Barriers to initiation of hepatitis C virus therapy in Germany: A retrospective, case-controlled study.

Despite the availability of highly effective and well-tolerated direct-acting antivirals, not all patients with chronic hepatitis C virus infection receive treatment. This retrospective, multi-centre, noninterventional, case-control study identified patients with chronic hepatitis C virus infection initiating (control) or not initiating (case) treatment at 43 sites in Germany from September 2017 to June 2018. It aimed to compare characteristics of the two patient populations and to identify factors involved in patient/physician decision to initiate/not initiate chronic hepatitis C virus treatment, with a particular focus on historical barriers. Overall, 793 patients were identified: 573 (72%) who received treatment and 220 (28%) who did not. In 42% of patients, the reason for not initiating treatment was patient wish, particularly due to fear of treatment (17%) or adverse events (13%). Other frequently observed reasons for not initiating treatment were in accordance with known historical barriers for physicians to initiate therapy, including perceived or expected lack of compliance (14.5%), high patient age (10.9%), comorbidities (15.0%), alcohol abuse (9.1%), hard drug use (7.7%), and opioid substitution therapy (4.5%). Patient wish against therapy was also a frequently reported reason for not initiating treatment in the postponed (35.2%) and not planned (47.0%) subgroups; of note, known historical factors were also common reasons for postponing treatment. Real-world and clinical trial evidence is accumulating, which suggests that such historical barriers do not negatively impact treatment effectiveness. Improved education is key to facilitate progress towards the World Health Organization target of eliminating viral hepatitis as a major public health threat by 2030.

Glecaprevir/pibrentasvir is safe and effective in hepatitis C patients with cirrhosis: Real-world data from the German Hepatitis C-Registry.

Glecaprevir/pibrentasvir is a pangenotypic direct-acting antiviral regimen approved for treating chronic hepatitis C virus. Real-world use of protease-inhibitor-containing regimens requires further evaluation in patients with cirrhosis. We evaluated the real-world safety and effectiveness of glecaprevir/pibrentasvir in patients with cirrhosis from the German Hepatitis C-Registry who initiated treatment between 2 August 2017 and 30 June 2019. Overall, 131 patients received 12-week (on-label) treatment and 51 received 8-week (off-label) treatment. No patient discontinued treatment due to adverse events. Four patients had serious adverse events; none were considered related to glecaprevir/pibrentasvir. Two patients had total bilirubin > 5 × upper limit of normal (ULN) during treatment. Three patients had alanine aminotransferase and three patients had aspartate aminotransferase > 3 × ULN. Rates of sustained virologic response were 100% (86/86) for 86 patients with available data. Glecaprevir/pibrentasvir treatment was well-tolerated and highly effective in patients with chronic hepatitis C and cirrhosis in real-world practice.

High-phosphorus diets reduce aortic lesions and cardiomyocyte size and modify lipid metabolism in Ldl receptor knockout mice.

The consumption of phosphorus in Western populations largely exceeds the recommended intake, while vitamin D supply is often insufficient. Both situations are linked to an increased cardiovascular risk. A 17-week two-factorial study with Ldl receptor-/- mice was conducted to investigate the cardiovascular impact of dietary phosphorus [adequate (0.3%; P0.3) vs. high (1.5%; P1.5)] in combination with a low (50 IU/kg; D50) or adequate vitamin D diet (1000 IU/kg; D1000). The data demonstrate that mice fed the P1.5 vs. P0.3 diets developed smaller vascular lesions (p = 0.013) and cardiac hypotrophy (p = 0.011), which were accompanied by diminished IGF1 and insulin signalling activity in their hearts. Vitamin D showed no independent effect on atherogenesis and heart morphology. Feeding P1.5 vs. P0.3 diets resulted in markedly reduced serum triacylglycerols (p < 0.0001) and cholesterol (p < 0.0001), higher faecal lipid excretion (p < 0.0001) and a reduced mRNA abundance of hepatic sterol exporters and lipoprotein receptors. Minor hypocholesterolaemic and hypotriglyceridaemic effects were also found in mice fed the D1000 vs. D50 diets (p = 0.048, p = 0.026). To conclude, a high phosphorus intake strongly affected the formation of vascular lesions, cardiac morphology, and lipid metabolism, although these changes are not indicative of an increased cardiovascular risk.

Vitamin D Receptor Deficiency Does Not Affect Blood Pressure and Heart Function.

