Omentectomy Prevents Metabolic Syndrome By Reducing Appetite and Body Weight In A Diet-Induced Obesity Rat Model.

Visceral fat deposition is associated with impairment of glucose and lipid metabolism while leptin levels are frequently related to subcutaneous fat area. At present, there is considerable controversy regarding the role of visceral adipose tissue accumulation in the development of metabolic syndrome (MS). Here we show the effects of omentectomy on the liver and MS in a diet induced obesity rat model. Our results reveal that undergoing omentectomy previously the establishment of the diet-induced-obesity reduced significantly body weight gain and avoid the development of MS, including non-alcoholic fatty liver disease. Intriguingly, the significantly lower body weight gain was due to decreased food intake. Omentum drives obesity progression through leptin resistance mediated by C-reactive protein, Interleucin (IL)-6 and high lipolysis activity. Omentum removal reversed immediately the increased plasma levels of CRP and IL-6 and gradually food intake, weight gain, and features of MS in diet-induced-obesity. Omentectomy caused no changes in normal-weigh-rats. This report displays causal mechanism by which omentum promotes obesity and propose omentectomy as a promising procedure in MS prevention.

NADPH oxidase is implicated in the pathogenesis of oxidative phosphorylation dysfunction in mice fed a high-fat diet.

The aim of this study was to evaluate the role of NADPH oxidase (NADPHox) in the pathogenesis of oxidative phosphorylation (OXPHOS) dysfunction as found in mice fed a high-fat diet (HFD). C57BL/6J mice were distributed in four groups: WT/SCD: six wild-type (WT) mice fed a standard chow diet (SCD); WT/HFD, six WT mice fed a HFD; NOX2(-/-)/SCD, six NADPHox-deficient mice on a SCD; (4) NOX2(-/-)/HFD, six NADPHox-deficient mice on a HFD. After 32 weeks, we studied the liver for: histology; OXPHOS complex activity; fully assembled OXPHOS complexes and their subunits; gene expression of OXPHOS subunits; oxidative and nitrosative stress; and oxidative DNA damage. In the liver of WT/HFD mice, we found a significant decreased in the activity of all OXPHOS complexes, in fully assembled complexes, in the amount of OXPHOS subunits, and in gene expression of mitochondrial DNA-encoded subunits. 8-hydroxy-2'-deoxyguanosine was only increased in mitochondrial DNA. The liver of NOX(-/-)/HFD mice showed mild steatosis but no non-alcoholic steatohepatitis (NASH) lesions were found. OXPHOS activity, OXPHOS subunits, and assembly of subunits into OXPHOS complexes were normal in these mice. We conclude that this study shows that NADPH deficiency protects mice from developing OXPHOS dysfunction and NASH caused by a HFD.

In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis.

Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFß1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50-60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.

Pioglitazone leads to an inactivation and disassembly of complex I of the mitochondrial respiratory chain.

BACKGROUND: Thiazolidinediones are antidiabetic agents that increase insulin sensitivity but reduce glucose oxidation, state 3 respiration, and activity of complex I of the mitochondrial respiratory chain (MRC). The mechanisms of the latter effects are unclear. The aim of this study was to determine the mechanisms by which pioglitazone (PGZ), a member of the thiazolidinedione class of antidiabetic agents, decreases the activity of the MRC. In isolated mitochondria from mouse liver, we measured the effects of PGZ treatment on MRC complex activities, fully-assembled complex I and its subunits, gene expression of complex I and III subunits, and [3H]PGZ binding to mitochondrial complexes. RESULTS: In vitro, PGZ decreased activity of complexes I and III of the MRC, but in vivo only complex I activity was decreased in mice treated for 12 weeks with 10 mg/kg/day of PGZ. In vitro treatment of isolated liver mitochondria with PGZ disassembled complex I, resulting in the formation of several subcomplexes. In mice treated with PGZ, fully assembled complex I was increased and two additional subcomplexes were found. Formation of supercomplexes CI+CIII2+CIVn and CI+CIII2 decreased in mouse liver mitochondria exposed to PGZ, while formation of these supercomplexes was increased in mice treated with PGZ. Two-dimensional analysis of complex I using blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) showed that in vitro PGZ induced the formation of four subcomplexes of 600 (B), 400 (C), 350 (D), and 250 (E) kDa, respectively. Subcomplexes B and C had NADH:dehydrogenase activity, while subcomplexes C and D contained subunits of complex I membrane arm. Autoradiography and coimmunoprecipitation assays showed [3H]PGZ binding to subunits NDUFA9, NDUFB6, and NDUFA6. Treatment with PGZ increased mitochondrial gene transcription in mice liver and HepG2 cells. In these cells, PGZ decreased intracellular ATP content and enhanced gene expression of specific protein 1 and peroxisome-proliferator activated receptor (PPAR)γ coactivator 1α (PGC-1α). CONCLUSIONS: PGZ binds complex I subunits, which induces disassembly of this complex, reduces its activity, depletes cellular ATP, and, in mice and HepG2 cells, upregulates nuclear DNA-encoded gene expression of complex I and III subunits.

