Effect of starvation on the activity of the mitochondrial tricarboxylate carrier

AbstractThe effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35–40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the Vmax of this process decreases while there is no change in the Km. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein

Diets based on virgin olive oil or fish oil but not on sunflower oil prevent age-related alvolar bone resorption by mitochondrial-related mechanisms

Background/Objectives: Aging enhances frequency of chronic diseases like cardiovascular diseases or periodontitis. Here we reproduced an age-dependent model of the periodontium, a fully physiological approach to periodontal conditions, to evaluate the impact of dietary fat type on gingival tissue of young (6 months old) and old (24 months old) rats.Methods/Findings: Animals were fed life-long on diets based on monounsaturated fatty acids (MUFA) as virgin olive oil, n-6 polyunsaturated fatty acids (n-6PUFA), as sunflower oil, or n-3PUFA, as fish oil. Age-related alveolar bone loss was higher in n-6PUFA fed rats, probably as a consequence of the ablation of the cell capacity to adapt to aging. Gene expression analysis suggests that MUFA or n-3PUFA allowed mitochondria to maintain an adequate turnover through induction of biogenesis, autophagy and the antioxidant systems, and avoiding mitochondrial electron transport system alterations.Conclusions: The main finding is that the enhanced alveolar bone loss associated to age may be targeted by an appropriate dietary treatment. The mechanisms involved in this phenomenon are related with an ablation of the cell capacity to adapt to aging. Thus, MUFA or n-3PUFA might allow mitochondrial maintaining turnover through biogenesis or autophagy. They might also be able to induce the corresponding antioxidant systems to counteract age-related oxidative stress, and do not inhibit mitochondrial electron transport chain. From the nutritional and clinical point of view, it is noteworthy that the potential treatments to attenuate alveolar bone loss (a feature of periodontal disease) associated to age could be similar to some of the proposed for the prevention and treatment of cardiovascular diseases, a group of pathologies recently associated with age-related periodontitis.This study was supported by I+D grants from the Spanish Ministry of Education and Science (AGL2008-01057) and the Autonomous Government of Andalusia (AGR832)

The tricarboxylate carrier of eel liver mitochondria: characteristics and partial purification

P-27

Extra virgin olive oil phenols down-regulate lipid synthesis in primarycultured rat- hepatocytes

Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil

Expression of citrate carrier gene is activated by ER stress effectors XBP1 and ATF6α, binding to an UPRE in its promoter

The Unfolded Protein Response (UPR) is an intracellular signaling pathway which is activated when unfolded or misfolded proteins accumulate in the Endoplasmic Reticulum (ER), a condition commonly referred to as ER stress. It has been shown that lipid biosynthesis is increased in ER-stressed cells. The N(ε)-lysine acetylation of ER-resident proteins, including chaperones and enzymes involved in the post-translational protein modification and folding, occurs upon UPR activation. In both ER proteins acetylation and lipid synthesis, acetyl-CoA is the donor of acetyl group and it is transported from the cytosol into the ER. The cytosolic pool of acetyl-CoA is mainly derived from the activity of mitochondrial citrate carrier (CiC). Here, we have demonstrated that expression of CiC is activated in human HepG2 and rat BRL-3A cells during tunicamycin-induced ER stress. This occurs through the involvement of an ER stress responsive region identified within the human and rat CiC proximal promoter. A functional Unfolded Protein Response Element (UPRE) confers responsiveness to the promoter activation by UPR transducers ATF6α and XBP1. Overall, our data demonstrate that CiC expression is activated during ER stress through the binding of ATF6α and XBP1 to an UPRE element located in the proximal promoter of Cic gene. The role of ER stress-mediated induction of CiC expression has been discussed

Dietary fat types differently modulate the activity and expression of mitochondrialcarnitine/acylcarnitine translocase in rat liver

The carnitine/acylcarnitine translocase (CACT), an integral protein of the mitochondrial innermembrane, belongs to the carnitine-dependent system of fatty acid transport into mitochondria, where beta-oxidation occurs. CACT exchanges cytosolic acylcarnitine or free carnitine for carnitine in the mitochondrial matrix. The object of this study was to investigate in rat liver the effect, if any, of diets enriched with saturated fatty acids (beef tallow, BT, the control), n−3 polyunsaturated fatty acids (PUFA) (fish oil, FO), n−6 PUFA (safflower oil, SO), and mono-unsaturated fatty acids (MUFA) (olive oil,OO) on the activity and expression of CACT. Translocase exchange rates increased, in parallelwith CACTmRNA abundance, upon FO-feeding,whereas OO-dietary treatment induced a decrease in both CACT activity and expression. No changes were observed upon SO-feeding. Nuclear run-on assay revealed that FO-treatment increased the transcriptional rate of CACT mRNA. On the other hand, only in the nuclei of hepatocytes fromOO-fed rats splicing of the last intron of CACT pre-mRNA and the rate of formation of the 3′-endwere affected. Overall, these findings suggest that compared to the BT‐enriched diet, the SO-enriched diet did not influence CACT activity and expression, whereas FO- and OO-feeding alters CACT activity in an opposite fashion, i.e. modulating its expression at transcriptional and post-transcriptional levels, respectively

