Associations Between rs9939609 FTO Polymorphism With Nutrient and Food Intake and Adherence to Dietary Patterns in an Urban Argentinian Population.

BACKGROUND: The A allele of rs9939609 polymorphism at the FTO gene has been consistently associated with higher body mass index in different populations, but conflicting results have been found regarding its contribution to food intake variability. OBJECTIVE: This study aimed to investigate the association between this genetic variant and nutrient and food intake in an urban Argentinian population. DESIGN: A cross-sectional, analytic investigation was performed between October 2018 and February 2020. PARTICIPANTS/SETTINGS: Adults of both sexes residing in La Plata, Argentina, were recruited through social networks (Instagram and Facebook). Of 179 eligible adults, a total of 173 adults were included in the final analyses. OUTCOME MEASURES: Nutrient and food group intake data were obtained by an interview-administered food frequency questionnaire. Height and weight were measured, and genotypes were obtained by real-time polymerase chain reaction. STATISTICAL ANALYSES: The per-allele effect on nutrient and food group intake was assessed by general linear models, adjusting for age, sex, educational level, total energy intake, and body mass index. Dietary patterns were derived by principal component analysis. The association of the A allele with adherence to each dietary pattern was also evaluated by the general linear model. RESULTS: The frequency of the risk allele was 27%. A-carriers showed a higher total fat (1.88 [0.55, 3.21] % of total energy intake), saturated fatty acids (0.82 [0.25-1.39] % of total energy intake), and monounsaturated fatty acids (0.66 [0.08, 1.24] % of total energy intake), and a lower carbohydrate (-1.99 [-3.48, -0.50] % of total energy intake) intake than TT homozygous. A-carriers also reported a higher "milk and yogurt" (1.08 [0.24, 1.91] % of total energy intake), "animal fats" (1.09 [0.14-2.03] % of total energy intake), and fat-rich ultraprocessed foods (2.10 [0.52, 3.67] % of total energy intake) intake in comparison with TT homozygous. Furthermore, A-carriers showed higher adherence to the Western dietary pattern. CONCLUSION: The A allele contributed to nutrient and food intake variability in the studied population and was associated with the consumption of saturated fatty acids-enriched foods.

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases -3 and -4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition.

BACKGROUND AND AIMS: The transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). METHODS: We used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). RESULTS: We showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4-/- BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. CONCLUSIONS: Our results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages.

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.

Small non-coding RNA landscape is modified by GPAT2 silencing in MDA-MB-231 cells.

Glycerol-3-phosphate acyltransferase-2 is a member of "cancer-testis gene" family. Initially linked to lipid metabolism, this gene has been recently found involved also in PIWI-interacting RNAs biogenesis in germline stem cells. To investigate its role in piRNA metabolism in cancer, the gene was silenced in MDA-MB-231 breast cancer cells and small RNA sequencing was applied. PIWI-interacting RNAs and tRNA-derived fragments expression profiles showed changes following GPAT2 silencing. Interestingly, a marked shift in length distribution for both small RNAs was detected in GPAT2-silenced cells. Most downregulated PIWI-interacting RNAs are single copy in the genome, intragenic, hosted in snoRNAs and previously found to be upregulated in cancer cells. Putative targets of these PIWI-interacting RNAs are linked to lipid metabolism. Downregulated tRNA derived fragments derived from, so-called 'differentiation tRNAs', whereas upregulated ones derived from proliferation-linked tRNAs. miRNA amounts decrease after Glycerol-3-phosphate acyltransferase-2 silencing and functional enrichment analysis of deregulated miRNA putative targets point to mitochondrial biogenesis, IGF1R signaling and oxidative metabolism of lipids and lipoproteins. In addition, miRNAs known to be overexpressed in breast cancer tumors with poor prognosis where found downregulated in GPAT2-silenced cells. In conclusion, GPAT2 silencing quantitatively and qualitatively affects the population of PIWI-interacting RNAs, tRNA derived fragments and miRNAs which, in combination, result in a more differentiated cancer cell phenotype.

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis.

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.

Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis.

Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.

Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the breast cancer MDA-MB-231 cell line.

The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2' deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.