Melatonin improves mitochondrial respiratory chain activity and liver morphology in ob/ob mice.

In previous studies, we have shown that mitochondrial respiratory chain (MRC) activity is decreased in patients with nonalcoholic steatohepatitis and in ob/ob mice and that peroxynitrite plays a pathogenic role. The present study examined whether melatonin, a peroxynitrite scavenger, prevents: (i) the in vitro effects of peroxynitrite on normal mitochondrial proteins and (ii) the development of nonalcoholic liver disease, MRC dysfunction and proteomic changes found in the mitochondrial complexes from ob/ob mice. We studied MRC activity, assembly of mitochondrial complexes and its subunits in normal mitochondrial proteins exposed to peroxynitrite in the absence and presence of melatonin. The same studies were done in mitochondrial proteins from ob/ob mice untreated and treated with melatonin. Preincubation of mitochondrial proteins from wild-type mice with melatonin prevented 3-tyrosine nitration of these proteins, eliminated the reduction in the MRC activity, the defect in the assembly of mitochondrial complexes and degradation of their subunits induced by peroxynitrite in vitro. Moreover, treatment of ob/ob mice with 10 mg/kg/day melatonin for 12 wk reduced oxidative and nitrosative stress, prevented the loss of MRC activity, protected their complexes and subunits from degradation, and favored assembling of mitochondrial complexes. In addition, this treatment improved fatty liver, decreased hepatic triglyceride concentration and increased apolipoprotein B100 in liver tissue. In conclusion, melatonin prevents the effects of peroxynitrite on mitochondrial proteins in vitro and administration of melatonin to ob/ob mice normalizes liver morphology, mitochondrial dysfunction and assembly of MRC complexes.

Mitochondrial complex I subunits are decreased in murine nonalcoholic fatty liver disease: implication of peroxynitrite.

We investigate the cause of the low activity of mitochondrial complex I found in ob/ob mice with nonalcoholic fatty liver disease. In mitochondrial proteins from ob/ob mice, we assessed complex I activity, fully assembled complex I, and its subunits, oxygen consumption, gene expression of complex I subunits, and oxidative damage to DNA. In mitochondrial proteins from the liver of ob/ob mice, complex I activity, fully assembly of this complex and complex I subunits were markedly reduced. Likewise, gene expression of mitochondrial DNA-encoded subunits was significantly decreased in obese mice, but not nuclear DNA-encoded subunits. Treatment of obese mice with uric acid, anti-TNFalpha antibody or a mimic of manganese superoxide dismutase normalized all these abnormalities. "In vitro" addition of peroxynitrite to mitochondrial proteins from wild-type mice reproduced the abnormalities found in ob/ob mice (decreased complex I activity, the amount of fully assembled complex I, and its subunits, and mitochondrial oxygen consumption). Low activity of complex I found in ob/ob mice can be ascribed to a reduced amount of fully assembled complex, which may be attributed to degradation and reduced synthesis of its subunits by peroxynitrite. Exposure of mitochondrial proteins from normal mice to peroxynitrite reproduced the proteomic abnormalities present in ob/ob mice.

Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA.

The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.

X-linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathy.

