257 research outputs found
Mechanistic insights into AMP-activated protein kinase-dependent gene expression
No full textAMP-activated protein kinase (AMPK) is a fundamental enzyme that controls energy homeostasis, through orchestrating the cellular response to a reduction in energy availability. Under conditions of cellular energy stress AMPK senses the decrease in ATP levels and responds by activating catabolic pathways, which will generate ATP, and switching off ATP-consuming ones, in order to restore the energy balance. AMPK regulates several signaling cascades linked to metabolism, overall favoring cellular consumption of glucose and lipid, allowing the cell to adapt to sustained energetic challenges through modulation of gene transcription. The aim of this thesis is to investigate the role that AMPK plays in the adaptive reprogramming of metabolism through transcriptional control. To identify genes and pathways regulated in an AMPK-dependent mechanism, we performed a whole-genome transcriptome profiling using microarray technology and compared the effects of two small molecule AMPK activators acting via distinct mechanisms, namely 991, which binds at the allosteric drug and metabolite site, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). The impact on gene expression of 991 and AICAR was investigated using two cellular and genetic models, mouse embryonic fibroblasts (MEFs) and mouse primary hepatocytes, either wild-type or AMPK-deficient. Statistical analysis of differential gene expression, followed by pathway analysis, revealed compound- and model-specific gene expression signatures. Notably we found that in contrast to AICAR, 991 affected gene expression almost exclusively in an AMPK-dependent manner. Interestingly, we identified that 991 modulated genes involved in the metabolic and lysosomal pathways, and that a number of these genes are under the control of the sterol regulatory element-binding protein (SREBP) and transcription factor EB (TFEB).
We identified the tumor suppressor folliculin (Flcn) and its binding partners, folliculin interacting protein (Fnip), as novel transcriptional targets of AMPK. We confirmed the upregulated expression of Flcn in response to pharmacological activation of AMPK in MEFs and primary hepatocytes. Furthermore, by taking advantage of a novel zebrafish whole-body knockout model of AMPK, we confirmed that physiological activation of AMPK partly mediates the increase in expression of Flcn and Fnip. We further identified TFEB as a mediator of the AMPK transcriptional response, accounting for the increase in Flcn expression through modulating its promoter activity. Moreover, we revealed the existence of a novel mechanism by which AMPK regulates TFEB through promoting dephosphorylation and nuclear translocation, independently of mammalian target of rapamycin complex 1 (mTORC1).
Taken together, we identified several new AMPK-dependent/-regulated genes and pathways that are differentially modulated in a cell type- and compound-specific manner. Most importantly, we discovered a novel and conserved AMPK-TFEB-FLCN axis in cellular and in vivo models. This work contributes to advance our understanding of AMPK-mediated regulation of transcriptional programs, nevertheless future studies will be required to elucidate the physiological relevance of the AMPK-TFEB-FLCN cascade.SSV-ENSS
AMP-activated Protein Kinase (AMPK): New molecular insights and novel downstream targets
No full textAMP-activated Protein Kinase (AMPK) is a central regulator of energy homeostasis and a promising drug target for metabolic disorders. It exists as complexes of three subunits, a catalytic alpha, and two regulatory beta and gamma subunits. The regulation of AMPK involves reversible phosphorylation and allosteric regulation by adenine nucleotides. It is activated by phosphorylation of Thr172 on the catalytic alpha subunit as a consequence of various energy-depleting conditions. Once activated, AMPK regulates a plethora of metabolic processes through the phosphorylation of target proteins to maintain energy homeostasis. The beta subunit has a vital role as a structural scaffold stabilising the AMPK heterotrimeric complex. It is also known to regulate AMPK activity through different posttranslational modifications (i.e., phosphorylation and myristoylation). Although myristoylation of Glycine-2 (Gly2) of the beta subunit has been shown to be required for sensing stress signals and achieving maximum AMPK activity in vitro, its physiological relevance at the cellular and organismal levels remains unknown. Critically, the underlying molecular mechanism by which beta subunit myristoylation controls AMPK activity is elusive.
The primary aim of this thesis was to investigate the molecular basis of AMPK regulation by the beta subunit myristoyl switch. I showed that mouse embryonic fibroblasts (MEFs) isolated from knock-in (KI) mice carrying Gly2 to Ala point mutation of beta1 and beta2 isoforms (beta1/2 G2A double knock-in (DKI)) displayed increased activity and phosphorylation of Thr172 in the beta subunit. Using proximity ligation assay, I found that the loss of beta1 myristoylation impedes the interaction/proximity of the phosphatases PPM1A/B with AMPK in cells. In vivo, beta1 G2A KI mice showed increased AMPK activity in the liver and were protected from high-fat diet-induced obesity, hepatic lipid accumulation, and insulin resistance.
