem, where organisms that are able to substitute these for phospholipids may have a competitive advantage.

Water chemistry, and isotopic ratios and fatty acids and alcohols in Calanus finmarchicus CV, norther Mid-Atlantic Ridge

Fatty acid and alcohol profiles and stable nitrogen and carbon isotope values, d15N and d13C, of Calanus finmarchicus CV were studied in June 2004 to estimate their trophic status along the northern Mid-Atlantic Ridge i.e. the Reykjanes Ridge (RR), extending from Iceland in the north to the productive region of the Sub-Polar Front (SPF) in the south. Two main groups of stations were defined in the study area based on fatty acid (FA) and fatty alcohol compositions, the stations in the RR area constituted one group and the stations in the frontal area constituted another. The sum of relative amounts of the dietary FAs was significantly higher in the RR area than in the frontal area. Conversely, the long-chained FAs, 20:1 and 22:1, were found in significantly lower relative amounts in the RR area than in the frontal area, thus indicating later ascent of the animals in the frontal area. Further support of this is provided by the fatty alcohols ratio 20:1/22:1 which differed significantly between the two areas. The d15N values were significantly higher in the frontal area compared to the RR area indicating higher trophic position and/or different pelagic-POM baseline in these areas.