Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2.

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2.HIGHLIGHTSFAD synthase deficiency alters mitochondrial morphology and bioenergetics;FAD synthase deficiency triggers a mitochondrial retrograde response;FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

Amelioration of hydrolyzed guar gum on high-fat diet-induced obesity: Integrated hepatic transcriptome and metabolome.

Hydrolyzed guar gum has gained attention as an anti-obesity agent; however, few studies have focused on its role in amelioration of hepatic-associated metabolic processes. Here, the anti-obesity effect of low molecular weight hydrolyzed guar gum (GMLP, 1-10 kDa) on high-fat diet (HFD)-fed C57BL/6 J mice was investigated via transcriptome and metabolome in liver. GMLP reduced body weight gain and hepatic lipid accumulation dose-dependently, regulated blood lipid levels, and improved liver damage in HFD-fed mice. Integrated transcriptome and metabolome indicated that GMLP mainly altered lipid metabolism pathways (glycerophospholipid metabolism, glycerolipid metabolism, and fatty acid degradation), reduced disease biomarkers of ethyl glucuronide and neopterin, and increased levels of choline, flavin adenine dinucleotide, and pantetheine metabolites. Real-time quantitative PCR showed that GMLP downregulated key genes involved in de novo lipogenesis and triacylglycerol synthesis, while promoting fatty acid oxidation and choline synthesis. This study provides a theoretical basis for GMLP treatment in future clinical applications.

Roseoflavin, a Natural Riboflavin Analogue, Possesses In Vitro and In Vivo Antiplasmodial Activity.

The ability of the human malaria parasite Plasmodium falciparum to access and utilize vital nutrients is critical to its growth and proliferation. Molecules that interfere with these processes could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilization of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focusing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4 to 5 days. Our data are consistent with riboflavin metabolism and/or the utilization of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

The effect of citalopram treatment on amyloid-ß precursor protein processing and oxidative stress in human hNSC-derived neurons.

Selective Serotonin Reuptake Inhibitors (SSRIs) may hold therapeutic benefits for people with Alzheimer's disease (AD). SSRIs may perturb AD progression, or the conversion from MCI to AD, via increased neurogenesis, reduced oxidative stress and/or favourable Amyloid-ß Precursor Protein (AßPP) processing. This study used iPSC derived cortical neuronal cells carrying 3 different PSEN1 mutations, to investigate the effect of treatment with the SSRI, Citalopram on AßPP processing and oxidative stress. Control and PSEN1 mutation (L286V, A246E, M146L) iPSC-derived neurons were treated with Citalopram for 45 days. ADAM10 activity, AßPP processing and Aß generation was measured in addition to cellular redox status. Citalopram treatment reduced the Aß1-42:40 ratio in control but not in fAD PSEN1 cells. ADAM10 activity was increased with Citalopram treatments in fAD PSEN1 cell lines, which was also seen for sAßPPα secretion. Lower superoxide generation in fAD PSEN1 cells following Citalopram treatment was identified, although there was no effect on end markers of oxidative stress. Treatment with Citalopram appears to have little effect on Aß generation in fADPSEN1 cells, but our findings suggest that treatment can significantly increase non-amyloidogenic AßPP processing and reduce oxidative stress. These changes may explain why SSRIs appear most effective in the prodromal period of the disease progression, as opposed to reducing established AD pathology. Further investigation of specific pathways conferring the beneficial effects of SSRIs treatment are warranted.

Flavin adenine dinucleotide ameliorates hypertensive vascular remodeling via activating short chain acyl-CoA dehydrogenase.

AIMS: Flavin adenine dinucleotide (FAD), participates in fatty acid ß oxidation as a cofactor, which has been confirmed to enhance SCAD activity and expression. However, the role of FAD on hypertensive vascular remodeling is unclear. In this study, we investigated the underlying mechanisms of FAD on vascular remodeling and endothelial homeostasis. MAIN METHODS: Morphological examination of vascular remodeling were analyzed with hematoxylin and eosin (HE) staining, Verhoeff's Van Gieson (EVG) staing, Dihydroethidium (DHE) staining and Sirius red staining. HUVECs apoptotic rate was detected by flow cytometry and HUVECs reactive oxygen species (ROS) was detected by DHE-probe. Enzymatic reactions were used to detect SCAD enzyme activity. The protein level was detected by Western Blots, the mRNA level was detected by quantitative real-time PCR. KEY FINDINGS: In vivo experiments, FAD significantly decreased blood pressure and ameliorated vascular remodeling by increasing SCAD expression, Nitric Oxide (NO) production and reducing ROS production. In vitro experiments, FAD protected against the tBHP induced injury in HUVEC, by increasing the activity of SCAD, increasing the elimination of free fatty acid (FFA), scavenging ROS, reducing apoptotic rate, thereby improving endothelial cell function. SIGNIFICANCE: FAD has a new possibility for preventing and treating hypertensive vascular remodeling.