The second aim of the thesis focused on the identification of novel AMPK substrates to expand our understanding of the AMPK system/signalling in the control of metabolic and also in non-metabolic processes. We performed an unbiased phosphoproteomics analysis which revealed that AMPK phosphorylates several proteins involved in regulating Golgi structure and function. I observed that pharmacological activation of AMPK induces Golgi fragmentation in wild-type but not in AMPK-deficient human U2OS cells and MEFs. We identified AMPK-dependent phosphorylation of three Golgi-related proteins and focused on the Oxysterol-binding protein like 9 (OSBPL9), a novel AMPK substrate phosphorylated on a threonine residue (Thr335). Interestingly, knockdown of OSBPL9 in cells induced Golgi fragmentation, linking the AMPK-OSBPL9 pathway to Golgi regulation.
Collectively, this study expands our understanding of the regulation and novel biological roles of AMPK. This will advance future studies to elucidate the significance of AMPK in the treatment of metabolic as well as non-metabolic disorders.SSV-EN
Common genetic variation and the control of HIV-1 in humans
No full textTo extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians
Investigation of physiological roles of AMPK-activated protein kinase y3
No full textClinical trials have shown that direct activators of an evolutionary-conserved metabolic sensor, AMP-activated protein kinase (AMPK), are beneficial in preventing/treating a range of metabolic disorders, including type 2 diabetes. These activators, including 991/MK-8722, bind at regulatory site, termed the allosteric drug and metabolite (ADaM-site) at the interface between the catalytic alpha subunit and regulatory beta subunit. In addition, another approach for direct activation of AMPK is through mimicking its natural ligands, AMP/ADP, by binding to the nucleotide site in the y3 subunit, e.g. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Interestingly, their ability to influence glucose homeostasis is reported to be through activation of AMPK complexes found within the skeletal muscle. The AMPKy3 is unique as an AMPK subunit isoform since it is selectively expressed in glycolytic skeletal muscle fibres.
To gain more insight into the y3 isoform-specific regulation of glucose metabolism, we used a y3 knock-out (y3-/-) mouse model to investigate the effects of the AICAR and 991/MK-8722. We confirm that y3 isoform expression and activity is high glycolytic muscles, including extensor digitorum longus (EDL), whereas there is very little detected in oxidative soleus (SOL) muscle. We show that AICAR and 991/MK-8722 stimulated glucose uptake in both EDL and SOL muscles. Interestingly, only the ability of AICAR to induce glucose uptake was blunted in EDL muscle taken from y3-/- mice. Consistent with this, whole-body glucose clearance was also impaired in y3-/- mice in response to an AICAR, whilst MK-8722 lowered blood glucose similarly between WT and y3-/- mice. Despite the presence of y1-containing complexes, which are the most predominant complexes in both EDL and SOL muscles, this suggests that different AMPKy isoforms play different roles in glucose homeostasis in response to AICAR.
We further looked at de novo glycogen synthesis and glucose utilisation in skeletal muscle. Despite similar basal glucose uptake between muscles from WT and y3-/-, y3-deficiency results in lower basal glycogen content only in glycolytic muscles. We show that this is not due to an inability to synthesise glycogen de novo, since MK-8722 was able to increase glycogen levels in EDL. Instead, we show that the decrease in basal glycogen in y3-/- EDL, may be linked lower expression of UDP-glucose pyrophosphorylase 2 (UGP2). This suggests that AMPKy3 may play a role in steering the molecular fate of glucose inside the cell.