Rol de la isoforma 2 de la glicerol-3-fosfatoaciltransferasa en el metabolismo lipídico testicular / Role of isoform 2 of glycerol-3-phosphate acyltransferase in testicular lipid metabolism / FunþÒo da isoforma 2 da glicerol-3-fosfatoaciltransferase no metabolismo lipídico testicular

El primer paso en la síntesis de novo de glicerolípidos es la acilación del glicerol-3-fosfato, la cual es catalizada por la enzima glicero-3-fosfato aciltransferasa (GPAT). En mamíferos han sido descriptas hasta el momento cuatro isoformas de GPAT, las cuales pueden diferenciarse por su localización tisular y subcelular, y por su sensibilidad a reactivos que reaccionan con los grupos sulfhidrilos. El objetivo de este trabajo fue estudiar la localización y función de GPAT2, una isoforma mitocondrial que se expresa principalmente en testículo utilizando como modelo células CHO-K1 que sobreexpresan GPAT2. Se observó que esta isoforma utiliza como sustrato específicamente al ácido araquidónico, estimula la síntesis de triacilgliceroles (TAG) con alto contenido de ácido araquidónico, y favorece la incorporación de [1-14C] ácido araquidónico en los TAG. Utilizando modelos animales se observó por un lado que el ARNm de GPAT2 se expresa sólo en los espermatocitos primarios, mientras que la expresión de la proteína se mantiene a lo largo de la espermatogénesis; y por otro lado que la expresión de GPAT2 varía durante el desarrollo sexual, alcanzando un máximo de expresión y actividad a los 30 días de edad en la rata. A esta edad se observó también un máximo en el contenido de TAG y de AA esterificado en los TAG del testículo. Se concluye que los resultados sugieren que GPAT2 sería la enzima responsable de esterificar el ácido araquidónico en los TAG de las células de la línea espermática.(AU)

De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. The objective of the present work was to study localization and function of GPAT2, a mitochondrial isoform that is mainly expressed in testis, using as a model CHO-K1 cells that overexpress GPAT2. Incubation of GPAT2-transfected CHO-K1 cells with arachidonoyl-CoA increased GPAT activity 2-fold and the incorporation of [1-14C] arachidonate into TAG. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.(AU)

O primeiro avanþo na síntese de novo de glicerolipídios é a acidulaþÒo do glicerol-3-fosfato, a qual é catalisada pela enzima glicerol-3-fosfato aciltransferase (GPAT). Em mamíferos tem sido descritas até o momento quatro isoformas de GPAT, as quais se podem distinguir pela sua localizaþÒo tissular e subcelular, e pela sua sensibilidade aos reagentes que reagem com os grupos sulfidrilos. O objetivo deste trabalho foi estudar a localizaþÒo e funþÒo de GPAT2, uma isoforma mitocondrial que se expressa principalmente no testículo, utilizando como modelo células CHO-K1 que superexpressam GPAT2. Observou-se que esta isoforma utiliza como substrato especificamente o ácido araquid¶nico, estimula a síntese de triacilgliceróis (TAG) com alto conteúdo de ácido araquid¶nico, e favorece a incorporaþÒo de [1-14C] ácido araquid¶nico nos TAG. Utilizando modelos animais se observou de um lado que o ARNm de GPAT2 se expressa apenas nos espermatócitos primários, enquanto que a expressÒo da proteína se mantém ao longo da espermatogÛnese; e do outro que a expressÒo de GPAT2 varia durante o crescimento sexual, atingindo um máximo de expressÒo e atividade aos 30 dias de idade no camundongo. Nessa idade foi observado também um máximo no conteúdo de TAG e de AA esterificado nos TAG do testículo. Conclui-se que os resultados sugerem que GPAT2 seria a enzima responsável por esterificar o ácido araquid¶nico nos TAG das células da linha espermática.(AU)

Rol de la isoforma 2 de la glicerol-3-fosfatoaciltransferasa en el metabolismo lipídico testicular / Role of isoform 2 of glycerol-3-phosphate acyltransferase in testicular lipid metabolism / Função da isoforma 2 da glicerol-3-fosfatoaciltransferase no metabolismo lipídico testicular