Efficacy and safety of human placental extract for alcoholic and nonalcoholic steatohepatitis: an open-label, randomized, comparative study.

Human placental extract (HPE) is a traditional medicine that has been used for the symptomatic treatment of liver disease without any verifying clinical evidence. This study aimed to evaluate the efficacy and safety of HPE in patients with alcoholic or nonalcoholic steatohepatitis (ASH or NASH). We designed this clinical trial as a multicenter, open-label, randomized, comparative noninferiority study to improve the reliability of analyses. The enrollment criteria were limited to ASH or NASH patients with serum alanine aminotransferase (ALT) 1.5-fold higher than the normal level. Patients in the control group were treated with a commercially available mixture of liver extract and flavin adenine dinucleotide (LE­FAD). Intention-to-treat (ITT) analysis was applied to 194 patients, and per-protocol (PP) analysis was available for 154 patients. The rate of primary goal achievement of treatment efficacy was arbitrarily defined as 20% or greater improvement in ALT level compared with the pretreatment level and did not differ significantly between the HPE and control groups [62.9% (44/70) vs. 48.8% (41/84); p=0.0772]. ITT and modified ITT analysis showed results similar to those of PP analysis. Adverse drug reactions (ADRs) of minimal to moderate degree occurred in 3.1% of patients. The ADR and treatment compliance rates were similar in both groups. In conclusion, the clinical value of HPE in the treatment of ASH and NASH is equivalent to that of LE­FAD.

Additive therapeutic effects of the liver extract preparation mixture adelavin-9 on interferon-beta treatment for chronic hepatitis C.

BACKGROUND/AIMS: We previously reported that intravenous interferon-beta administration was effective in treating patients with chronic hepatitis C and that there was no significant correlation between the response to therapy and host in vitro immunoglobulin production. The aims of this study were to evaluate the additive effect of a liver extract preparation and flavin adenine dinucleotide mixture and to reevaluate the correlation. METHODOLOGY: 65 patients with chronic hepatitis C received intravenously 6 million units of interferon-beta and 2 mL of a liver extract preparation and flavin adenine dinucleotide mixture. The results of this study were compared with those of our previous study, on interferon-beta monotherapy in 91 patients with chronic hepatitis C. In addition, peripheral blood mononuclear cells were obtained before interferon-beta administration and cultured. Immunoglobulin concentrations in their supernatants were measured and the correlation with the interferon response was evaluated. RESULTS: The virological end-of-treatment or sustained response occurred in 49 of 58 cases (84.5%), and 16 of 58 cases (27.6%), respectively. Biochemical end-of-treatment or sustained responses occurred in 22 of 58 cases (37.9%), and 30 of 58 cases (51.7%), respectively. These response rates were higher than those reported in our previous study of interferon monotherapy. Monovariate analysis indicated that the use of the liver extract preparation and flavin adenine dinucleotide mixture was a significant predictor of the virological end-of-treatment response and the sustained biochemical response, but by multivariate analysis these relationships were not significant. Immunoglobulin production was not correlated with the virological and biochemical responses. CONCLUSIONS: This study indicated that combination therapy with intravenous interferon-beta and the liver extract preparation and flavin adenine dinucleotide mixture was more effective than intravenous interferon-beta monotherapy. However, no correlation between the interferon response and in vitro immunoglobulin production was observed.

Enhancing effect of the liver extract and flavin adenin dinucleotide mixture on anti-viral efficacy of interferon in patients with chronic hepatitis C.

A combined preparation of liver extract and flavin adenin dinucleotide (FAD) (Adelavin) has been widely used in patients with chronic liver diseases in Japan. One milliliter of this agent contains 15 microliters of phenol-soluble phase of liver nucleic acid fraction and 10 mg of FAD. To examine the advantages of using this preparation in the elimination of hepatitis C virus (HCV) from patients with chronic hepatitis (CH)-C receiving interferon (IFN), 2 ml of this preparation was intravenously (n = 9) or intramuscularly (n = 8) administered daily for 5 days before 6 million units of IFN-alpha was intramuscularly injected once. Before and 48 hours after the injection of IFN, serum ALT, 2'5'-oligoadenylate synthetase (2'5-AS) activity, and HCV RNA levels were measured. The daily administration of this preparation alone for 5 days did not significantly change serum ALT, 2'5-AS activities, and HCV RNA levels. The 2'5-AS activities were significantly increased by IFN after the intravenous injection of this preparation (p < 0.01), while an injection of IFN alone of this dose did not change its activities (n = 10). HCV RNA levels were significantly decreased by IFN only after the administration of the preparation (intramuscular, p < 0.01; intravenous, p < 0.01). The effect of intravenous injection of this preparation was also elicited in patients with HCV genotype II and with HCV more than 10(5) copies/ml. These results suggest that this preparation may enhance the 2'5-AS production by IFN as a result of the increase in mitochondrial adenosin triphosphate production and may be a potent agent to enhance the anti-viral efficacy of IFN in patients with CH-C.

