Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis but the underlying mechanism remains unclear. In this study we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischaemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin10 (IL10). Therefore we hypothesised that ketosis-stabilised HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, eight-week-old rats were fed either the standard chow diet (SD) or the ketogenic (KG) diet for 4 weeks before ischaemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet fed group.

Chronic intake of high dietary sucrose induces sexually dimorphic metabolic adaptations in mouse liver and adipose tissue.

Almost all effective treatments for non-alcoholic fatty liver disease (NAFLD) involve reduction of adiposity, which suggests the metabolic axis between liver and adipose tissue is essential to NAFLD development. Since excessive dietary sugar intake may be an initiating factor for NAFLD, we have characterized the metabolic effects of liquid sucrose intake at concentrations relevant to typical human consumption in mice. We report that sucrose intake induces sexually dimorphic effects in liver, adipose tissue, and the microbiome; differences concordant with steatosis severity. We show that when steatosis is decoupled from impairments in insulin responsiveness, sex is a moderating factor that influences sucrose-driven lipid storage and the contribution of de novo fatty acid synthesis to the overall hepatic triglyceride pool. Our findings provide physiologic insight into how sex influences the regulation of adipose-liver crosstalk and highlight the importance of extrahepatic metabolism in the pathogenesis of diet-induced steatosis and NAFLD.

Integrated omics analysis reveals sirtuin signaling is central to hepatic response to a high fructose diet.

BACKGROUND: Dietary high fructose (HFr) is a known metabolic disruptor contributing to development of obesity and diabetes in Western societies. Initial molecular changes from exposure to HFr on liver metabolism may be essential to understand the perturbations leading to insulin resistance and abnormalities in lipid and carbohydrate metabolism. We studied vervet monkeys (Clorocebus aethiops sabaeus) fed a HFr (n=5) or chow diet (n=5) for 6 weeks, and obtained clinical measures of liver function, blood insulin, cholesterol and triglycerides. In addition, we performed untargeted global transcriptomics, proteomics, and metabolomics analyses on liver biopsies to determine the molecular impact of a HFr diet on coordinated pathways and networks that differed by diet. RESULTS: We show that integration of omics data sets improved statistical significance for some pathways and networks, and decreased significance for others, suggesting that multiple omics datasets enhance confidence in relevant pathway and network identification. Specifically, we found that sirtuin signaling and a peroxisome proliferator activated receptor alpha (PPARA) regulatory network were significantly altered in hepatic response to HFr. Integration of metabolomics and miRNAs data further strengthened our findings. CONCLUSIONS: Our integrated analysis of three types of omics data with pathway and regulatory network analysis demonstrates the usefulness of this approach for discovery of molecular networks central to a biological response. In addition, metabolites aspartic acid and docosahexaenoic acid (DHA), protein ATG3, and genes ATG7, and HMGCS2 link sirtuin signaling and the PPARA network suggesting molecular mechanisms for altered hepatic gluconeogenesis from consumption of a HFr diet.

Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis, but the underlying mechanism remains unclear. In this study, we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain, and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin 10 (IL10). Therefore, we hypothesized that ketosis-stabilized HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, 8-week-rats were fed either the standard chow diet (SD) or the KG diet for 4 weeks before ischemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet-fed group.

TGR5 signaling mitigates parenteral nutrition-associated liver disease.