El primer paso en la síntesis de novo de glicerolípidos es la acilación del glicerol-3-fosfato, la cual es catalizada por la enzima glicero-3-fosfato aciltransferasa (GPAT). En mamíferos han sido descriptas hasta el momento cuatro isoformas de GPAT, las cuales pueden diferenciarse por su localización tisular y subcelular, y por su sensibilidad a reactivos que reaccionan con los grupos sulfhidrilos. El objetivo de este trabajo fue estudiar la localización y función de GPAT2, una isoforma mitocondrial que se expresa principalmente en testículo utilizando como modelo células CHO-K1 que sobreexpresan GPAT2. Se observó que esta isoforma utiliza como sustrato específicamente al ácido araquidónico, estimula la síntesis de triacilgliceroles (TAG) con alto contenido de ácido araquidónico, y favorece la incorporación de [1-14C] ácido araquidónico en los TAG. Utilizando modelos animales se observó por un lado que el ARNm de GPAT2 se expresa sólo en los espermatocitos primarios, mientras que la expresión de la proteína se mantiene a lo largo de la espermatogénesis; y por otro lado que la expresión de GPAT2 varía durante el desarrollo sexual, alcanzando un máximo de expresión y actividad a los 30 días de edad en la rata. A esta edad se observó también un máximo en el contenido de TAG y de AA esterificado en los TAG del testículo. Se concluye que los resultados sugieren que GPAT2 sería la enzima responsable de esterificar el ácido araquidónico en los TAG de las células de la línea espermática.

De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. The objective of the present work was to study localization and function of GPAT2, a mitochondrial isoform that is mainly expressed in testis, using as a model CHO-K1 cells that overexpress GPAT2. Incubation of GPAT2-transfected CHO-K1 cells with arachidonoyl-CoA increased GPAT activity 2-fold and the incorporation of [1-14C] arachidonate into TAG. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

O primeiro avanço na síntese de novo de glicerolipídios é a acidulação do glicerol-3-fosfato, a qual é catalisada pela enzima glicerol-3-fosfato aciltransferase (GPAT). Em mamíferos tem sido descritas até o momento quatro isoformas de GPAT, as quais se podem distinguir pela sua localização tissular e subcelular, e pela sua sensibilidade aos reagentes que reagem com os grupos sulfidrilos. O objetivo deste trabalho foi estudar a localização e função de GPAT2, uma isoforma mitocondrial que se expressa principalmente no testículo, utilizando como modelo células CHO-K1 que superexpressam GPAT2. Observou-se que esta isoforma utiliza como substrato especificamente o ácido araquidônico, estimula a síntese de triacilgliceróis (TAG) com alto conteúdo de ácido araquidônico, e favorece a incorporação de [1-14C] ácido araquidônico nos TAG. Utilizando modelos animais se observou de um lado que o ARNm de GPAT2 se expressa apenas nos espermatócitos primários, enquanto que a expressão da proteína se mantém ao longo da espermatogênese; e do outro que a expressão de GPAT2 varia durante o crescimento sexual, atingindo um máximo de expressão e atividade aos 30 dias de idade no camundongo. Nessa idade foi observado também um máximo no conteúdo de TAG e de AA esterificado nos TAG do testículo. Conclui-se que os resultados sugerem que GPAT2 seria a enzima responsável por esterificar o ácido araquidônico nos TAG das células da linha espermática.

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation.

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols.

BACKGROUND: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. METHODS AND RESULTS: Incubation of GPAT2-transfected CHO-K1 cells with [1-(14)C]arachidonate for 3 h increased incorporation of [(14)C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. CONCLUSIONS: These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

Photosensitization of DNA by ß-carbolines: kinetic analysis and photoproduct characterization.

