Fat from Hermetia illucens Alters the Cecal Gut Microbiome and Lowers Hepatic Triglyceride Concentration in Comparison to Palm Oil in Obese Zucker Rats.

BACKGROUND: Palm oil (PO) is the most widely utilized plant oil for food production. Owing to the great ecologic problems associated with PO production, sustainably produced fats, such as insect fat, might be a suitable alternative. OBJECTIVES: The hypothesis was tested that fat from Hermetia illucens larvae (HF) compared with PO and soybean oil (SO) has no adverse effects on hepatic lipid metabolism, plasma metabolome, and cecal microbiome in obese Zucker rats. METHODS: Thirty male obese Zucker rats were randomly assigned to 3 groups (SO, PO, HF; n = 10 rats/group) and fed 3 different semisynthetic diets containing either SO, PO, or HF as the main fat source for 4 wk. The effects were evaluated by measurement of liver and plasma lipid concentrations, liver transcriptomics, targeted plasma metabolomics, and cecal microbiomics. RESULTS: Supplementation of HF reduced hepatic triglyceride concentration and messenger ribonucleic acid concentrations of selected genes involved in fatty acid and triglyceride synthesis in comparison to PO (P < 0.05). Pairwise comparison of the Simpson index and Jaccard index showed a higher cecal microbial α- and ß-diversity in rats fed the HF diet than in rats fed the PO diet (P = 0.015 and P = 0.027), but no difference between rats fed the diets with SO or PO. Taxonomic analysis of the cecal microbial community revealed a lower abundance of Clostridium_sensu_stricto_1 and a higher abundance of Blautia, Mucispirillum, Anaerotruncus, Harryflintia, and Peptococcus in rats supplemented with HF than in rats supplemented with PO (P < 0.05). CONCLUSIONS: HF, compared with PO, has liver lipid-lowering effects in obese Zucker rats, which may be caused by a shift in the gut microbial community. Thus, HF might serve as a sustainably produced fat alternative to PO for food production.

Effect of Hermetia illucens Fat, Compared with That of Soybean Oil and Palm Oil, on Hepatic Lipid Metabolism and Plasma Metabolome in Healthy Rats.

Palm oil (PO) is currently the most widely used fat source for food production, but insect fat from Hermetia illucens larvae (HF) might be a suitable alternative fat source, because its production is less harmful to the environment. The present study investigated the effect of HF, as compared to PO and soybean oil (SO), on the hepatic lipid metabolism and the plasma metabolome of healthy rats, which were randomly assigned to three groups (n = 10 rats/group), and fed three different semi-synthetic diets containing either SO, PO, or HF as the main fat source for 4 weeks. Feed intake, body weight gain, liver and plasma lipid concentrations, and the hepatic mRNA levels of genes involved in lipid metabolism and inflammation did not differ between groups. Targeted plasma metabolomics revealed 294 out of 630 metabolites analyzed to be different between groups. Principal component analysis showed a clear separation of the plasma metabolomes of the SO group and the other two groups, but no separation of those of the PO and the HF groups. The present study shows that HF exerts no adverse metabolic effects in healthy rats, compared to PO or SO, indicating that HF is a safe alternative fat source to PO for food production.

Feeding of cuticles from Tenebrio molitor larvae modulates the gut microbiota and attenuates hepatic steatosis in obese Zucker rats.

Insect biomass obtained from large-scale mass-rearing of insect larvae has gained considerable attention in recent years as an alternative and sustainable source of food and feed. A byproduct from mass-rearing of insect larvae is the shed cuticles - the most external components of insects which are a relevant source of the polysaccharide chitin. While it has been shown that chitin modulates the gut microbiota and ameliorates lipid metabolic disorders in obese rodent models, feeding studies dealing with isolated insects' cuticles are completely lacking. Thus, the present study tested the hypothesis that dietary insects' cuticles modulate the gut microbiome and improve hepatic lipid metabolism in obese Zucker rats. To test this hypothesis, three groups of obese Zucker rats were fed a nutrient-adequate, semisynthetic basal diet which was supplemented with either 0% (group O), 1.5% (group O1.5) or 3.0% (group O3.0) Tenebrio molitor cuticles at the expense of cellulose. Oil red O-stained liver sections showed a marked lipid accumulation, but lipid accumulation was clearly less in group O3.0 than in groups O and O1.5. In line with this, hepatic lipid concentrations were 30% lower in group O3.0 than in group O (p < 0.05). No differences were observed across the obese groups regarding liver concentrations of methionine, S-adenosylmethionine and homocysteine. Analysis of cecal microbial community at the family level revealed that the relative abundances of Bifidobacteriaceae, Coriobacteriaceae Erysipelotrichaceae, Lactobacillaceae, Prevotellaceae, Sutterellaceae, unknown Deltaproteobacteria and unknown Firmicutes were higher and those of Anaeroplasmataceae, Desulfovibrionaceae, Eubacteriaceae, Ruminococcaceae, Saccharibacteria and unknown Clostridiales were lower in group O3.0 compared to group O (p < 0.05). Cecal digesta concentrations of total short-chain fatty acids, acetate and butyrate were higher in group O3.0 than in group O (p < 0.05). Targeted plasma metabolomics revealed 53 metabolites differing between groups, amongst which two indole metabolites, indole-3-propionic acid and 3-indoxylsulfate, were markedly elevated in group O3.0 compared to groups O1.5 and O. Regarding that increased abundances of bacteria of the Actinobacteria phylum and Lactobacillaceae family in the gut have been reported to be associated with antisteatotic, hepatoprotective and antiinflammatory effects, the pronounced increases of Bifidobacteriaceae and Coriobacteriaceae (both Actinobacteria), and of Lactobacillaceae in group O3.0 might have contributed to the amelioration of fatty liver.