Bile acid receptors regulate the metabolic and immune functions of circulating enterohepatic bile acids. This process is disrupted by administration of parenteral nutrition (PN), which may induce progressive hepatic injury for unclear reasons, especially in the newborn, leading to PN-associated liver disease. To explore the role of bile acid signaling on neonatal hepatic function, we initially observed that Takeda G protein receptor 5 (TGR5)-specific bile acids were negatively correlated with worsening clinical disease markers in the plasma of human newborns with prolonged PN exposure. To test our resulting hypothesis that TGR5 regulates critical liver functions to PN exposure, we used TGR5 receptor deficient mice (TGR5-/-). We observed PN significantly increased liver weight, cholestasis, and serum hepatic stress enzymes in TGR5-/- mice compared with controls. Mechanistically, PN reduced bile acid synthesis genes in TGR5-/-. Serum bile acid composition revealed that PN increased unconjugated primary bile acids and secondary bile acids in TGR5-/- mice, while increasing conjugated primary bile acid levels in TGR5-competent mice. Simultaneously, PN elevated hepatic IL-6 expression and infiltrating macrophages in TGR5-/- mice. However, the gut microbiota of TGR5-/- mice compared with WT mice following PN administration displayed highly elevated levels of Bacteroides and Parabacteroides, and possibly responsible for the elevated levels of secondary bile acids in TGR5-/- animals. Intestinal bile acid transporters expression was unchanged. Collectively, this suggests TGR5 signaling specifically regulates fundamental aspects of liver bile acid homeostasis during exposure to PN. Loss of TGR5 is associated with biochemical evidence of cholestasis in both humans and mice on PN.NEW & NOTEWORTHY Parenteral nutrition is associated with deleterious metabolic outcomes in patients with prolonged exposure. Here, we demonstrate that accelerated cholestasis and parental nutrition-associated liver disease (PNALD) may be associated with deficiency of Takeda G protein receptor 5 (TGR5) signaling. The microbiome is responsible for production of secondary bile acids that signal through TGR5. Therefore, collectively, these data support the hypothesis that a lack of established microbiome in early life or under prolonged parenteral nutrition may underpin disease development and PNALD.

Bile Diversion Improves Metabolic Phenotype Dependent on Farnesoid X Receptor (FXR).

OBJECTIVE: The current study investigated whether bile diversion (BD) improves metabolic phenotype under farnesoid X receptor (FXR) deficiency. METHODS: BD was performed in high-fat diet (HFD)-fed FXR knockout (FXRko) and wild-type (WT) animals. Metabolic phenotypes, circulating enteroendocrine hormones, total bile acids (BAs) and BA composition, and cecal gut microbiota were analyzed. RESULTS: FXR-deficient mice were resistant to HFD-induced obesity; however, FXR-deficient mice also developed hyperglycemia and exhibited increased liver weight, liver steatosis, and circulating triglycerides. BD increased circulating total BAs and taurine-b-muricholic acid, which were in line with normalized hyperglycemia and improved glucose tolerance in HFD-fed WT mice. FXR deficiency also increased total BAs and taurine-b-muricholic acid, but these animals remained hyperglycemic. While BD improved metabolic phenotype in HFD-fed FXRko mice, these improvements were not as effective as in WT mice. BD increased liver expression of fibroblast growth factor 21 and peroxisome proliferator-activated receptor γ coactivator-1ß and elevated circulating glucagon-like peptide-1 levels in WT mice but not in FXRko mice. FXR deficiency altered gut microbiota composition with a specific increase in phylum Proteobacteria that may act as a possible microbial signature of some diseases. These cellular and molecular changes in FXRko mice may contribute to resistance toward improved metabolism. CONCLUSIONS: FXR signaling plays a pivotal role in improved metabolic phenotype following BD surgery.

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Proteomic characterization of high-density lipoprotein particles in patients with non-alcoholic fatty liver disease.

BACKGROUND: Metabolic diseases such as obesity and diabetes are associated with changes in high-density lipoprotein (HDL) particles, including changes in particle size and protein composition, often resulting in abnormal function. Recent studies suggested that patients with non-alcoholic fatty liver disease (NAFLD), including individuals with non-alcoholic steatohepatitis (NASH), have smaller HDL particles when compared to individuals without liver pathologies. However, no studies have investigated potential changes in HDL particle protein composition in patients with NAFLD, in addition to changes related to obesity, to explore putative functional changes of HDL which may increase the risk of cardiovascular complications. METHODS: From a cohort of morbidly obese females who were diagnosed with simple steatosis (SS), NASH, or normal liver histology, we selected five matched individuals from each condition for a preliminary pilot HDL proteome analysis. HDL particles were enriched using size-exclusion chromatography, and the proteome of the resulting fraction was analyzed by liquid chromatography tandem mass spectrometry. Differences in the proteomes between the three conditions (normal, SS, NASH) were assessed using label-free quantitative analysis. Gene ontology term analysis was performed to assess the potential impact of proteomic changes on specific functions of HDL particles. RESULTS: Of the 95 proteins identified, 12 proteins showed nominally significant differences between the three conditions. Gene ontology term analysis revealed that severity of the liver pathology may significantly impact the anti-thrombotic functions of HDL particles, as suggested by changes in the abundance of HDL-associated proteins such as antithrombin III and plasminogen. CONCLUSIONS: The pilot data from this study suggest that changes in the HDL proteome may impact the functionality of HDL particles in NAFLD and NASH patients. These proteome changes may alter cardio-protective properties of HDL, potentially contributing to the increased cardiovascular disease risk in affected individuals. Further validation of these protein changes by orthogonal approaches is key to confirming the role of alterations in the HDL proteome in NAFLD and NASH. This will help elucidate the mechanistic effects of the altered HDL proteome on cardioprotective properties of HDL particles.

HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges.

Analysis of phagosomal proteomes: from latex-bead to bacterial phagosomes.

Phagosomal proteome characterization has contributed significantly to the understanding of host-pathogen interaction and the mechanism of infectious diseases caused by intracellular bacteria. The latex bead-containing phagosome has been widely used as a model system to study phagosomal proteomes at a global level. In contrast, the study of bacteria-containing phagosomes at a similar level has just begun. A number of intracellular microbial species are studied for their proteomes during the invasion of a host, providing insight into their metabolic adaptation in host cells and interaction with host-cell antimicrobial environments. In this review, we attempt to summarize the most recent advancements in the proteomic study of microbial phagosomes, especially those originating from mouse or human cells. We also briefly describe the proteomics of latex bead-containing phagosomes because they are often used as model phagosomes for study. We provide descriptions on major biological and technological components in phagosomal proteome studies. We also discuss the role of phagosomal proteome study in the broader horizon of systems biology and the technological challenges in phagosomal proteome characterization.

A systems biology approach to study the phagosomal proteome modulated by mycobacterial infections.

Systems biology and proteomics have recently contributed significantly to the insight into the biogenesis and immunity-related functions of the phagosome. To gain insight into the modulation of the phagosomal proteome by the wild-type Mycobacterium tuberculosis H37Rv reference strain, an attenuated mutant of the H37Rv strain, and the BCG Pasteur vaccine strain, we employed the nano-liquid chromatography/LTQ-FTMS based proteomics approach and a systems biology approach to analyze the bacillus-containing phagosomes purified from the bone-marrow-derived BMA3.A3 macrophages infected with the three different mycobacterial strains. We identified 322 proteins at a false-discovery rate of 2%. These proteins were quantified with a label-free proteomics method. All but one of these proteins is mouse proteins. The gene ontology analysis of these mouse proteins suggests that lysosomal proteins represented <3% of the detected proteins, supporting the observation that these mycobacterial strains inhibit or limit the phagosome maturation process. The results also indicate that the endoplasmic reticulum (ER) proteins do not constitute a major part of the phagosome proteome, supporting the phagosome maturation model of the role of ER in phagosome biogenesis. This phagosome maturation model is in contrast to the phagocytosis model which predicts that half of the phagosome membrane is derived from ER. This pilot study demonstrates that a combination of proteomics, multivariate analysis, and systems biology promises to bring forward new insights into the mycobacterial pathogenesis and the interconnected phagosome biology.

Protein turnover in mycobacterial proteomics.

Understanding the biology of Mycobacterium tuberculosis is one of the primary challenges in current tuberculosis research. Investigation of mycobacterial biology using the systems biology approach has deciphered much information with regard to the bacilli and tuberculosis pathogenesis. The modulation of its environment and the ability to enter a dormant phase are the hallmarks of M. tuberculosis. Until now, proteome studies have been able to understand much about the role of various proteins, mostly in growing M. tuberculosis cells. It has been difficult to study dormant M. tuberculosis by conventional proteomic techniques with very few proteins being found to be differentially expressed. Discrepancy between proteome and transcriptome studies lead to the conclusion that a certain aspect of the mycobacterial proteome is not being explored. Analysis of protein turnover may be the answer to this dilemma. This review, while giving a gist of the proteome response of mycobacteria to various stresses, analyzes the data obtained from abundance studies versus data from protein turnover studies in M. tuberculosis. This review brings forth the point that protein turnover analysis is capable of discerning more subtle changes in protein synthesis, degradation, and secretion activities. Thus, turnover studies could be incorporated to provide a more in-depth view into the proteome, especially in dormant or persistent cells. Turnover analysis might prove helpful in drug discovery and a better understanding of the dynamic nature of the proteome of mycobacteria.