OBJECTIVE: Mitochondrial complex I deficiency is the commonest diagnosed respiratory chain defect, being genetically heterogeneous. The male preponderance of previous patient cohorts suggested an X-linked underlying genetic defect. We investigated mutations in the X-chromosomal complex I structural genes, NDUFA1 and NDUFB11, as a novel cause of mitochondrial encephalomyopathy. METHODS: We sequenced 12 nuclear genes and the mitochondrial DNA-encoded complex I genes in 26 patients with respiratory chain complex I defect. Novel mutations were confirmed by polymerase chain reaction restriction length polymorphism. Assembly/stability studies in fibroblasts were performed using two-dimensional blue native gel electrophoresis. RESULTS: Two novel p.Gly8Arg and p.Arg37Ser hemizygous mutations in NDUFA1 were identified in two unrelated male patients presenting with Leigh's syndrome and with myoclonic epilepsy and developmental delay, respectively. Two-dimensional blue native gel electrophoresis showed decreased levels of intact complex I with no accumulation of lower molecular weight subcomplexes, indicating that assembly, stability, or both are compromised. INTERPRETATION: Mutations in the X-linked NDUFA1 gene result in complex I defect and encephalomyopathy. Assembly/stability analysis might give an explanation for the different clinical phenotypes and become useful for future diagnostic purposes.

Association of novel POLG mutations and multiple mitochondrial DNA deletions with variable clinical phenotypes in a Spanish population.

BACKGROUND: Both dominant and recessive mutations were reported in the gene encoding the mitochondrial (mt) DNA polymerase gamma (POLG) in patients with progressive external ophthalmoplegia (PEO). Phenotypes other than PEO were recently documented in patients with mutations in the POLG gene. OBJECTIVE: To screen patients with mitochondrial disease and multiple mtDNA deletions in muscle for mutations in the coding regions of the POLG, PEO1, and SLC25A4 genes. DESIGN: To identify the underlying molecular defect in a group of patients with multiple mtDNA deletions comparing their molecular genetic findings with those of healthy controls. PATIENTS: Twenty-four patients (16 men and 8 women) diagnosed with mitochondrial disease and having multiple mtDNA deletions in muscle by Southern blot analysis. Thirteen patients had PEO; 2 had PEO alone, 4 had PEO and myopathy, and 5 had PEO and multisystem involvement. Four patients had multisystem disease without PEO. The remaining 9 patients had isolated myopathy. DNA from 100 healthy individuals was also studied. RESULTS: No mutation was identified in the PEO1 or SLC25A4 genes. Nine POLG mutations were observed in 6 of 24 patients. Four novel mutations were detected and mapped in the linker region (M603L) and in the pol domain of the enzyme (R853W; D1184N; R1146C). Five patients with PEO had mutations: 2 were compound heterozygotes, 1 was homozygous, and another showed a mutation in a single allele. The remaining patient also showed a sole mutation and had an unusual phenotype lacking ocular involvement. CONCLUSIONS: POLG molecular defects were found in 25% of our patients with multiple mtDNA deletions and mitochondrial disease. The uncommon phenotype found in 1 of these patients stresses the clinical variability of patients harboring POLG mutations. Molecular studies in the POLG gene should be addressed in patients with mitochondrial disease, particularly in those with PEO, and multiple mtDNA deletions.

Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene.

BACKGROUND: Mutations in the nuclear-encoded subunits of complex I of the mitochondrial respiratory chain are a recognized cause of Leigh syndrome (LS). Recently, 6 mutations in the NDUFS1 gene were identified in 3 families. OBJECTIVE: To describe a Spanish family with LS, complex I deficiency in muscle, and a novel mutation in the NDUFS1 gene. DESIGN: Using molecular genetic approaches, we identified the underlying molecular defect in a patient with LS with a complex I defect. PATIENT: The proband was a child who displayed the clinical features of LS. RESULTS: Muscle biochemistry results showed a complex I defect of the mitochondrial respiratory chain. Sequencing analysis of the mitochondrial DNA-encoded ND genes, the nuclear DNA-encoded NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, and NDUFAB1 genes, and the complex I assembly factor CIA30 gene revealed a novel homozygous L231V mutation (c.691C-->G) in the NDUFS1 gene. The parents were heterozygous carriers of the L231V mutation. CONCLUSIONS: Identifying nuclear mutations as a cause of respiratory chain disorders will enhance the possibility of prenatal diagnosis and help us understand how molecular defects can lead to complex I deficiency.