Janina Dose

Anti‐inflammatory potential of allyl‐isothiocyanate – role of Nrf2, NF‐κB and microRNA‐155

In this study, the underlying mechanisms of the potential anti‐inflammatory properties of allyl‐isothiocyanate (AITC) were analysed in vitro and in vivo. Murine RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were supplemented with increasing concentrations of AITC. In addition, C57BL/6 mice (n= 10 per group) were fed a pro‐inflammatory high‐fat diet and AITC was administered orally via gavage for 7 days. Biomarkers of inflammation were determined both in cultured cells and in mice. AITC significantly decreased tumour necrosis factor α mRNA levels and its secretion in LPS stimulated RAW264.7 macrophages. Furthermore, gene expression of other pro‐inflammatory markers including interleukin‐1β and inducible nitric oxide synthase were down‐regulated following AITC treatment. AITC decreased nuclear p65 protein levels, a subunit of the transcription factor NF‐κB. Importantly, our data indicate that AITC significantly attenuated microRNA‐155 levels in LPS‐stimulated RAW264.7 macrophages in a dose‐dependent manner. The anti‐inflammatory effects of AITC were accompanied by an increase in Nrf2 nuclear translocation and consequently by an increase of mRNA and protein levels of the Nrf2 target gene heme‐oxygenase 1. AITC was slightly less potent than sulforaphane (used as a positive control) in down‐regulating inflammation in LPS‐stimulated macrophages. A significant increase in nuclear Nrf2 and heme‐oxygenase 1 gene expression and only a moderate down‐regulation of interleukin‐1β and microRNA‐155 levels due to AITC was found in mouse liver. Present data suggest that AITC exhibits potent anti‐inflammatory activity in cultured macrophages in vitro but has only little anti‐inflammatory activity in mice in vivo.

Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health.

Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization-Studies in gene-targeted replacement mice

SCOPE Of the three human apolipoprotein E (APOE) alleles, the ε3 allele is most common, which may be a result of adaptive evolution. In this study, we investigated whether the APOE genotype affects body weight and energy metabolism through regulation of fatty acid utilization. METHODS AND RESULTS Targeted replacement mice expressing the human APOE3 were significantly heavier on low- and high-fat diets compared to APOE4 mice. Particularly on high-fat feeding, food intake and dietary energy yields as well as fat mass were increased in APOE3 mice. Fatty acid mobilization determined as activation of adipose tissue lipase and fasting plasma nonesterified fatty acid levels were significantly lower in APOE3 than APOE4 mice. APOE4 mice, in contrast, exhibited higher expression of proteins involved in fatty acid oxidation in skeletal muscle. CONCLUSION Our data suggest that APOE3 is associated with the potential to more efficiently harvest dietary energy and to deposit fat in adipose tissue, while APOE4 carriers tend to increase fatty acid mobilization and utilization as fuel substrates especially under high-fat intake. The different handling of dietary energy may have contributed to the evolution and worldwide distribution of the ε3 allele.

Beneficial effects of a 6-month dietary restriction are time-dependently abolished within 2 weeks or 6 months of refeeding—genome-wide transcriptome analysis in mouse liver

Dietary restriction (DR) has been shown to exert a number of beneficial effects including the prolongation of life span. One of the mechanisms by which DR leads to these advantages seems to be the induction of endogenous antioxidant defense and stress response mechanisms. However, little is known about the persistence of DR benefits after return to an ad libitum diet. In this study, male C57BL/6 mice were fed 75% of a normal diet for 6 months (DR) followed by 6 months of ad libitum refeeding (RF) and compared to a continuously ad libitum fed control group. To study the impact of DR and RF on the liver transcriptome, a global gene expression profile was generated using microarray technology. In comparison, the DR group showed lower body weight, lower triglyceride and cholesterol levels, reduced lipid peroxidation, and a changed hepatic fatty acid pattern. mRNA transcription and activity of antioxidant and phase II enzymes, as well as metallothionein 1 gene expression, were increased and autophagy was induced. Shifting from long-term DR to RF abolished 96% of the DR-mediated changes in differential gene expression within 2 weeks, and after 6 months of refeeding all of the previously differentially expressed genes were similar in both groups. These results indicate that DR has to be maintained continuously to keep its beneficial effects.

Dietary Tocotrienol/γ-Cyclodextrin Complex Increases Mitochondrial Membrane Potential and ATP Concentrations in the Brains of Aged Mice

Brain aging is accompanied by a decrease in mitochondrial function. In vitro studies suggest that tocotrienols, including γ- and δ-tocotrienol (T3), may exhibit neuroprotective properties. However, little is known about the effect of dietary T3 on mitochondrial function in vivo. In this study, we monitored the effect of a dietary T3/γ-cyclodextrin complex (T3CD) on mitochondrial membrane potential and ATP levels in the brain of 21-month-old mice. Mice were fed either a control diet or a diet enriched with T3CD providing 100 mg T3 per kg diet for 6 months. Dietary T3CD significantly increased mitochondrial membrane potential and ATP levels compared to those of controls. The increase in MMP and ATP due to dietary T3CD was accompanied by an increase in the protein levels of the mitochondrial transcription factor A (TFAM). Furthermore, dietary T3CD slightly increased the mRNA levels of superoxide dismutase, γ-glutamyl cysteinyl synthetase, and heme oxygenase 1 in the brain. Overall, the present data suggest that T3CD increases TFAM, mitochondrial membrane potential, and ATP synthesis in the brains of aged mice.

