Muscle-Specific Loss of \u3cem\u3eBmal1\u3c/em\u3e Leads to Disrupted Tissue Glucose Metabolism and Systemic Glucose Homeostasis

Background: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 −/− mouse (iMSBmal1 −/−). Results: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 −/− mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 −/− muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 −/− muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle. Conclusions: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism

Genotyping of Apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I ( Clostridium formicoaceticum ), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine clinical analysis of apo E. © 1998 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35073/1/281_ftp.pd

The development of separations and mass spectrometry technologies for the analysis of biomolecules.

The goal of this work was to develop liquid phase separations and mass spectrometric detection methods for the analysis of DNA and proteins. The methods are intended to replace gel electrophoresis in order to improve the speed, reproducibility and resolving power for molecular biological procedures used in genomic and proteomic studies. Four projects were undertaken which illustrate different aspects of this approach. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) has been applied to the detection of DNA restriction fragments for a common diagnostic test for apolipoprotein E, a risk factor for Alzheimer's disease. A reversed-phase HPLC method for the separation of DNA restriction fragments and Polymerase Chain Reaction (PCR) products was also developed. Compared with gel electrophoresis, both methods reduce analysis times from several hours to a few minutes or less. A two-dimensional (2-D) liquid phase separation technique that separates proteins according to isoelectric point (pI) in the first dimension and hydrophobicity using reversed phase HPLC in the second dimension was developed as an alternative to 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE). The liquid phase technique produces a map of cellular proteins similar to that produced by 2-D PAGE where the proteins in the liquid phase after separation, thus simplifying further analysis. This method requires 8 hours to complete, compared with 3 days to complete a 2-D PAGE gel, and is highly reproducible. A three-dimensional separation technique was developed where the same 2-D liquid phase separation (pl vs. hydrophobicity) is used, after which the proteins are injected on-line into an Electrospray Ionization Time of Flight Mass Spectrometer (ESI-TOF MS) that gives highly accurate masses for the intact proteins. The use of three parameters (pI, hydrophobicity and protein mass) as a basis for comparison of proteins expressed in cells under different conditions (i.e normal vs. diseased) ensures that the majority of common proteins can be confidently identified, allowing researchers to quickly assess the presence of biologically significant changes in proteins expressed in different cell types. This method can also reveal post-translational modifications (PTMs), because mass shifts due to PTMs are much larger than the variance in protein masses detected by this method.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129721/2/3042094.pd

Multi-Omics Approach Profiling Metabolic Remodeling in Early Systolic Dysfunction and in Overt Systolic Heart Failure

Metabolic remodeling plays an important role in the pathophysiology of heart failure (HF). We sought to characterize metabolic remodeling and implicated signaling pathways in two rat models of early systolic dysfunction (MOD), and overt systolic HF (SHF). Tandem mass tag-labeled shotgun proteomics, phospho-(p)-proteomics, and non-targeted metabolomics analyses were performed in left ventricular myocardium tissue from Sham, MOD, and SHF using liquid chromatography–mass spectrometry, n = 3 biological samples per group. Mitochondrial proteins were predominantly down-regulated in MOD (125) and SHF (328) vs. Sham. Of these, 82% (103/125) and 66% (218/328) were involved in metabolism and respiration. Oxidative phosphorylation, mitochondrial fatty acid β-oxidation, Krebs cycle, branched-chain amino acids, and amino acid (glutamine and tryptophan) degradation were highly enriched metabolic pathways that decreased in SHF > MOD. Glycogen and glucose degradation increased predominantly in MOD, whereas glycolysis and pyruvate metabolism decreased predominantly in SHF. PKA signaling at the endoplasmic reticulum–mt interface was attenuated in MOD, whereas overall PKA and AMPK cellular signaling were attenuated in SHF vs. Sham. In conclusion, metabolic remodeling plays an important role in myocardial remodeling. PKA and AMPK signaling crosstalk governs metabolic remodeling in progression to SHF

Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat

Excess Ω-6 Fatty Acids Influx in Aging Drives Metabolic Dysregulation, Electrocardiographic Alterations, and Low-Grade Chronic Inflammation

Maintaining a balance of ω-6 and ω-3 fatty acids is essential for cardiac health. Current ω-6 and ω-3 fatty acids in the American diet have shifted from the ideal ratio of 2:1 to almost 20:1; while there is a body of evidence that suggests the negative impact of such a shift in younger organisms, the underlying age-related metabolic signaling in response to the excess influx of ω-6 fatty acids is incompletely understood. In the present study, young (6 mo old) and aging (≥18 mo old) mice were fed for 2 mo with a ω-6-enriched diet. Excess intake of ω-6 enrichment decreased the total lean mass and increased nighttime carbohydrate utilization, with higher levels of cardiac cytokines indicating low-grade chronic inflammation. Dobutamine-induced stress tests displayed an increase in PR interval, a sign of an atrioventricular defect in ω-6-fed aging mice. Excess ω-6 fatty acid intake in aging mice showed decreased 12-lipoxygenase with a concomitant increase in 15-lipoxygenase levels, resulting in the generation of 15(S)-hydroxyeicosatetraenoic acid, whereas cyclooxygenase-1 and -2 generated prostaglandin E2, leukotriene B4, and thromboxane B2. Furthermore, excessive ω-6 fatty acids led to dysregulated nuclear erythroid 2-related factor 2/antiox-idant-responsive element in aging mice. Moreover, ω-6 fatty acid-mediated changes were profound in aging mice with respect to the eicosanoid profile while minimal changes were observed in the size and shape of cardiomyocytes. These findings provide compelling evidence that surplus consumption of ω-6 fatty acids, coupled with insufficient intake of ω-3 fatty acids, is linked to abnormal changes in ECG. These manifestations contribute to functional deficiencies and expansion of the inflammatory mediator milieu during later stages of aging. NEW & NOTEWORTHY Aging has a profound impact on the metabolism of fatty acids to maintain heart function. The excess influx of ω-6 fatty acids in aging perturbed electrocardiography with marked signs of inflammation and a dysregulated oxidative-redox balance. Thus, the quality and quantity of fatty acids determine the cardiac pathology and energy utilization in aging

Glioblastoma Therapy Can Be Augmented by Targeting IDH1-Mediated NADPH Biosynthesis

NADPH is a critical reductant needed in cancer cells to fuel the biosynthesis of deoxynucleotides and antioxidants and to sustain stress-survival responses after radiation-induced DNA damage. Thus, one rational strategy to attack cancer cells is to target their heavy reliance on NADPH. Here, we report that the isocitrate dehydrogenase IDH1 is the most strongly upregulated NADPH-producing enzyme in glioblastoma (GBM). IDH1 silencing in GBM cells reduced levels of NADPH, deoxynucleotides, and glutathione and increased their sensitivity to radiation-induced senescence. Rescuing these metabolic restrictions was sufficient to reverse IDH1-mediated radiosensitization. In a murine xenograft model of human GBM, we found that IDH1 silencing significantly improved therapeutic responses to fractionated radiotherapy, when compared with either treatment alone. In summary, our work offers a mechanistic rationale for IDH1 inhibition as a metabolic strategy to improve the response of GBM to radiotherapy. Cancer Res; 77(4); 960-70. ©2016 AACR