Branched-Chain Fatty Acids as Mediators of the Activation of Hepatic Peroxisome Proliferator-Activated Receptor Alpha by a Fungal Lipid Extract.

The study aimed to test the hypothesis that monomethyl branched-chain fatty acids (BCFAs) and a lipid extract of Conidiobolus heterosporus (CHLE), rich in monomethyl BCFAs, are able to activate the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Rat Fao cells were incubated with the monomethyl BCFAs 12-methyltridecanoic acid (MTriA), 12-methyltetradecanoic acid (MTA), isopalmitic acid (IPA) and 14-methylhexadecanoic acid (MHD), and the direct activation of PPARalpha was evaluated by reporter gene assay using a PPARalpha responsive reporter gene. Furthermore, Fao cells were incubated with different concentrations of the CHLE and PPARalpha activation was also evaluated by using the reporter gene assay, and by determining the mRNA concentrations of selected PPARalpha target genes by real-time RT-PCR. The reporter gene assay revealed that IPA and the CHLE, but not MTriA, MHD and MTA, activate the PPARalpha responsive reporter gene. CHLE dose-dependently increased mRNA concentrations of the PPARalpha target genes acyl-CoA oxidase (ACOX1), cytochrome P450 4A1 (CYP4A1), carnitine palmitoyltransferase 1A (CPT1A) and solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5). In conclusion, the monomethyl BCFA IPA is a potent PPARalpha activator. CHLE activates PPARalpha-dependent gene expression in Fao cells, an effect that is possibly mediated by IPA.

1,25-hydroxyvitamin D3 decreases endoplasmic reticulum stress-induced inflammatory response in mammary epithelial cells.

Recent studies indicated that intramammary administration of active vitamin D3 hormone (1,25D3) inhibits the inflammatory process associated with mastitis. We hypothesized that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in mammary epithelial cells (MECs) is an important cellular mechanism contributing to this beneficial effect of intramammary treatment with 1,25D3. To test this hypothesis, the effect of 1,25D3 was studied on induction of ER stress in a transformed human MEC line, MCF-7 cells. Treatment with two different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), caused a dose-dependent induction of ER stress as evident from up-regulation of protein kinase RNA-like ER kinase (PERK), heat shock protein family A (Hsp70) member 5 (HSPA5), activating transcription factor (ATF4), ATF6, DNA damage inducible transcript 3 (DDIT3) and spliced X-box binding protein 1 (sXBP1) and impaired cell viability and decreased expression of vitamin D receptor (VDR) in MCF-7 cells (P < 0.05). Treatment with 1,25D3 (100 nM) inhibited TG (10 nM)- and TM (1 µg/mL)-induced mRNA and/or protein levels of ATF4, ATF6, DDIT3 and HSPA5 in MCF-7 cells (P < 0.05). In addition, 1,25D3 (100 nM) antagonized the effect of TG (10 nM) and TM (1 µg/mL) on mRNA and protein levels of VDR and mRNA levels of genes involved in production and degradation of 1,25D3 in MCF-7 cells (P < 0.05). Moreover, 1,25D3 (100 nM) inhibited nuclear factor-κB (NF-κB) activation in response to TM (10 nM) and TG (1 µg/mL) in MCF-7 cells. In conclusion, the present findings show that 1,25D3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis.

Decreased All- trans Retinoic Acid-Induced Expression of Sodium-Iodide Transporter in Mammary Epithelial Cells Caused by Conjugated Linoleic Acid Isomers.

