Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C₂C₁₂ myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.     

Exercise and obesity in fibromyalgia: beneficial roles of IGF-1 and resistin?

Introduction

Severe fatigue is a major health problem in fibromyalgia (FM). Obesity is common in FM, but the influence of adipokines and growth factors is not clear. The aim was to examine effects of exercise on fatigue, in lean, overweight and obese FM patients.

Methods

In a longitudinal study, 48 FM patients (median 52 years) exercised for 15 weeks. Nine patients were lean (body mass index, BMI 18.5 to 24.9), 26 overweight (BMI 25 to 29.9) and 13 obese. Fatigue was rated on a 0 to 100 mm scale (fibromyalgia impact questionnaire [FIQ] fatigue) and multidimensional fatigue inventory (MFI-20) general fatigue (MFIGF). Higher levels in FIQ fatigue and MFIGF indicate greater degree of fatigue. Free and total IGF-1, neuropeptides, adipokines were determined in serum and cerebrospinal fluid (CSF).

Results

Baseline FIQ fatigue correlated negatively with serum leptin (r = -0.345; P = 0.016) and nerve growth factor (NGF; r = -0.412; P = 0.037). In lean patients, baseline MFIGF associated negatively with serum resistin (r = -0.694; P = 0.038). FIQ Fatigue associated negatively with CSF resistin (r = -0.365; P = 0.073). Similarly, FIQ fatigue (r = -0.444; P = 0.026) and MFIGF correlated negatively with CSF adiponectin (r = -0.508; P = 0.01). In lean patients, FIQ fatigue (P = 0.046) decreased after 15 weeks. After 30 weeks, MFIGF decreased significantly in lean (MFIGF: P = 0.017), overweight (MFIGF: P = 0.001), and obese patients (MFIGF: P = 0.016). After 15 weeks, total IGF-1 increased in lean (P = 0.043) patients. ∆Total IGF-1 differed significantly between lean and obese patients (P = 0.010). ∆Total IGF-1 related negatively with ∆MFIGF after 15 weeks (r = -0.329; P = 0.050). After 30 weeks, ∆FIQ fatigue negatively correlated with ∆NGF (r = -0.463; P = 0.034) and positively with ∆neuropeptide Y (NPY) (r = 0.469; P = 0.032). Resistin increased after 30 weeks (P = 0.034). ∆MFIGF correlated negatively with ∆resistin (r = -0.346; P = 0.031), being strongest in obese patients (r = -0.815; P = 0.007). In obese patients, ∆FIQ fatigue after 30 weeks correlated negatively with ∆free IGF-1 (r = -0.711; P = 0.032).

Conclusions

Exercise reduced fatigue in all FM patients, this effect was achieved earlier in lean patients. Baseline levels of resistin in both serum and CSF associated negatively with fatigue. Resistin was increased after the exercise period which correlated with decreased fatigue. Changes in IGF-1 indicate similar long-term effects in obese patients. This study shows reduced fatigue after moderate exercise in FM and indicates the involvement of IGF-1 and resistin in these beneficial effects.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00643006","term_id":"NCT00643006"}}NCT00643006     

Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and β1-integrin

A spheroid is an in vitro multicellular aggregate that provides a microenvironment resembling that of normal tissue in vivo. Although cell adhesion molecules such as integrins and cadherins have been implicated in participating in the process of spheroid formation, little is known about the timing of their action. In this study, we have employed an image-based quantitative method to investigate the compactness of cell aggregates during hepatoma spheroid formation in a dynamic fashion. By modulating β1-integrin and E-cadherin activity with specific blocking antibodies, ion chelators, and RGD-sequence-containing peptides, we show that these cell adhesion molecules mediate the formation of spheroids through the establishment of complex cell-cell and cell-extracellular matrix (ECM) interactions. The dynamics of spheroid formation can be separated into three stages. In the first stage, ECM fibers act as a long-chain linker for the attachment of dispersed single-cells to form loose aggregations through the binding of integrins. This is followed by a delay period in which cell aggregates pause in compaction, presumably because of the accumulation of sufficient amounts of E-cadherins. In the third stage, strong homophilic interaction of E-cadherins is a major factor for the morphological transition from loose cell aggregates to compact spheroids. These findings thus provide comprehensive information on the molecular mechanisms and dynamics of hepatoma spheroid formation.This work was supported by the National Program of Genome Medicine, ROC (NSC 93-3112-B007-001) and Veteran General Hospitals University System of Taiwan Joint Research Program (VGHUST94-G6-06-3).     