ß-Carbolines (ßCs) are a group of alkaloids present in many plants and animals. It has been suggested that these alkaloids participate in a variety of significant photosensitized processes. Despite their well-established natural occurrence, the main biological role of these alkaloids and the mechanisms involved are, to date, poorly understood. In the present work, we examined the capability of three important ßCs (norharmane, harmane and harmine) and two of its derivatives (N-methyl-norharmane and N-methyl-harmane) to induce DNA damage upon UV-A excitation, correlating the type and extent of the damage with the photophysical characteristics and DNA binding properties of the compounds. The results indicate that DNA damage is mostly mediated by a direct type-I photoreaction of the protonated ßCs after non-intercalative electrostatic binding. Reactive oxygen species such as singlet oxygen and superoxide are not involved to a major extent, as indicated by the only small influence of D(2)O and of superoxide dismutase on damage generation. An analysis with repair enzymes revealed that oxidative purine modifications such as 8-oxo-7,8-dihydroguanine, sites of base loss and single-strand breaks (SSB) are generated by all ßCs, while only photoexcited harmine gives rise to the formation of cyclobutane pyrimidine dimers as well.

Photosensitized cleavage of plasmidic DNA by norharmane, a naturally occurring beta-carboline.

UV-A radiation (320-400 nm) induces damages to the DNA molecule and its components through photosensitized reactions. Beta-carbolines (betaCs), heterocyclic compounds widespread in biological systems, participate in several biological processes and are able to act as photosensitizers. The photosensitization of plasmidic DNA by norharmane in aqueous solution under UV-A radiation was studied. The effect of pH was evaluated and the participation of reactive oxygen species (ROS), such as hydroxyl radical (HO*), superoxide anion (O(2)(*-)) and singlet oxygen ((1)O(2)) was investigated. A strong dependence of the photosensitized DNA relaxation on the pH was observed. The extent of the reaction was shown to be higher in the experiments performed at pH 4.7 than those performed at pH 10.2. As was expected, an intermediate extent of the reaction was observed at physiological pH (pH 7.4). Kinetic studies using ROS scavengers revealed that the chemical reactions between ROS and DNA are not the main pathways responsible for the damage of DNA. Consequently, the predominant mechanism yielding the DNA strand break takes place most probably via a type I mechanism (electron transfer) from the single excited state (S(1)) of the protonated form of norharmane ((1)[nHoH(+)]*). Additional information about the nature of the norharmane electronic excited states involved in the photocleavage reaction was obtained by using the N-methyl derivative of norharmane (N-methyl-norharmane).

Nuclear receptors and hepatic lipidogenic enzyme response to a dyslipidemic sucrose-rich diet and its reversal by fish oil n-3 polyunsaturated fatty acids.

A sucrose-rich diet (SRD), compared with a starch diet, induces time-dependent metabolic disorders and insulin resistance with hypertriglyceridemia, similar to type 2 diabetes. In this study, we examined the effect of SRD, after 8 mo, on nuclear receptors peroxisome proliferator-activated receptor-alpha (PPARalpha), and liver X receptor-alpha (LXRalpha), stearoyl-CoA desaturase-1 (SCD-1), and Delta6 and Delta5 desaturases mRNA and activity, hepatic enzymes involved in lipid metabolism, and fatty acid (FA) composition as well as the reversal produced by cod liver oil. SRD induced triglyceride increase in plasma and liver, increasing the anabolic FA synthase, malic enzyme, and glucose-6-phosphate dehydrogenase, but not the prooxidative enzymes FA oxidase and carnitine palmitoyltransferase I, and correspondingly decreased PPARalpha and increased LXRalpha expressions. Results suggest a contribution of both nuclear receptors' interaction on these enzymatic activities. SRD depressed SCD-1 without altering oleic acid proportion and increased Delta6 and Delta5 desaturases and the proportion of n-6 arachidonic acid. Therefore, the data do not support that SRD hypertriglyceridemia is produced by increased SCD-1-dependent oleic acid biosynthesis. The administration of 7% cod liver oil for 2 mo depressed LXRalpha, enhancing PPARalpha in control and SRD-fed rats, reversing the activity of the hepatic enzymes involved in lipid metabolism and therefore the hyperlipidemia produced by the SRD. Fish oil increased n-3 PUFA and depressed n-6 PUFA of liver lipids without altering the 18:1/18:0 ratio, suggesting that its effects were produced mainly by competition of dietary n-6 and n-3 FA and not through desaturase activity modification.

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV).

Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity.

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.