Expression of sodium-iodide symporter (NIS) is stimulated by sterol-regulatory-element-binding transcription factors (SREBFs) in mammary epithelial MCF-7 cells. Because conjugated linoleic acid (CLA) isomers have been shown to inhibit transcriptional activity of SREBFs in the mammary gland, the hypothesis was tested that CLA isomers inhibit NIS expression induced by all- trans retinoic acid (ATRA) in MCF-7 cells through inhibiting SREBF activity. c9t11-CLA and t10c12-CLA decreased ATRA-induced NIS-mRNA expression from 1.00 (ATRA alone) to 0.80 ± 0.12 (200 µM c9t11-CLA, P < 0.05) and 0.62 ± 0.10 (200 µM t10c12-CLA, P < 0.05), NIS-protein expression from 1.00 (ATRA alone) to 0.77 ± 0.08 (200 µM c9t11-CLA, P < 0.05) and 0.63 ± 0.05 (200 µM t10c12-CLA, P < 0.05), and NIS-promoter activity from 1.00 (ATRA alone) to 0.74 ± 0.13 (200 µM c9t11-CLA, P < 0.05) and 0.76 ± 0.13 (200 µM t10c12-CLA, P < 0.05); however, c9t11-CLA and t10c12-CLA increased the mRNA levels of SREBF isoforms and their target genes. In contrast, the mRNA expression of peroxisome-proliferator-activated receptor γ (PPARG) was strongly induced by ATRA alone but decreased by CLA isomers from 1.00 (ATRA alone) to 0.80 ± 0.06 (200 µM c9t11-CLA, P < 0.05) and 0.86 ± 0.06 (200 µM t10c12-CLA, P < 0.05). Overexpression of PPARγ in MCF-7 cells increased basal NIS-promoter activity, and treatment with the PPARγ ligand troglitazone stimulated ATRA-induced NIS-promoter activity. In conclusion, the results suggest that CLA isomers exert their effect on the expression of NIS by decreasing PPARG expression in MCF-7 cells.

The Antisteatotic and Hypolipidemic Effect of Insect Meal in Obese Zucker Rats is Accompanied by Profound Changes in Hepatic Phospholipid and 1-Carbon Metabolism.

SCOPE: The hypothesis is tested that insect meal, which has a low methionine content, reduces the hepatic phosphatidylcholine (PC):phosphatidylethanolamine (PE) ratio, which is a critical determinant of hepatic lipid synthesis, by decreasing availability of the methionine metabolite S-adenosylmethionine (SAM). METHODS AND RESULTS: Obese rats (n = 24) are randomly divided into two groups (Obese Casein and Obese Insect) of 12 rats each. In addition, lean rats (n = 12) are used as control group (LC). Groups LC and OC receive a control diet with casein as protein source, whereas in the OI group, casein is replaced isonitrogenously by insect meal, which is found to be less digestible (-12% units). Plasma and liver concentrations of lipids and hepatic expression of lipid synthesizing genes are reduced in the OI group compared to the OC group. Plasma and liver concentration of PC and the PC:PE ratio are decreased in the OI group compared to the OC group, while hepatic concentration of SAM and the hepatic SAM:S-adenosylhomocysteine (SAH) ratio is lower in the OI group than in the OC group. CONCLUSION: The decrease of the hepatic PC:PE ratio is probably a key mechanism explaining the pronounced antisteatotic and hypolipidemic action of insect meal in obese rats.

Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis.

Transcriptional regulation of the human, porcine and bovine OCTN2 gene by PPARα via a conserved PPRE located in intron 1.

BACKGROUND: The novel organic cation transporter 2 (OCTN2) is the physiologically most important carnitine transporter in tissues and is responsible for carnitine absorption in the intestine, carnitine reabsorption in the kidney and distribution of carnitine between tissues. Genetic studies clearly demonstrated that the mouse OCTN2 gene is directly regulated by peroxisome proliferator-activated receptor α (PPARα). Despite its well conserved role as an important regulator of lipid catabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle, and human. In the present study we examined the hypothesis that the porcine, bovine, and human OCTN2 gene are also PPARα target genes. RESULTS: Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene. Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene. CONCLUSIONS: The results of the present study show that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, is directly regulated by PPARα. This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

Sterol regulatory element-binding proteins are regulators of the rat thyroid peroxidase gene in thyroid cells.