Taken together, whilst we show that AMPKy3 is dispensable for ex vivo glucose transport and in vivo glucose homeostasis in response to ADaM-site AMPK activators, nucleotide-mediated activation of y3 by AICAR is impaired. Further research is required to understand the nucleotide regulation of different y-containing AMPK complexes. This differential activation mechanism could be exploited to specifically target AMPKy3 complexes in the muscle, and potentially avoid deleterious effects of activation of AMPK complexes in other tissues.SSV-EN
DNA methylation during human adipogenesis and the impact of fructose
No full textBackground Increased adipogenesis and altered adipocyte function contribute to the development of obesity and associated comorbidities. Fructose modified adipocyte metabolism compared to glucose, but the regulatory mechanisms and consequences for obesity are unknown. Genome-wide methylation and global transcriptomics in SGBS pre-adipocytes exposed to 0, 2.5, 5, and 10 mM fructose, added to a 5-mM glucose-containing medium, were analyzed at 0, 24, 48, 96, 192, and 384 h following the induction of adipogenesis. Results Time-dependent changes in DNA methylation compared to baseline (0 h) occurred during the final maturation of adipocytes, between 192 and 384 h. Larger percentages (0.1% at 192 h, 3.2% at 384 h) of differentially methylated regions (DMRs) were found in adipocytes differentiated in the glucose-containing control media compared to adipocytes differentiated in fructose-supplemented media (0.0006% for 10 mM, 0.001% for 5 mM, and 0.005% for 2.5 mM at 384 h). A total of 1437 DMRs were identified in 5237 differentially expressed genes at 384 h post-induction in glucose-containing (5 mM) control media. The majority of them inversely correlated with the gene expression, but 666 regions were positively correlated to the gene expression. Conclusions Our studies demonstrate that DNA methylation regulates or marks the transformation of morphologically differentiating adipocytes (seen at 192 h), to the more mature and metabolically robust adipocytes (as seen at 384 h) in a genome-wide manner. Lower (2.5 mM) concentrations of fructose have the most robust effects on methylation compared to higher concentrations (5 and 10 mM), suggesting that fructose may be playing a signaling/regulatory role at lower concentrations of fructose and as a substrate at higher concentrations
Centenarians as extreme phenotypes: An ecological perspective to get insight into the relationship between the genetics of longevity and age-associated diseases
No full textIn this review, we address the genetic continuum between aging and age-related diseases, with particular attention to the ecological perspective. We describe the connections between genes that promote longevity and genes associated with age-related diseases considering tradeoff mechanisms in which the same genetic variants could have different effects according to the tissue considered and could be involved in several biological pathways. Then we describe mechanisms of antagonistic pleiotropy, focusing on the complex interplay between genetic variants and environmental changes (internal or external). We sustain the use of centenarians as "super-controls" for the study of the major age-related diseases, starting from the concept that the maximization of the phenotypic differences in the considered cohort, achieved by selecting the most divergent phenotypes, could be useful for increasing the significant differences observed in the genetic association study. We describe the potential impact of the population genetic variability in the study of human longevity and the possible contribution of the past selective pressures in shaping the current genomic background of individuals. In conclusion, we illustrate recent findings emerged from whole-genome sequencing of long-lived individuals and future perspectives for interpreting the huge amount of genetic data that will be generated in the next future
C3H7NO2S effect on concrete steel-rebar corrosion in 0.5 M H2SO4 simulating industrial/microbial environment
No full textThis paper investigates C3H7NO2S (Cysteine) effect on the inhibition of reinforcing steel corrosion in concrete immersed in 0.5 M H2SO4, for simulating industrial/microbial environment. Different C3H7NO2S concentrations were admixed, in duplicates, in steel-reinforced concrete samples that were partially immersed in the acidic sulphate environment. Electrochemical monitoring techniques of open circuit potential, as per ASTM C876-91 R99, and corrosion rate, by linear polarization resistance, were then employed for studying anticorrosion effect in steel-reinforced concrete samples by the organic hydrocarbon admixture. Analyses of electrochemical test-data followed ASTM G16-95 R04 prescriptions including probability distribution modeling with significant testing by Kolmogorov-Smirnov and student's t-tests statistics. Results established that all datasets of corrosion potential distributed like the Normal, the Gumbel and the Weibull distributions but that only the Weibull model described all the corrosion rate datasets in the study, as per the Kolmogorov-Smirnov test-statistics. Results of the student's t-test showed that differences of corrosion test-data between duplicated samples with the same C3H7NO2S concentrations were not statistically significant. These results indicated that 0.06878 M C3H7NO2S exhibited optimal inhibition efficiency η = 90.52±1.29% on reinforcing steel corrosion in the concrete samples immersed in 0.5 M H2SO4, simulating industrial/microbial service-environment
A Liver-Enriched Transcriptional Activator Protein, LAP, and a Transcriptional Inhibitory Protein, LIP, Are Translated from the Same mRNA
No full textLAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerlzation helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP/LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP/LIP ratio increases about 5-fold during terminal rat liver differentlation and Is thus likely to modulate the activity of LAP in the intact animal
Convex geodesic bicombings and hyperbolicity
No full textA geodesic bicombing on a metric space selects for every pair of points a geodesic connecting them. We prove existence and uniqueness results for geodesic bicombings satisfying different convexity conditions. In combination with recent work by the second author on injective hulls, this shows that every word hyperbolic group acts geometrically on a proper, finite dimensional space X with a unique (hence equivariant) convex geodesic bicombing of the strongest type. Furthermore, the Gromov boundary of X is a Z -set in the closure of X , and the latter is a metrizable absolute retract, in analogy with the Bestvina-Mess theorem on the Rips complex
Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity
No full textMitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum–bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function
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