Quercetin Reduces Lipid Accumulation in a Cell Model of NAFLD by Inhibiting De Novo Fatty Acid Synthesis through the Acetyl-CoA Carboxylase 1/AMPK/PP2A Axis

Dysregulation of de novo lipogenesis (DNL) has recently gained strong attention as being one of the critical factors that contribute to the assessment of non-alcoholic fatty liver disease (NAFLD). NAFLD is often diagnosed in patients with dyslipidemias and type 2 diabetes; thus, an interesting correlation can be deduced between high hematic free fatty acids and glucose excess in the DNL dysregulation. In the present study, we report that, in a cellular model of NAFLD, the coexistence of elevated glucose and FFA conditions caused the highest cellular lipid accumulation. Deepening the molecular mechanisms of the DNL dysregulation—RT-qPCR and immunoblot analysis demonstrated increased expression of mitochondrial citrate carrier (CiC), cytosolic acetyl-CoA carboxylase 1 (ACACA), and diacylglycerol acyltransferase 2 (DGAT2) involved in fatty acids and triglycerides synthesis, respectively. XBP-1, an endoplasmic reticulum stress marker, and SREBP-1 were the transcription factors connected to the DNL activation. Quercetin (Que), a flavonoid with strong antioxidant properties, and noticeably reduced the lipid accumulation and the expression of SREBP-1 and XBP-1, as well as of their lipogenic gene targets in steatotic cells. The anti-lipogenic action of Que mainly occurs through a strong phosphorylation of ACACA, which catalyzes the committing step in the DNL pathway. The high level of ACACA phosphorylation in Que-treated cells was explained by the intervention of AMPK together with the reduction of enzymatic activity of PP2A phosphatase. Overall, our findings highlight a direct anti-lipogenic effect of Que exerted through inhibition of the DNL pathway by acting on ACACA/AMPK/PP2A axis; thus, suggesting this flavonoid as a promising molecule for the NAFLD treatment

Sterol-Regulatory Binding Protein-1 (SREBP-1) mRNA contains an internal ribosome entry site that regulates its translation in response to cellular stress.

Sterol-regulatory-element-binding proteins (SREBPs) are a family of transcription factors that modulate the expression of several enzymes implicated in endogenous cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. Here, evidences for SREBP-1 regulation at the translational level are reported. We demonstrated that the 5’-UTR (untranslated region) of the SREBP-1a mRNA contains an IRES (internal ribosome entry site). Cellular stress conditions, such as serum starvation caused an increase in the level of SREBP-1 precursor and mature form in Hep G2 cells, despite the overall reduction in protein synthesis, whereas mRNA level for SREBP-1 was unaffected by cellular stress. Transfection experiments carried out with a dicistronic construct demonstrated that the cap-dependent translation was affected more than IRES mediated translation by serum starvation. Unfolded protein response (UPR) also increased SREBP-1 expression in Hep G2 cells, through the capindependent translation mediated by IRES. Our fi ndings indicate that the presence of IRES in the SREBP-1a 5’-UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation

n-3 and n-6 polyunsaturated fatty acids differently affect citrate carrier promoter activity.

Citrate carrier (CiC), a mitochondrial membrane protein, plays an important metabolic role by transporting, in the form of citrate, acetyl-CoA from mitochondria into the cytosol for fatty acid and cholesterol synthesis. A PUFA (polyunsaturated fatty acids) response region, composed of a NF-Y site, an E-box like site, a SRE1 like site and four Sp1 sites, has been identified within the CiC promoter. Transcription factor SRE-Binding Protein-1 (SREBP-1c) is target for PUFA down-regulation of CiC transcription. Transfection and gel mobility shift assays indicated that a functional E-box like confers responsiveness to SREBP-1c. In H4IIE cells overexpression of SREBP-1c overrides arachidonic acid suppression but does not prevent the repression by docosahexaenoic acid. ChIP assay showed that docosahexaenoic acid affects the binding of NF-Y, Sp1 and SREBP-1 to PUFA response region whereas arachidonic acid alters only the binding of SREBP-1. PUFA inhibition of Cic gene transcription is mediated not only by the SREBP-1c but might also involve a reduction in Sp1 and NF-Y DNA binding, suggesting differential mechanisms in the Cic gene regulation by different PUFA