Vitamin D is thought to play a role in blood pressure regulation, which in turn can influence cardiovascular risk. Several meta-analyses of cohort studies found low serum levels of 25-hydroxyvitamin D to be associated with increased blood pressure or increased cardiovascular morbidity and mortality in the general population. Active vitamin D mediates its function via the vitamin D receptor (Vdr), which is a ligand-activated transcription factor. A suitable model to examine the causal role of vitamin D in blood pressure regulation and heart function is the Vdr knockout (Vdr-/-) mouse. To elucidate the role of vitamin D on blood pressure, heart function, and cardiac myocyte size, we conducted a long-term study using Vdr-/- mice and well-defined diets. Group 1 comprised Vdr-/- mice that received a high-calcium, high-phosphorus rescue diet to prevent hypocalcemia and a rickets phenotype. Groups 2 and 3 included Vdr+/+ mice that were fed either the rescue diet or a control diet containing normal amounts of these minerals. As Vdr is a nuclear factor that regulates transcription, we analyzed the renal mRNA expression and serum concentration of renin and found that the Vdr-/- group had an almost 50% higher renin mRNA expression in the kidney compared to both groups of Vdr+/+ mice. Additionally, serum concentration of renin in Vdr-/- mice was significantly higher than that of Vdr+/+ mice that received the rescue or control diet (+ 17%,+ 32%; P < 0.05). In contrast, renin activity was lower in Vdr-/- mice than in both groups of Vdr+/+ mice (P < 0.05). However, blood pressure, heart rate, cardiac myocyte sizes, and the expression of renal renin receptor, hepatic angiotensinogen and angiotensin II receptor, type 1, in kidney, liver and heart, did not differ between the three groups of mice. Additionally, data from transthoracic echocardiography did not indicate the role of Vdr on heart function, as the left ventricular ejection fraction, fractional shortening, and velocity of blood flow were comparable between the three groups. To conclude, the roles of Vdr and therefore most probably of vitamin D, in blood pressure regulation and heart function, were not confirmed by our findings.

Real-world effectiveness and safety of glecaprevir/pibrentasvir for the treatment of chronic hepatitis C infection: data from the German Hepatitis C-Registry.

BACKGROUND: Glecaprevir/pibrentasvir is a pangenotypic direct-acting antiviral regimen approved for treating adults chronically infected with hepatitis C virus (HCV). There are limited real-world data on glecaprevir/pibrentasvir to date. AIM: To evaluate the effectiveness and safety of glecaprevir/pibrentasvir under real-world conditions in the German Hepatitis C-Registry (DHC-R). METHODS: The DHC-R is an ongoing, non-interventional, multicentre, prospective, observational cohort study that monitors patients with chronic HCV infection. Data were collected from patients who initiated glecaprevir/pibrentasvir and completed a screening visit on or after 2 August 2017. The primary effectiveness endpoint was sustained virological response at post-treatment Week 12 (SVR12). Safety and tolerability were also assessed. RESULTS: As of 15 July 2018, 586 patients received glecaprevir/pibrentasvir and had documented SVR12 data, treatment discontinuation, loss to follow-up or HCV reinfection. Five hundred and fifty-two patients (94%) received on-label treatment. At baseline, most on-label patients were infected with HCV genotype 1 (53%) or 3 (33%), HCV treatment-naïve (90%), without cirrhosis (94%), and treated for 8 weeks (93%). Five hundred and thirty-four patients (96.7%) achieved SVR12 (intention-to-treat [ITT] analysis). By modified ITT analysis (excluding patients who discontinued and did not achieve SVR12 or patients lost to follow-up), the SVR12 rate was 99.4% (n/N = 534/537). There was one documented virological failure (relapse) and two documented HCV reinfections. One hundred and forty-two (26%) adverse events (AEs) and 9 (2%) serious AEs occurred; 2 (<1%) AEs led to treatment discontinuation. All patients treated off-label (N = 34) achieved SVR12. CONCLUSION: Glecaprevir/pibrentasvir was highly effective and well tolerated under real-world conditions. Clinical trial number: DRKS00009717 (German Clinical Trials Register, DRKS).

Metabolic footprint and intestinal microbial changes in response to dietary proteins in a pig model.