R-α lipoic acid γ-cyclodextrin complex increases energy expenditure: a 4-month feeding study in mice.

OBJECTIVE A high-fat diet (HFD) affects energy expenditure in laboratory rodents. R-α lipoic acid cyclodextrin (RALA-CD) complex is a stable form of lipoic acid (LA) and may improve energy expenditure. The aim of this study was to determine the effect of RALA-CD on energy expenditure and underlying molecular targets in female laboratory mice. METHODS Female C57BL/6J mice were fed a HFD containing 0.1% LA for about 16 wk. The effects on energy expenditure, gene and protein expression were assessed using indirect calorimetry, real-time reverse transcriptase polymerase chain reaction, and Western blot, respectively. RESULTS Supplementing mice with RALA-CD resulted in a significant increase in energy expenditure. However, both RALA per se (without γ-cyclodextrin) and S-α lipoic acid cyclodextrin did not significantly alter energy expenditure. Furthermore RALA-CD changed expression of genes encoding proteins centrally involved in energy metabolism. Transcriptional key regulators sirtuin 3 and peroxisome proliferator-activated receptor-γ, coactivator 1 alpha, as well as thyroid related enzyme type 2 iodothyronine deiodinase were up-regulated in brown adipose tissue (BAT) of RALA-CD-fed mice. Importantly, mRNA and/or protein expression of downstream effectors uncoupling protein (Ucp) 1 and 3 also were elevated in BAT from RALA-CD-supplemented mice. CONCLUSION Overall, present data suggest that RALA-CD is a regulator of energy expenditure in laboratory mice.

Identification and characterization of two functional variants in the human longevity gene FOXO3

FOXO3 is consistently annotated as a human longevity gene. However, functional variants and underlying mechanisms for the association remain unknown. Here, we perform resequencing of the FOXO3 locus and single-nucleotide variant (SNV) genotyping in three European populations. We find two FOXO3 SNVs, rs12206094 and rs4946935, to be most significantly associated with longevity and further characterize them functionally. We experimentally validate the in silico predicted allele-dependent binding of transcription factors (CTCF, SRF) to the SNVs. Specifically, in luciferase reporter assays, the longevity alleles of both variants show considerable enhancer activities that are reversed by IGF-1 treatment. An eQTL database search reveals that the alleles are also associated with higher FOXO3 mRNA expression in various human tissues, which is in line with observations in long-lived model organisms. In summary, we present experimental evidence for a functional link between common intronic variants in FOXO3 and human longevity.FOXO3 is one of the few established longevity genes. Here, the authors fine-map the FOXO3-longevity association to two intronic SNPs and, using luciferase assays and EMSAs, show that these SNPs affect binding of transcription factors CTCF and SRF and associate with FOXO3 expression.

Influence of the APOE genotype on hepatic stress response: Studies in APOE targeted replacement mice and human liver cells

Apolipoprotein E (APOE) is a multifunctional plasma protein mainly acting in lipid metabolism. Human APOE is polymorphic with three major isoforms (APOE2, APOE3 and APOE4). Up to 75% of the bodys APOE is produced by the liver. There is increasing evidence from studies in brain-derived cells that APOE4 affects mitochondrial function and biogenesis as well as stress and inflammatory responses - processes, whose disturbances are considered hallmarks of the ageing process. However, although the liver is the main production site of APOE, knowledge about the impact of the APOE genotype on hepatic stress response-related processes is rather limited. Therefore, we studied biomarkers of oxidative status (glutathione levels, 3-nitrotyrosine adducts, protein carbonyl concentration), ER stress (XBP1(S), BiP, DDIT3), proteasome activity, mitochondrial function (respiratory complexes, ATP levels and mitochondrial membrane potential as well as biomarkers of mitochondrial biogenesis, fission and fusion), autophagy (LC3, LAMP2A), apoptosis (BCL2, BAX, CYCS) and DNA damage in the liver of APOE targeted replacement (TR) mice and in Huh7 hepatocytes overexpressing the APOE3 and the APOE4 isoform, respectively. APOE4 mice exhibited a lower chymotrypsin-like and a higher trypsin-like proteasome activity. Levels of protein carbonyls were moderately higher in liver tissue of APOE4 vs. APOE3 mice. Other biomarkers of oxidative stress were similar between the two genotypes. Under basal conditions, the stress-response pathways investigated appeared largely unaffected by the APOE genotype. However, upon stress induction, APOE4 expressing cells showed lower levels of adenosine triphosphate (ATP) and lower mRNA levels of the ATP-generating complex V of the mitochondrial respiratory chain. Overall, our findings provide evidence for a rather low influence of the APOE genotype on the hepatic stress response processes investigated in this study.

On a Western diet, APOE ɛ4 is associated with low innate immune sensing, but not APOE ɛ3

Under conditions reflecting the Western lifestyle, APOE ε4 vs. APOE ε3 may be associated with a lower expression of markers important in innate immune activation. The results encourage further investigations into the role of the APOE genotype in metabolic diseases associated with chronic inflammation.

Publisher Correction: Identification and characterization of two functional variants in the human longevity gene FOXO3

The original version of this Article contained an error in the spelling of the author Robert Häsler, which was incorrectly given as Robert Häesler. This has now been corrected in both the PDF and HTML versions of the Article.