Purification and enzymatic characterization of PgcM: a β-phosphoglucomutase and glucose-1-phosphate phosphodismutase of Bacillus subtilis

Phosphoglucomutases catalyze the reversible conversion of D-glucose 1-phosphate to D-glucose 6-phosphate, a key metabolic step in all cells. Two classes of phosphoglucomutases have been described so far, using either the alpha- or beta-forms of the phosphorylated sugars. The pgcM gene of Bacillus subtilis was cloned and used to construct a plasmid-based overexpression system for PgcM in Bacillus megaterium. The obtained protein was purified and its enzymatic activities were characterized. PgcM exhibits beta-phosphoglucomutase activity, transforming mainly beta-glucose 1-phosphate to beta-glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Nevertheless, alpha-glucose 1-phosphate can also serve as a substrate, but with a seven-fold lower affinity than that observed for the beta-form. Additionally, PgcM exhibits a glucose-1-phosphate phosphodismutase activity using the alpha- and beta-forms as substrates, with affinities comparable to those observed for the phosphoglucomutase activity. Conformational changes of PgcM triggered by cofactors (MgCl2, glucose 1,6-bisphosphate) and substrate (glucose 1-phosphate) were detected by fluorescence spectra. Insertional mutagenesis of pgcM resulted in an inactivation of beta-phosphoglucomutase activity in B. subtilis. These mutants showed growth deficiency on minimal medium containing starch or maltodextrins (maltose to maltoheptaose) compared either to the wild-type or to growth on minimal medium containing glucose.     

Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.     

Synthesis and properties of alkylglucosides with mild detergent action: improved synthesis and purification of β-1-octyl-, -nonyl-, and -decyl-glucose. Synthesis of β-1-undecylglucose and β-1-dodecylmaltose

Medium chain β-1-alkylglycosides show interesting mild detergent properties. Therefore, their synthesis and purification have been investigated and improved so as to permit preparation of 50–100 g amounts. Preparatory methods are presented for the already known compounds β-1-octyl-, β-1-nonyl and β-1-decyl-glucose and for the new compounds β-1-undecylglucose and β-1-dodecylmaltose. Some relevant properties such as melting point, optical rotation, critical micelle concentration and NMR-spectra have been determined. They illustrate the suitability of this class of detergents for membrane research.     

The α1-adrenergic receptors: diversity of signaling networks and regulation

The α(1)-adrenergic receptor (AR) subtypes (α(1a), α(1b), and α(1d)) mediate several physiological effects of epinephrine and norepinephrine. Despite several studies in recombinant systems and insight from genetically modified mice, our understanding of the physiological relevance and specificity of the α(1)-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that β arrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α(1)-AR subtypes in various organs.     

SUVi and BACH1: a new subfamily of mammalian helicases?

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes and eukaryotes. Members of this family are genes responsible for cancer predisposition disorders like Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. Here, we show the sequence homologies between two recently identified mammalian helicases, namely SUVi and BACH1. These two genes also share strong homologies with other members of the RecQ family. On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.     

Assessment of bioburden on human and animal tissues: Part 1—Results of method development and validation studies