Epidemiological studies revealed that dietary proteins can contribute to the modulation of the cardiovascular disease risk. Still, direct effects of dietary proteins on serum metabolites and other health-modulating factors have not been fully explored. Here, we compared the effects of dietary lupin protein with the effects of beef protein and casein on the serum metabolite profile, cardiovascular risk markers and the fecal microbiome. Pigs were fed diets containing 15% of the respective proteins for 4 weeks. A classification analysis of the serum metabolites revealed six biomarker sets of two metabolites each that discriminated between the intake of lupin protein, lean beef or casein. These biomarker sets included 1- and 3-methylhistidine, betaine, carnitine, homoarginine and methionine. The study revealed differences in the serum levels of the metabolites 1- and 3- methylhistidine, homoarginine, methionine and homocysteine, which are involved in the one-carbon cycle. However, these changes were not associated with differences in the methylation capacity or the histone methylation pattern. With the exception of serum homocysteine and homoarginine levels, other cardiovascular risk markers, such as the homeostatic model assessment index, trimethylamine-N-oxide and lipids, were not influenced by the dietary protein source. However, the composition of the fecal microorganisms was markedly changed by the dietary protein source. Lupin-protein-fed pigs exhibited more species from the phyla Bacteroidetes and Firmicutes than the other two groups. In conclusion, different dietary protein sources induce distinct serum metabolic fingerprints, have an impact on the cardiovascular risk and modulate the composition of the fecal microbiome.

Impact of a high-protein diet during lactation on milk composition and offspring in a pig model.

PURPOSE: Early postnatal nutrition not only holds relevance to infant growth, but also determines the risk of developing obesity and chronic diseases such as diabetes type 2 and cardiovascular diseases in adulthood. It is suggested that a high-protein (HP) diet in early childhood can predispose children to obesity. However, data concerning possible alterations in milk composition and the development of the offspring in response to a maternal HP diet are currently not available. To address this question, we conducted a study using pigs as a model organism. METHODS: At parturition, sows were assigned to two experimental groups. During lactation, the control group received a diet with a protein content of 16%, whereas the diet of the HP group contained 30% protein. After 28 days of lactation, samples were taken from sows and piglets for the quantification of free amino acids and other metabolites and for histology. RESULTS: Serum and milk urea showed the most marked differences between the two groups of sows, whereas serum urea concentration in piglets did not differ. Here, we found that the intake of an HP diet changed a series of metabolites in sows, but had only small effects on milk composition and virtually no effects on growth in the offspring. Interestingly, maternal protein intake during lactation shapes the microbiome of the offspring. CONCLUSION: From our current study, we conclude that even a very high maternal protein intake throughout lactation has no impact on growth and health parameters of the offspring.

Plant Oils as Potential Sources of Vitamin D.

To combat vitamin D insufficiency in a population, reliable diet sources of vitamin D are required. The recommendations to consume more oily fish and the use of UVB-treated yeast are already applied strategies to address vitamin D insufficiency. This study aimed to elucidate the suitability of plant oils as an alternative vitamin D source. Therefore, plant oils that are commonly used in human nutrition were first analyzed for their content of vitamin D precursors and metabolites. Second, selected oils were exposed to a short-term UVB irradiation to stimulate the synthesis of vitamin D. Finally, to elucidate the efficacy of plant-derived vitamin D to improve the vitamin D status, we fed UVB-exposed wheat germ oil (WGO) for 4 weeks to mice and compared them with mice that received non-exposed or vitamin D3 supplemented WGO. Sterol analysis revealed that the selected plant oils contained high amounts of not only ergosterol but also 7-dehydrocholesterol (7-DHC), with the highest concentrations found in WGO. Exposure to UVB irradiation resulted in a partial conversion of ergosterol and 7-DHC to vitamin D2 and D3 in these oils. Mice fed the UVB-exposed WGO were able to improve their vitamin D status as shown by the rise in the plasma concentration of 25-hydroxyvitamin D [25(OH)D] and the liver content of vitamin D compared with mice fed the non-exposed oil. However, the plasma concentration of 25(OH)D of mice fed the UVB-treated oil did not reach the values observed in the group fed the D3 supplemented oil. It was striking that the intake of the UVB-exposed oil resulted in distinct accumulation of vitamin D2 in the livers of these mice. In conclusion, plant oils, in particular WGO, contain considerable amounts of vitamin D precursors which can be converted to vitamin D via UVB exposure. However, the UVB-exposed WGO was less effective to improve the 25(OH)D plasma concentration than a supplementation with vitamin D3.