Protective effects of riboflavin and its derivatives against ischemic reperfused damage of rat heart.

The effects of riboflavin and its derivatives such as FAD, FMN and lumichrome on the levels of high energy phosphate compounds (ATP and creatine phosphate) and intracellular pH in ischemic reperfused rat hearts were investigated using a Langendorff perfusion technique. 31P-NMR study showed a decrease in the levels of high energy phosphate compounds and pH values in myocardium after 30 min global ischemia and a slight recovery of these levels after a 30 min reperfusion following ischemia. However, in all the hearts perfused with riboflavin and its derivatives during ischemia-reperfusion, a marked recovery of high energy phosphate compounds and pH values were observed. In addition, the cardiac mitochondrial respiratory function was protected from ischemia-reperfusion injury. These results suggest that riboflavin, FAD, FMN, and lumichrome have a protective effect against ischemia-reperfusion injury to rat myocardium in vitro. It is assumed that these substances exert their effect directly in the extracellular space.

Erythrocyte enzymes in polycythemia vera: a comparison to erythrocyte enzyme activities of patients with iron deficiency anemia.

Fifteen cytoplasmic erythrocyte enzyme activities were determined in patients with polycythemia vera (PV), iron deficiency anemia (IDA), and a group of healthy volunteers. Among the PV patients, the erythrocyte enzyme activities were compared between 2 groups: patients treated solely with phlebotomy and patients treated with phlebotomy, Myleran (busulfan) and/or radioactive phosphorus 32P. Significant reduction in glutathione reductase activity was found in the PV group of patients. This activity was normalized by the addition of flavin adenine dinucleotide. In contrast to previous reports, no other enzyme activity was found to be significantly reduced. The activities of the enzymes known to be age-dependent were significantly elevated in all the groups tested except for phosphofructokinase and 3-phosphoglycerate mutase. The former was not elevated in any of the groups studied, while the latter was elevated only in the group of patients treated with Myleran and/or 32P. It was concluded that glutathione reductase (GR) deficiency is the only acquired enzyme deficiency in our group of PV patients and that radiation and chemotherapy did not induce further reduction in the activities of any of the enzymes tested. The possible involvement of GR deficiency in the etiology of the red cell life span shortening was discussed.

[Effect of flavin coenzymes on the pyridoxine treatment of avitaminosis B6]. / Vliianie flavinovykh kofermentov na lechenie avitaminoza B6 piridoksinom.

Studies on rats with experimental vitamin B6 deficiency have shown that the combined use of pyridoxine and flavine coenzymes affects the supply of rats with these vitamins. Flavine coenzymes have been found to promote more efficient normalization of pyridoxine and riboflavin balance in the body and to improve the general status of the animals, to increase the excretion of riboflavin and 4-pyridoxic acid with urine, to prove more rapid cure of vitamin deficiency.

[Effect of the combined use of flavin coenzyme preparations and pyridoxine on the body vitamin balance in animals]. / Vliianie sochetannogo primeneniia preparatov flavinovykh kofermentov i piridoksina na vitaminnyi balans organizma zhivotnykh.

The effect of combined administration of flavin coenzymes and pyridoxine on B2-avitaminosis rats' supply with these vitamins has been studied. It has been disclosed that pyridoxine promotes more effective normalization of riboflavin and pyridoxine balance in the body, this balance being measured from the excretion of riboflavin and 4-pyridoxin acid with urine as well as from the content of total flavins in blood and tissues. In vitamin B2 lack, it is recommended that pyridoxin be combined with flavin mononucleotide and in particular with flavin adenine dinucleotide.

Protection of chlorpromazine-induced arrhythmia by flavin-adenine-dinucleotide in canine heart.

To investigate the mechanism of chlorpromazine(CPZ)-induced ventricular arrhythmia, the changes in ventricular fibrillation threshold (VFT) were followed after intravenous injection of CPZ (1 mg/Kg) in dogs. Following injection, VFT was decreased to 56.6 +/- 5.4% (mean +/- SE) of the initial level. Since flavin-adenine-dinucleotide (FAD) combines specifically with CPZ in vitro, we investigate whether or not prior treatment with FAD prevents the CPZ effect. With FAD (2 mg/Kg), the CPZ-induced decrease in VFT was significantly cancelled (92.2 +/- 4.2% of the initial level). Mitochondria isolated from canine heart after CPZ injection showed a significant decrease in respiratory control index and ADP/O. Effects of CPZ on canine heart mitochondria were also well cancelled by prior administration of FAD. The findings suggest that the arrhythmogenic action of CPZ might be associated in part with impaired function of heart mitochondria. These results also suggest that FAD might be useful in the treatment of the cardiac disturbances associated with overdosage of CPZ.