Recovered human and animal tissues are used extensively in surgery for wound repair and reconstruction. In preparation for the validation of chemical disinfection and radiation sterilization processes, studies were performed on the development and validation of quantitative bioburden recovery methods for human bone and soft tissue and also for porcine dermis. The use of a swab-based method was not considered due to the known poor efficiency of recovery for this technique. The “exhaustive extraction” and “inoculated product” approaches to validation of a bioburden recovery efficiency factor have inherent strengths and weaknesses; in this study, tissues were inoculated and also subjected to a series of extractions to determine if/when “exhaustion” occurred. Femoral and tibial shaft rings, iliac crest wedges, sections of Achilles tendon, a soft tissue composite sample, and porcine dermis, were inoculated at several sites with Bacillus atrophaeus spores, and then subjected to either shaking by hand, mechanical shaking, or sonication plus mechanical shaking. Each of these methods of agitation were performed in combination with three rinse (extraction) fluids: phosphate buffer (Butterfield’s buffer), phosphate buffer with 0.2% polysorbate 80 (a surfactant), and water with 1% peptone and 1% polysorbate 80 (Fluid D). The highest recovery efficiencies were observed with sonication plus mechanical shaking; of the three extraction media, Fluid D gave the highest first-rinse recovery efficiency (65%) and Butterfield’s buffer gave the lowest (39%). Each of the three recovery methods, however appeared to reach “exhaustion”, a subsequent rinse giving less than 10% of the recovery found in the first rinse. The results demonstrated the importance of performing bioburden method development and validation studies. The method validation strategy described here, using a combination of tissue inoculation and repetitive extraction, showed the superiority of sonication plus mechanical shaking using Fluid D as the rinse medium. In addition, the use of only the exhaustive extraction approach could have resulted in the development of a methodology that consistently underestimated the bioburden present on/in recovered tissue.     

Branched-chain sucroses: Synthesis and Wittig reaction of the 1′-aldehydo derivative of sucrose

The reaction of 2,3,4,3′,4′-penta-O-acetylsucrose (1) with 3.3 mol. equiv. of tert-butyldiphenylsilyl chloride in pyridine in the presence of 4-dimethylamino-pyridine gave the 6,1′,6′-tris(tert-butyldiphenylsilyl) derivative 2 (27%) and the 6,6′-bis(tert-butyldiphenylsilyl) derivative (67%). Oxidation of the HO-1′ in 3 with methyl sulphoxide and trifluoroacetic anhydride gave the 1′-aldehydo derivative 5, which reacted with the stabilised Wittig reagent (Ph₃PCHCO₂Et) to give the 1′-ethoxycarbonylmethylene derivative 6. Deacetylation of the hepta-acetate 7 of 6 with methanolic sodium methoxide was accompanied by a Michael addition reaction to give 2,1′-anhydro-1′-methoxycarbonylmethylsucrose.     

Synthesis and reactions of 1′,2:4,6-di-o-isopropylidene-sucrose

The reaction of sucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A) gave, after acetylation followed by chromatography, 1′,2:4,6-di-O-isopropylidenesucrose tetra-acetate (1) in 15% yield. The structure of 1 was determined on the basis of p.m.r. and mass spectrometry, and by chemical transformations. Treatment of 1 with aqueous acetic acid afforded sucrose 3,3′,4′,6′-tetra-acetate 2. Reacetalation of 2 using reagent A gave 1 in 80% yield. The p.m.r. spectrum of 2 confirmed the presence of hydroxyl groups at C-2 and C-4. The following sequence of reactions showed that the remaining two hydroxyl groups were located at C-6 and C-1′. Selective tritylation of 2 gave 1′,6-di-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (3) as the minor, and 6-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (4) as the major, product. When tritylation was carried out under forcing conditions, 2 gave 3 as the major product. Acetylation of 4 afforded 6-O-tritylsucrose hepta-acetate. Mesylation of 2 gave the tetramethanesulphonate 5, which afforded the 6-dcoxy-6-iodo derivative 6 on treatment with a refluxing solution of sodium iodide in butanone. Treatment of 3 with methanesulphonyl chloride in pyridine gave the disulphonate 7, which on detritylation followed by acetylation gave 2,4-di-O-methanesulphonylsucrose hexa-acetate (9). Treatment of 9 with sodium benzoate in hexamethylphosphoric triamide displaced the 4-sulphonate, with inversion of configuration, to give the galacto derivative 10.     

The α1-adrenergic receptors: diversity of signaling networks and regulation

The α₁-adrenergic receptor (AR) subtypes (α_1a, α_1b, and α_1d) mediate several physiological effects of epinephrine and norepinephrine. Despite several studies in recombinant systems and insight from genetically modified mice, our understanding of the physiological relevance and specificity of the α₁-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that βarrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α₁-AR subtypes in various organs.     

Autophagy: an overlooked mechanism of HIV-1 pathogenesis and neuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4(+) lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4(+) T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.     

CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene–gene interactions

Gene–environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene–gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene–gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene–gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9–8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5–15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.