High Leucine Diets Stimulate Cerebral Branched-Chain Amino Acid Degradation and Modify Serotonin and Ketone Body Concentrations in a Pig Model.

In addition to its role as an essential protein component, leucine (Leu) displays several other metabolic functions such as activation of protein synthesis. This property makes it an interesting amino acid for the therapy of human muscle atrophy and for livestock production. However, Leu can stimulate its own degradation via the branched-chain keto acid dehydrogenase complex (BCKDH). To examine the response of several tissues to excessive Leu, pigs were fed diets containing two- (L2) and four-fold (L4) higher Leu contents than the recommended amount (control). We found that the L4 diet led to a pronounced increase in BCKDH activity in the brain (2.5-fold, P < 0.05), liver (1.8-fold, P < 0.05) and cardiac muscle (1.7-fold, P < 0.05), whereas we found no changes in enzyme activity in the pancreas, skeletal muscle, adipose tissue and intestinal mucosa. The L2 diet had only weak effects on BCKDH activity. Both high Leu diets reduced the concentrations of free valine and isoleucine in nearly all tissues. In the brain, high Leu diets modified the amount of tryptophan available: for serotonin synthesis. Compared to the controls, pigs treated with the high Leu diets consumed less food, showed increased plasma concentrations of 3-hydroxybutyrate and reduced levels of circulating serotonin. In conclusion, excessive Leu can stimulate BCKDH activity in several tissues, including the brain. Changes in cerebral tryptophan, along with the changes in amino acid-derived metabolites in the plasma may limit the use of high Leu diets to treat muscle atrophy or to increase muscle growth.

Tissue-specific expression of monocarboxylate transporters during fasting in mice.

Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs). Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4), thyroid hormones (MCT8, -10) and aromatic amino acids (MCT10). Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR)-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT) and PPARα knockout (KO) mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1-4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms.

Vitamin D receptor regulates intestinal proteins involved in cell proliferation, migration and stress response.

BACKGROUND: Genome-wide association studies found low plasma levels of 25-hydroxyvitamin D and vitamin D receptor (VDR) polymorphisms associated with a higher prevalence of pathological changes in the intestine such as chronic inflammatory bowel diseases. METHODS: In this study, a proteomic approach was applied to understand the overall physiological importance of vitamin D in the small intestine, beyond its function in calcium and phosphate absorption. RESULTS: In total, 569 protein spots could be detected by two-dimensional-difference in-gel electrophoresis (2D-DIGE), and 82 proteins were considered as differentially regulated in the intestinal mucosa of VDR-deficient mice compared to that of wildtype (WT) mice. Fourteen clearly detectable proteins were identified by MS/MS and further analyzed by western blot and/or real-time RT-PCR. The differentially expressed proteins are functionally involved in cell proliferation, cell adhesion and cell migration, stress response and lipid transport. Mice lacking VDR revealed higher levels of intestinal proteins associated with proliferation and migration such as the 37/67 kDa laminin receptor, collagen type VI (alpha 1 chain), keratin-19, tropomyosin-3, adseverin and higher levels of proteins involved in protein trafficking and stress response than WT mice. In contrast, proteins that are involved in transport of bile and fatty acids were down-regulated in small intestine of mice lacking VDR compared to WT mice. However, plasma and liver concentrations of cholesterol and triglycerides were not different between the two groups of mice. CONCLUSION: Collectively, these data imply VDR as an important factor for controlling cell proliferation, migration and stress response in the small intestine.

Treatment with pharmacological PPARα agonists stimulates the ubiquitin proteasome pathway and myofibrillar protein breakdown in skeletal muscle of rodents.

