Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H₂O₂, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNFα and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.     

IL-10- and TGFβ-mediated Th9 Responses in a Human Helminth Infection

Background

Th9 cells are a subset of CD4⁺ T cells that express the protoypical cytokine, IL-9. Th9 cells are known to effect protective immunity in animal models of intestinal helminth infections. However, the role of Th9 cells in human intestinal helminth infections has never been examined.

Methodology

To examine the role of Th9 cells in Strongyloidis stercoralis (Ss), a common intestinal helminth infection, we compared the frequency of Th9 expressing IL-9 either singly (mono-functional) or co-expressing IL-4 or IL-10 (dual-functional) in Ss-infected individuals (INF) to frequencies in uninfected (UN) individuals.

Principal Findings

INF individuals exhibited a significant increase in the spontaneously expressed and/or antigen specific frequencies of both mono- and dual-functional Th9 cells as well as Th2 cells expressing IL-9 compared to UN. The differences in Th9 induction between INF and UN individuals was predominantly antigen-specific as the differences were no longer seen following control antigen or mitogen stimulation. In addition, the increased frequency of Th9 cells in response to parasite antigens was dependent on IL-10 and TGFx since neutralization of either of these cytokines resulted in diminished Th9 frequencies. Finally, following successful treatment of Ss infection, the frequencies of antigen-specific Th9 cells diminished in INF individuals, suggesting a role for the Th9 response in active Ss infection. Moreover, IL-9 levels in whole blood culture supernatants following Ss antigen stimulation were higher in INF compared to UN individuals.

Conclusion

Thus, Ss infection is characterized by an IL-10- and TGFβ dependent expansion of Th9 cells, an expansion found to reversible by anti-helmintic treatment.     

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2-driven experimental evolution

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O₂-evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD⁺/NADH, leading to an optimized O₂ reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.     

Function of oxygen resistance proteins in the anaerobic,sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.     

Oxygen defense in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) are strict anaerobes that are often found in biotopes where oxic conditions can temporarily exist. The bacteria have developed several defense strategies in order to survive exposure to oxygen. These strategies includes peculiar behaviors in the presence of oxygen, like aggregation or aerotaxis, and enzymatic systems dedicated to the reduction and the elimination of oxygen and its reactive species. Sulfate-reducing bacteria, and specially Desulfovibrio species, possess a variety of enzymes acting together to achieve an efficient defense against oxidative stress. The function and occurrence of these enzymatic systems are described.     

Accelerated aging as evidenced by increased telomere shortening and mitochondrial DNA depletion in patients with type 2 diabetes

Although shortened telomeres were shown associated with several risk factors of diabetes, there is lack of data on their relationship with mitochondrial dysfunction. Therefore, we compared the relationship between telomere length and mitochondrial DNA (mtDNA) content in patients with type 2 diabetes mellitus (T2DM; n = 145) and in subjects with normal glucose tolerance (NGT; n = 145). Subjects were randomly recruited from the Chennai Urban Rural Epidemiology Study. mtDNA content and telomere length were assessed by Real-Time PCR. Malonodialdehyde, a marker of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) using fluorescence methodology. Adiponectin levels were measured by radioimmunoassay. Oxidative stress as determined by lipid peroxidation (TBARS) was significantly (p < 0.001) higher in patients with T2DM compared to NGT subjects. In contrast, the mean telomere length, adiponectin and mtDNA content were significantly (p < 0.001) lower in patients with T2DM compared to NGT subjects. Telomere length was positively correlated with adiponectin, HDL, mtDNA content and good glycemic/lipid control and negatively correlated with adiposity and insulin resistance. On regression analysis, shortened telomeres showed significant association with T2DM even after adjusting for waist circumference, insulin resistance, triglyceride, HDL, adiponectin, mtDNA & TBARS. mtDNA depletion showed significant association with T2DM after adjusting for waist circumference and adiponectin but lost its significance when further adjusted for telomere length, TBARS and insulin resistance. Our study emphasizes the clustering of accelerated aging features viz., shortened telomeres, decreased mtDNA content, hypoadiponectinemia, low HDL, and increased oxidative stress in Asian Indian type 2 diabetes patients.     

Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.