IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-β1-induced fibrotic responses

TGF-ß1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-ß1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localization of IHG-1 is pivotal in amplification of TGF-ß1 signaling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-ß1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (?mts-IHG-1) repressed TGF-ß1 fibrotic signaling in renal epithelial cells. In cells expressing ?mts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-ß1. ?mts-IHG-1 modulation of TGF-ß1 signaling was associated with increased Smad7 protein expression. ?mts-IHG-1 modulated TGF-ß1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-ß1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-ß1 signal transduction and that IHG-1 is a key player in this modulation.

Synthesis, spectroscopy and spectroelectrochemistry of chlorocarbonyl {1,2-bis[(2,6-diisopropylphenyl)imino]-acenaphthene-kappa(2)-N,N '}rhodium (I)

Reaction of the dinuclear complex [{Rh(CO)(2)}(2) (mu-Cl)(2)]with an alpha-diimine ligand, 1,2- bis[(2,6-diisopropylphenyl) imino] acenaphthene (iPr(2)Ph-bian), produces square-planar [RhCl(CO)(iPr(2)Ph-bian)]. For the first time, 2: 1 and 1: 1 alpha-diimine/dimer reactions yielded the same product. The rigidity of iPr(2)Ph-bian together with its flexible electronic properties and steric requirements of the 2,6-diisopropyl substituents on the benzene rings allow rapid closure of a chelate bond and replacement of a CO ligand instead of chloride. A resonance Raman study of [RhCl(CO)(iPr(2)Ph-bian)] has revealed a predominant Rh-to-bian charge transfer (MLCT) character of electronic transitions in the visible spectral region. The stabilisation of [RhCl(CO)(iPr(2)Ph-bian)] in lower oxidation states by the pi-acceptor iPr(2)Ph-bian ligand was investigated in situ by UV-VIS, IR and EPR spectroelectrochemistry at variable temperatures. The construction of the novel UV-VIS-NIR-IR low-temperature OTTLE cell used in these studies is described in the last part of the paper.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

Regressive logistic and proportional hazards disease models for within-family analyses of measured genotypes, with application to a CYP17 polymorphism and breast cancer

Various statistical methods have been proposed to evaluate associations between measured genetic variants and disease, including some using family designs. For breast cancer and rare variants, we applied a modified segregation analysis method that uses the population cancer incidence and population-based case families in which a mutation is known to be segregating. Here we extend the method to a common polymorphism, and use a regressive logistic approach to model familial aggregation by conditioning each individual on their mother's breast cancer history. We considered three models: 1) class A regressive logistic model; 2) age-of-onset regressive logistic model; and 3) proportional hazards familial model. Maximum likelihood estimates were calculated using the software MENDEL. We applied these methods to data from the Australian Breast Cancer Family Study on the CYP17 5UTR TC MspA1 polymorphism measured for 1,447 case probands, 787 controls, and 213 relatives of case probands found to have the CC genotype. Breast cancer data for first- and second-degree relatives of case probands were used. The three methods gave consistent estimates. The best-fitting model involved a recessive inheritance, with homozygotes being at an increased risk of 47% (95% CI, 28-68%). The cumulative risk of the disease up to age 70 years was estimated to be 10% or 22% for a CYP17 homozygote whose mother was unaffected or affected, respectively. This analytical approach is well-suited to the data that arise from population-based case-control-family studies, in which cases, controls and relatives are studied, and genotype is measured for some but not all subjects.

Striated muscle activator of Rho signalling (STARS) is a PGC-1α/oestrogen-related receptor-α target gene and is upregulated in human skeletal muscle after endurance exercise

Exercise improves the ability of skeletal muscle to metabolise fats and sugars. For these improvements to occur the muscle detects a signal caused by exercise, resulting in changes in genes and proteins that control metabolism. We show that endurance exercise increases the amount of a protein called striated muscle activator of Rho signalling (STARS) as well as several other proteins influenced by STARS.We also show that the amount of STARS can be increased by signals directed from proteins called peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) and oestrogen-related receptor-α (ERRα). We also observed that when we reduce the amount of STARS in muscle cells, we block the ability of PGC-1α/ERRα to increase a gene called carnitine palmitoyltransferase-1β (CPT-1β), which is important for fat metabolism. Our study has shown that the STARS pathway is regulated by endurance exercise. STARS may also play a role in fat metabolism in muscle.