BACKGROUND: Treatment of hyperlipidemic patients with fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), provokes muscle atrophy as a side effect. The molecular mechanism underlying this phenomenon is still unknown. We tested the hypothesis that activation of PPARα leads to an up-regulation of the ubiquitin proteasome system (UPS) which plays a major role in protein degradation in muscle. METHODS: Rats, wild-type and PPARα-deficient mice (PPARα(-/-)) were treated with synthetic PPARα agonists (clofibrate, WY-14,643) to study their effect on the UPS and myofibrillar protein breakdown in muscle. RESULTS: In rats and wild-type mice but not PPARα(-/-) mice, clofibrate or WY-14,643 caused increases in mRNA and protein levels of the ubiquitin ligases atrogin-1 and MuRF1 in muscle. Wild-type mice treated with WY-14,643 had a greater 3-methylhistidine release from incubated muscle and lesser muscle weights. In addition, wild-type mice but not PPARα(-/-) mice treated with WY-14,643 had higher amounts of ubiquitin-protein conjugates, a decreased activity of PI3K/Akt1 signalling, and an increased activity of FoxO1 transcription factor in muscle. Reporter gene and gel shift experiments revealed that the atrogin-1 and MuRF1 promoter do not contain functional PPARα DNA-binding sites. CONCLUSIONS: These findings indicate that fibrates stimulate ubiquitination of proteins in skeletal muscle which in turn stimulates protein degradation. Up-regulation of ubiquitin ligases is probably not mediated by PPARα-dependent gene transcription but by PPARα-dependent inhibition of the PI3K/Akt1 signalling pathway leading to activation of FoxO1. GENERAL SIGNIFICANCE: PPARα plays a role in the regulation of the ubiquitin proteasome system.

PPARα modulates the TSH ß-subunit mRNA expression in thyrotrope TαT1 cells and in a mouse model.

SCOPE: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis. METHODS AND RESULTS: Thyroid-stimulating hormone ß-subunit (TSHß) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.05), indicating an inhibitory transcriptional regulation of TSHß by PPARα. As expected, fasting significantly downregulated TSHß mRNA expression in a two-factorial study with fed or fasted wild-type (WT) and PPARα knockout mice (p < 0.05). In contrast to the in vitro data, fasted PPARα knockout mice revealed lower mRNA concentrations of pituitary TSHß (-64%) and TSH-regulated thyroid genes, and lower plasma concentrations of thyroxine (T4, -25%), triiodothyronine (T3, -25%), free T4 (-60%), and free T3 (-35%) than fasted WT mice (p < 0.05). Those differences were not observed in fed mice. CONCLUSIONS: Data from thyrotrope cells revealed that PPARα could contribute to the fasting-associated downregulation of the TSHß mRNA expression. In a mouse model, fasting led to a significant reduction in TSHß mRNA level, but unexpectedly this effect was stronger in mice lacking PPARα than in WT mice.

Monocarboxylate transporter 1 and CD147 are up-regulated by natural and synthetic peroxisome proliferator-activated receptor alpha agonists in livers of rodents and pigs.

Monocarboxylate transporter (MCT)-1 mediates the transport of ketone bodies and other monocarboxylic acids across the plasma membrane. MCT1 is up-regulated by peroxisome proliferator-activated receptor (PPAR)-alpha, a transcription factor that mediates the adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketogenesis. Here, we show for the first time that MCT1 is up-regulated by dietary natural PPAR-alpha agonists. Both, an oxidized fat and conjugated linoleic acids increased MCT1 mRNA concentration in the liver of rats. Also, in the liver of pigs as non-proliferating species MCT1 was up-regulated upon PPAR-alpha activation by clofibrate, oxidized fat and fasting. Concomitant with up-regulation of MCT1, mRNA level of CD147 was increased in livers of rats and pigs. CD147 is a plasma membrane glycoprotein that is required for translocation and transport activity of MCT1. CD147 mRNA increase upon PPAR-alpha activation could not be observed in mice lacking PPAR-alpha, which also fail in up-regulation of MCT1 indicating a co-regulation of MCT1 and CD147. Analysis of the 5'-flanking region of mouse MCT1 gene by reporter gene assay revealed that promoter activity of mouse MCT1 was not induced by PPAR-alpha, indicating that the 5'-flanking region is not involved in MCT1 regulation by PPAR-alpha.

Fasting Upregulates PPARalpha Target Genes in Brain and Influences Pituitary Hormone Expression in a PPARalpha Dependent Manner.

PPARalpha is a lipid-activable transcription factor that mediates the adaptive response to fasting. Recent data indicate an important role of brain PPARalpha in physiological functions. However, it has not yet been shown whether PPARalpha in brain can be activated in the fasting state. Here we demonstrate that fasting of rats increased mRNA concentrations of typical PPARalpha target genes implicated in beta-oxidation of fatty acids (acyl-CoA oxidase, carnitine palmitoyltransferase-1, medium chain acyl-CoA dehydrogenase) and ketogenesis (mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase) in pituitary gland and partially also in frontal cortex and diencephalon compared to nonfasted animals. These data strongly indicate that fasting activates PPARalpha in brain and pituitary gland. Furthermore, pituitary prolactin and luteinizing hormone-beta mRNA concentrations were increased upon fasting in wild-type mice but not in mice lacking PPARalpha. For proopiomelanocortin and thyrotropin-beta, genotype-specific differences in pituitary mRNA concentrations were observed. Thus, PPARalpha seems to be involved in transcriptional regulation of pituitary hormones.