Sterol regulatory element-binding proteins (SREBPs)-1c and -2, which were initially discovered as master transcriptional regulators of lipid biosynthesis and uptake, were recently identified as novel transcriptional regulators of the sodium-iodide symporter gene in the thyroid, which is essential for thyroid hormone synthesis. Based on this observation that SREBPs play a role for thyroid hormone synthesis, we hypothesized that another gene involved in thyroid hormone synthesis, the thyroid peroxidase (TPO) gene, is also a target of SREBP-1c and -2. Thyroid epithelial cells treated with 25-hydroxycholesterol, which is known to inhibit SREBP activation, had about 50% decreased mRNA levels of TPO. Similarly, the mRNA level of TPO was reduced by about 50% in response to siRNA mediated knockdown of both, SREBP-1 and SREBP-2. Reporter gene assays revealed that overexpression of active SREBP-1c and -2 causes a strong transcriptional activation of the rat TPO gene, which was localized to an approximately 80 bp region in the intron 1 of the rat TPO gene. In vitro- and in vivo-binding of both, SREBP-1c and SREBP-2, to this region in the rat TPO gene could be demonstrated using gel-shift assays and chromatin immunoprecipitation. Mutation analysis of the 80 bp region of rat TPO intron 1 revealed two isolated and two overlapping SREBP-binding elements from which one, the overlapping SRE+609/InvSRE+614, was shown to be functional in reporter gene assays. In connection with recent findings that the rat NIS gene is also a SREBP target gene in the thyroid, the present findings suggest that SREBPs may be possible novel targets for pharmacological modulation of thyroid hormone synthesis.

Sterol regulatory element-binding proteins are regulators of the NIS gene in thyroid cells.

The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS.

Supplementation of a grape seed and grape marc meal extract decreases activities of the oxidative stress-responsive transcription factors NF-κB and Nrf2 in the duodenal mucosa of pigs.

BACKGROUND: In pigs, enteric infections and the development of gut disorders such as diarrhoea are commonly observed, particularly after weaning. The present study investigated the hypothesis that feeding a grape seed and grape marc extract (GSGME) as a dietary supplement has the potential to suppress the inflammatory process in the small intestine of pigs by modulating the activities of NF-κB and Nrf2 due to its high content of flavonoids. METHODS: Twenty-four crossbred, 6 weeks old pigs were randomly assigned to 2 groups of 12 animals each and fed nutritionally adequate diets without or with 1% GSGME for 4 weeks. RESULTS: Pigs administered GSGME had a lower transactivation of NF-κB and Nrf2 and a lower expression of various target genes of these transcription factors in the duodenal mucosa than control pigs (P < 0.05). Concentrations of α-tocopherol and thiobarbituric acid reactive substances (TBARS) in liver and plasma and total antioxidant capacity of plasma and relative mRNA abundances of NF-κB and Nrf2 target genes in the liver did not differ between the two groups. However, the ratio of villus height:crypt depth and the gain:feed ratio was higher in the pigs fed GSGME than in control pigs (P < 0.05). CONCLUSIONS: This study shows that dietary supplementation of a polyphenol rich GSGME suppresses the activity of NF-κB in the duodenal mucosa of pigs and thus might provide a useful dietary strategy to inhibit inflammation in the gut frequently occurring in pigs. Feeding GSGME did not influence vitamin E status and the antioxidant system of the pigs but improved the gain:feed ratio. In overall, the study suggests that polyphenol-rich plant extracts such GSGME could be useful feed supplements in pig nutrition, in order to maintain animal health and improve performance.

13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages.

BACKGROUND: Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. METHODS: RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. RESULTS: Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells. CONCLUSION: 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

Mouse γ-butyrobetaine dioxygenase is regulated by peroxisome proliferator-activated receptor α through a PPRE located in the proximal promoter.

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5'-truncation BBD promoter reporter constructs revealed that one PPRE located at -75 to -87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.

Mouse OCTN2 is directly regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) via a PPRE located in the first intron.

Recent studies provided strong evidence to suggest that organic cation transporter 2 (OCTN2) is a direct target gene of peroxisome proliferator-activated receptor alpha (PPARalpha). However, subsequent studies failed to demonstrate a functional peroxisome proliferator response element (PPRE) in the promoter region of the OCTN2 gene. In the present study we hypothesized that the OCTN2 gene is transcriptionally induced by PPARalpha via a functional PPRE located in the first intron. In silico-analysis of the first intron of mouse OCTN2 revealed 11 putative PPRE with high similarity to the consensus PPRE. In addition, reporter gene assays using a mouse OCTN2 intron reporter construct containing a cluster of three partially overlapping PPRE (PPREint-1-8-10) revealed a marked response to exogenous mouse PPARalpha/RXRalpha and subsequent stimulation with PPARalpha agonist WY-14,643. Introduction of a selective mutation in either PPRE8 or PPRE10 in the PPREint-1-8-10 reporter constructs caused a substantial loss of the responsiveness to PPARalpha activation, but a selective mutation in PPRE1 resulted in a complete loss of responsiveness to PPARalpha activation. Moreover, gel shift assays revealed binding of PPARalpha/RXRalpha heterodimer to the PPRE1 of mouse OCTN2 first intron. In conclusion, the present study shows that mouse OCTN2 is a direct target gene of PPARalpha and that transcriptional upregulation of OCTN2 by PPARalpha is likely mediated via PPRE1 in its first intron.