Exercise early in life in rats born small does not normalize reductions in skeletal muscle PGC-1α in adulthood

We have previously shown that 4 wk of exercise training early in life normalizes the otherwise greatly reduced pancreatic β-cell mass in adult male rats born small. The aim of the current study was to determine whether a similar normalization in adulthood of reduced skeletal muscle mitochondrial biogenesis markers and alterations in skeletal muscle lipids of growth-restricted male rats occurs following early exercise training. Bilateral uterine vessel ligation performed on day 18 of gestation resulted in Restricted offspring born small (P < 0.05) compared with both sham-operated Controls and a sham-operated Reduced litter group. Offspring remained sedentary or underwent treadmill running from 5–9 (early exercise) or 20–24 (later exercise) wk of age. At 24 wk of age, Restricted and Reduced litter offspring had lower (P < 0.05) skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein expression compared with Control offspring. Early exercise training had the expected effect of increasing skeletal muscle markers of mitochondrial biogenesis, but, at this early age (9 wk), there was no deficit in Restricted and Reduced litter skeletal muscle mitochondrial biogenesis. Unlike our previous observations in pancreatic β-cell mass, there was no “reprogramming” effect of early exercise on adult skeletal muscle such that PGC-1α was lower in adult Restricted and Reduced litter offspring irrespective of exercise training. Later exercise training increased mitochondrial biogenesis in all groups. In conclusion, although the response to exercise training remains intact, early exercise training in rats born small does not have a reprogramming effect to prevent deficits in skeletal muscle markers of mitochondrial biogenesis in adulthood.

The role of PGC-1α and PGC-1β in myotube protein synthesis

Skeletal muscle mass is regulated by a balance between protein synthesis and protein degradation. Using molecular techniques, this PhD thesis identified that two proteins, PGC-1α and PGC-1β, can increase protein synthesis in myotubes in vitro. This has implications for the treatment of diseases associated with skeletal muscle atrophy.

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P<0.0125) and creatine uptake (1.8 and 1.6-fold, P<0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P<0.05), resulting in a 2.2-fold (P<0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between markers of mitochondrial biogenesis, cell signaling, and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats + DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise; and (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P < 0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P < 0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P < 0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) mRNA compared to the control animals that were exercised (P < 0.05). This study provides novel evidence that by lowering the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

New gene targets of PGC-1α and ERRα co-regulation in C2C12 myotubes

As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P < 0.001) were identified. From these, further genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P < 0.001) and of Mtfp1, Mrm1, Oxnad1 and Cluh (P < 0.05). Screening of the promoter and intron 1 regions also revealed all genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P < 0.05) except for Akr1b10. These findings provide preliminary evidence for the novel regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.

Expressão Imuno-histoquímica das proteínas GLUT-1 e HIF-1α em lesões vasculares de mucosa oral

The correct histological diagnosis of vascular lesions in the oral mucosa is critical, especially in defining the treatment and prognosis, as some vascular lesions show spontaneous involution and others do not show such behavior. This study analyzed the expression immunohistochemistry of human glucose transporter protein (GLUT-1), in oral benign vascular tumors and to reclassify such lesions according to with his immunoexpression. In addition, we evaluated the immunohistochemical expression of hypoxia-inducible factor 1 alpha (HIF-1α), the main transcription factor involved in cellular adaptation to hypoxia. We analyzed 60 cases of benign oral vascular tumors: 30 cases with histological diagnosis of HEM and 30 cases of oral pyogenic granuloma (PG). The results of this research showed that of the 30 lesions initially classified as HEM, only 7 showed immuno-positivity for GLUT-1, remaining with the initial diagnosis. The remaining 23 were reclassified as vascular malformation (VM) (13 cases) and PG (10 cases). All cases in the sample with an initial diagnosis of PG were negative for GLUT-1, demonstrating the accuracy of histological diagnosis of these lesions. Concerning to the immunoexpression of HIF-1α, the Mann-Whitney test revealed a statistically significant difference between the cases of GP and MV (p = 0.002), where the median of GP (m=78) was higher than the MV (m=53). Based on these results, this study showed that a histological diagnosis alone is not always sufficient for the correct diagnosis of oral HEM and that HIF-1α participates in the pathogenesis of vascular lesions

Curves in homogeneous spaces and their contact with 1-dimensional orbits

Let alpha be a C(infinity) curve in a homogeneous space G/H. For each point x on the curve, we consider the subspace S(k)(alpha) of the Lie algebra G of G consisting of the vectors generating a one parameter subgroup whose orbit through x has contact of order k with alpha. In this paper, we give various important properties of the sequence of subspaces G superset of S(1)(alpha) superset of S(2)(alpha) superset of S(3)(alpha) superset of ... In particular, we give a stabilization property for certain well-behaved curves. We also describe its relationship to the isotropy subgroup with respect to the contact element of order k associated with alpha.

Thermal behavior of Cu-Al alloys near the alpha-Cu-Al solubility limit

The thermal behavior of Cu-Al alloys with 17, 19 and 21 at.%Al was examined by differential thermal analysis (DTA), differential scanning calorimetry (DSC), X-ray diffractometry (XRD), optical microscopy (OM) and scanning electron microscopy (SEM). The presence of the gamma phase (Al4Cu9) was clearly detected for the Cu-19 at.%Al alloy and caused the alpha (2) phase disordering process in two stages. The tendency to increase the alpha (2) dissolution precipitates with the increase in the Al content seems to be reverted for compositions at about 21 at.%Al and the heating/cooling ratio seems to influence the thermal response of this process. The presence of the endothermic peak corresponding to the beta (1)--> beta transformation depends on an incomplete beta decomposition reaction. The variation of the heating rate showed that the beta (1)-->(alpha+gamma (1)) decomposition is the dominant reaction for alloys containing 19 and 21 at.%Al.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)