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.

Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

Activation of PPARalpha and PPARgamma reduces triacylglycerol synthesis in rat hepatoma cells by reduction of nuclear SREBP-1.

Fibrates and thiazolidinediones, agonists of PPARalpha and PPARgamma, respectively, reduce triglyceride concentrations in rat liver and plasma. Fatty acid and triacylglycerol synthesis in mammals is regulated by sterol regulatory element-binding protein (SREBP)-1c. Recently, it was shown that insulin-induced gene (Insig)-1, the key regulator of SREBP activity, is up-regulated by both activation of PPARalpha and PPARgamma. In order to elucidate whether inhibition of SREBP-1 activation may contribute to the triacylglycerol lowering effect of PPARalpha and PPARgamma agonists, we incubated rat hepatoma Fao cells with WY 14,643 and troglitazone, strong and selective agonists of PPARalpha and PPARgamma, respectively. Activation of both, PPARalpha and PPARgamma led to increased concentrations of Insig-1 and Insig-2a, with the most prominent effect on Insig-2a after troglitazone incubation. As a result, the amount of nuclear SREBP-1 was reduced in Fao cells by both WY 14,643 and troglitazone treatment. The reduction of nuclear SREBP-1 was associated with decreased mRNA concentrations of its target genes fatty acid synthase and glycerol-3-phosphate acyltransferase, implicated in fatty acid and triacylglycerol synthesis. This was finally reflected in reduced rates of newly synthesized triacylglycerols from de novo-derived fatty acids and decreased intracellular and secreted triacylglycerol concentrations in Fao cells treated with WY 14,643 and troglitazone, respectively. Thus, these data suggest that the triacylglycerol reducing effect of fibrates and thiazolidinediones is partially caused by inhibition of SREBP-1 activation via up-regulation of Insig.

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARalpha.

Peroxisome proliferator-activated receptor (PPAR)-alpha mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARalpha in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARalpha agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARgamma agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARalpha we treated wild-type and PPARalpha-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARalpha-null mice.

PPAR alpha mediates transcriptional upregulation of novel organic cation transporters-2 and -3 and enzymes involved in hepatic carnitine synthesis.

We tested the hypothesis that transcription of novel organic cation transporters (OCTNs) is directly regulated by peroxisome proliferator-activated receptor (PPAR)-alpha. Therefore, wild-type mice and mice deficient in PPAR alpha (PPAR alpha-/-) were treated with the PPAR alpha agonist WY 14,643. Wild-type mice treated with WY 14,643 had a greater abundance of OCTN2 mRNA in their liver, muscle, kidney, and small intestine and a greater abundance of OCTN3 mRNA in kidney and small intestine than did untreated wild-type mice (P < 0.05). Moreover, wild-type mice treated with WY 14,643 had greater mRNA abundances of enzymes involved in hepatic carnitine synthesis (4-N-trimethylaminobutyraldehyde dehydrogenase, gamma-butyrobetaine dioxygenase) and increased carnitine concentrations in liver and muscle than did untreated wild-type mice (P < 0.05). Untreated PPAR alpha-/- mice had a lower abundance of OCTN2 mRNA in liver, kidney, and small intestine and lower carnitine concentrations in plasma, liver, and kidney than did untreated wild-type mice (P < 0.05). In PPAR alpha-/- mice, treatment with WY 14,643 did not influence mRNA abundance of OCTN2 and OCTN3 and carnitine concentrations in all tissues analyzed. The abundance of OCTN1 mRNA in all the tissues analyzed was not changed by treatment with WY 14,643 in wild-type or PPAR alpha-/- mice. In conclusion, this study shows that transcriptional upregulation of OCTN2 and OCTN3 in tissues and of enzymes involved in hepatic carnitine biosynthesis are mediated by PPAR alpha. It also shows that PPAR alpha mediates changes of whole-body carnitine homeostasis in mice by upregulation of carnitine transporters and enzymes involved in carnitine synthesis.