Mouse carnitine-acylcarnitine translocase (CACT) is transcriptionally regulated by PPARalpha and PPARdelta in liver cells.

BACKGROUND: Hepatic PPARalpha acts as the primary mediator of the adaptive response to fasting by upregulation of a number of genes involved in fatty acid catabolism. Whether carnitine-acylcarnitine translocase (CACT), which mediates the import of acylcarnitines into the mitochondrial matrix for subsequent beta-oxidation of fatty acid moieties, is also regulated by PPARalpha in the liver has not yet been investigated. METHODS AND RESULTS: Herein, we observed that hepatic mRNA abundance of CACT was increased by both, fasting and treatment with PPARalpha agonist WY-14,643 in wild-type mice but not PPARalpha-knockout mice (P<0.05). Cell culture experiments revealed that CACT mRNA abundance was higher in liver cells treated with either WY-14,643 or PPARdelta agonist GW0742, but not with PPARgamma agonist troglitazone (TGZ) than in control cells (P<0.05). In addition, reporter assays revealed activation of mouse CACT promoter by WY-14,643 and GW0742, but not TGZ. Moreover, deletion and mutation analyses of CACT promoter and 5'-UTR revealed one functional PPRE in the 5'-UTR of mouse CACT. GENERAL SIGNIFICANCE: CACT is upregulated by PPARalpha and PPARdelta, probably by binding to a functional PPRE at position +45 to +57 relative to the transcription start site. The upregulation of CACT by PPARalpha and PPARdelta, which are both important for the regulation of fatty acid oxidation in tissues during fasting, may increase the import of acylcarnitine into the mitochondrial matrix during fasting.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.

Exploring the chemistry of uncultivated bacterial symbionts: antitumor polyketides of the pederin family.

Symbiotic bacteria have long been proposed as being responsible for the production of numerous natural products isolated from invertebrate animals. However, systematic studies of invertebrate-symbiont associations are usually associated with serious technical challenges, such as the general resistance of symbionts to culturing attempts and the complexity of many microbial consortia. Herein an overview is provided on the culture-independent, metagenomic strategies recently employed by our group to contribute to a better understanding of natural product symbiosis. Using terrestrial Paederus spp. beetles and the marine sponge Theonella swinhoei as model animals, the putative genes responsible for the production of pederin-type antitumor polyketides have been isolated. In Paederus fuscipes, which uses pederin for chemical defense, these genes belong to an as-yet unculturable symbiont closely related to Pseudomonas aeruginosa. To study the extremely complex association of T. swinhoei and its multispecies bacterial consortium, we used a phylogenetic approach that allowed the isolation of onnamide/theopederin polyketide synthase genes from an uncultured sponge symbiont. Analysis of the biosynthesis genes provided unexpected insights into a possible evolution of pederin-type pathways. Besides revealing new facets of invertebrate chemical ecology, these first gene clusters from uncultivated symbiotic producers suggest possible biotechnological strategies to solve the supply problem associated with the development of most marine drug candidates.

The DNA sequence of the human X chromosome.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei.

Bacterial symbionts have long been suspected to be the true producers of many drug candidates isolated from marine invertebrates. Sponges, the most important marine source of biologically active natural products, have been frequently hypothesized to contain compounds of bacterial origin. This symbiont hypothesis, however, remained unproven because of a general inability to cultivate the suspected producers. However, we have recently identified an uncultured Pseudomonas sp. symbiont as the most likely producer of the defensive antitumor polyketide pederin in Paederus fuscipes beetles by cloning the putative biosynthesis genes. Here we report closely related genes isolated from the highly complex metagenome of the marine sponge Theonella swinhoei, which is the source of the onnamides and theopederins, a group of polyketides that structurally resemble pederin. Sequence features of the isolated genes clearly indicate that it belongs to a prokaryotic genome and should be responsible for the biosynthesis of almost the entire portion of the polyketide structure that is correlated with antitumor activity. Besides providing further proof for the role of the related beetle symbiont-derived genes, these findings raise intriguing ecological and evolutionary questions and have important general implications for the sustainable production of otherwise inaccessible marine drugs by using biotechnological strategies.