Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis

Peer reviewedPublisher PD

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

AIMS/HYPOTHESIS Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. METHODS Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. RESULTS Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. CONCLUSIONS/INTERPRETATION Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects

An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline

BACKGROUND: The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation. RESULTS: The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics. CONCLUSION: We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process

Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice

BACKGROUND: Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain( NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT. METHODS: BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post- infection (wpi) by Western blotting and RT-PCR. RESULTS: Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain. CONCLUSION: Further studies are needed to determine whether there is an increased risk of CT progression into neurodegenerative disease because neurodegeneration-associated AbetaPP and phosphorylated tau emerged in the brain. DOI: 10.1186/1471-2334-8-8

Investigating Bacterial Sources of Toxicity as an Environmental Contributor to Dopaminergic Neurodegeneration

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as α-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD

Oxytocin receptor gene polymorphisms are associated with human directed social behavior in dogs (Canis familiaris)

The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (−212AG, 19131AG) and one known (rs8679684) single nucleotide polymorphisms (SNPs) in the regulatory regions (5′ and 3′ UTR) of the oxytocin receptor gene in German Shepherd (N = 104) and Border Collie (N = 103) dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3′ and 5′ UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene–behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i) proximity seeking towards an unfamiliar person, as well as their owner, and on (ii) how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system

Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition

Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity

Huntington's disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database.

BACKGROUND: Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. METHODS: To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. RESULTS: Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling). For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are associated with HD, at http://hdtt.sysbiolab.eu Additionally, we derived a candidate set of 24 novel genetic modifiers, including histone deacetylase 3 (HDAC3), metabotropic glutamate receptor 1 (GRM1), CDK5 regulatory subunit 2 (CDK5R2), and coactivator 1ß of the peroxisome proliferator-activated receptor gamma (PPARGC1B). CONCLUSIONS: The results of our study give us an intriguing picture of the molecular complexity of HD. Our analyses can be seen as a first step towards a comprehensive list of biological processes, molecular functions, and pathways involved in HD, and may provide a basis for the development of more holistic disease models and new therapeutics

p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

p21(WAF1/CIP1) is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown.We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis.We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1