Principal Component Analysis of Proteome Dynamics in Iron-starved Mycobacterium Tuberculosis.

The goal of this study is to use principal component analysis (PCA) for multivariate analysis of proteome dynamics based on both protein abundance and turnover information generated by high-resolution mass spectrometry. We previously reported assessing protein dynamics in iron-starved Mycobacterium tuberculosis, revealing interesting interconnection among the cellular processes involving protein synthesis, degradation, and secretion (Anal. Chem. 80, 6860-9). In this study, we use target-decoy database search approach to select peptides for quantitation at a false discovery rate of 4.2%. We further use PCA to reduce the data dimensions for simpler interpretation. The PCA results indicate that the protein turnover and relative abundance properties are approximately orthogonal in the data space defined by the first three principal components. We show the potential of the Hotelling's T2 (T2) value as a quantifiable index for comparing changes between protein functional categories. The T2 value represents the gross change of a protein in both abundance and turnover. Close examination of the antigen 85 complex demonstrates that T2 correctly predicts the coordinated changes of the antigen 85 complex proteins. The multi-dimensional protein dynamics data further reveal the secretion of the antigen 85 complex. Overall, this study demonstrates PCA as an effective means to facilitate interpretation of the multivariate proteome dynamics dataset which otherwise would remain a significant challenge using traditional methods.

Protein dynamics in iron-starved Mycobacterium tuberculosis revealed by turnover and abundance measurement using hybrid-linear ion trap-Fourier transform mass spectrometry.

To study the proteome response of Mycobacterium tuberculosis H37Rv to a change in iron level, iron-starved late-log-phase cells were diluted in fresh low- and high-iron media containing [ (15)N]-labeled asparagine as the sole nitrogen source for labeling the proteins synthesized upon dilution. We determined the relative protein abundance and protein turnover in M. tuberculosis H37Rv under these two conditions. For measurements, we used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation. While relative protein abundance analysis shows that only 5 proteins were upregulated by high iron, 24 proteins had elevated protein turnover for the cells in the high-iron medium. This suggests that protein turnover is a sensitive parameter to assess the proteome dynamics. Cluster analysis was used to explore the interconnection of protein abundance and turnover, revealing coordination of the cellular processes of protein synthesis, degradation, and secretion that determine the abundance and allocation of a protein in the cytosol and the extracellular matrix of the cells. Further potential utility of the approach is discussed.

Determination of global protein turnover in stressed mycobacterium cells using hybrid-linear ion trap-fourier transform mass spectrometry.

We determined the global protein turnover profiles for Mycobacterium smegmatis under acid shock and iron starvation conditions using a simple (15)N isotope doping technique and a complete medium replacement method for chasing. We used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation to measure protein turnover values for 151 proteins over a dynamic range of 3 orders of magnitude ranging from about 0.2 to 500. Of these 151 proteins, 31 had significant protein turnover changes (p <0.05) at both stress conditions and had protein turnover values increased or decreased by more than 2-fold under at least one stress condition. Protein turnover increased under acid shock for 28 of the 31 proteins but decreased under iron starvation for all the 31 proteins. Only two proteins had protein turnover lowered by more than 2-fold (p <0.05) under both stress conditions, including an ATP synthase F1 beta subunit (MSMEG4921; AtpD) and a catalase/peroxidase (MSMEG6346; KatG). KatG is required for in vivo activation of isoniazid to be bacterialcidal. Decrease of KatG protein turnover under both stress conditions supports the view that isoniazid may induce a dormancy program in mycobacteria, which in turn limits the efficacy of this drug against dormant subpopulation of mycobacteria. Thus, measuring protein turnover in stressed Mycobacterium cells has implications in understanding drug action and resistance mechanisms.