Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling

NIA-AA Research Framework: Toward a Biological Definition of Alzheimer\u27s Disease

In 2011, the National Institute on Aging and Alzheimer\u27s Association created separate diagnostic recommendations for the preclinical, mild cognitive impairment, and dementia stages of Alzheimer\u27s disease. Scientific progress in the interim led to an initiative by the National Institute on Aging and Alzheimer\u27s Association to update and unify the 2011 guidelines. This unifying update is labeled a “research framework” because its intended use is for observational and interventional research, not routine clinical care. In the National Institute on Aging and Alzheimer\u27s Association Research Framework, Alzheimer\u27s disease (AD) is defined by its underlying pathologic processes that can be documented by postmortem examination or in vivo by biomarkers. The diagnosis is not based on the clinical consequences of the disease (i.e., symptoms/signs) in this research framework, which shifts the definition of AD in living people from a syndromal to a biological construct. The research framework focuses on the diagnosis of AD with biomarkers in living persons. Biomarkers are grouped into those of β amyloid deposition, pathologic tau, and neurodegeneration [AT(N)]. This ATN classification system groups different biomarkers (imaging and biofluids) by the pathologic process each measures. The AT(N) system is flexible in that new biomarkers can be added to the three existing AT(N) groups, and new biomarker groups beyond AT(N) can be added when they become available. We focus on AD as a continuum, and cognitive staging may be accomplished using continuous measures. However, we also outline two different categorical cognitive schemes for staging the severity of cognitive impairment: a scheme using three traditional syndromal categories and a six-stage numeric scheme. It is important to stress that this framework seeks to create a common language with which investigators can generate and test hypotheses about the interactions among different pathologic processes (denoted by biomarkers) and cognitive symptoms. We appreciate the concern that this biomarker-based research framework has the potential to be misused. Therefore, we emphasize, first, it is premature and inappropriate to use this research framework in general medical practice. Second, this research framework should not be used to restrict alternative approaches to hypothesis testing that do not use biomarkers. There will be situations where biomarkers are not available or requiring them would be counterproductive to the specific research goals (discussed in more detail later in the document). Thus, biomarker-based research should not be considered a template for all research into age-related cognitive impairment and dementia; rather, it should be applied when it is fit for the purpose of the specific research goals of a study. Importantly, this framework should be examined in diverse populations. Although it is possible that β-amyloid plaques and neurofibrillary tau deposits are not causal in AD pathogenesis, it is these abnormal protein deposits that define AD as a unique neurodegenerative diseaseamong different disorders that can lead to dementia. We envision that defining AD as a biological construct will enable a more accurate characterization and understanding of the sequence of events that lead to cognitive impairment that is associated with AD, as well as the multifactorial etiology of dementia. This approach also will enable a more precise approach to interventional trials where specific pathways can be targeted in the disease process and in the appropriate people

DJ-1 contributes to adipogenesis and obesity-induced inflammation

Adipose tissue functions as an endocrine organ, and the development of systemic inflammation in adipose tissue is closely associated with metabolic diseases, such as obesity and insulin resistance. Accordingly, the fine regulation of the inflammatory response caused by obesity has therapeutic potential for the treatment of metabolic syndrome. In this study, we analyzed the role of DJ-1 (PARK7) in adipogenesis and inflammation related to obesity in vitro and in vivo. Many intracellular functions of DJ-1, including oxidative stress regulation, are known. However, the possibility of DJ-1 involvement in metabolic disease is largely unknown. Our results suggest that DJ-1 deficiency results in reduced adipogenesis and the down-regulation of pro-inflammatory cytokines in vitro. Furthermore, DJ-1-deficient mice show a low-level inflammatory response in the high-fat diet-induced obesity model. These results indicate previously unknown functions of DJ-1 in metabolism and therefore suggest that precise regulation of DJ-1 in adipose tissue might have a therapeutic advantage for metabolic disease treatment.open0

AMP-Activated Protein Kinase Plays an Important Evolutionary Conserved Role in the Regulation of Glucose Metabolism in Fish Skeletal Muscle Cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP∶ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish

Leaf Trait-Environment Relationships in a Subtropical Broadleaved Forest in South-East China

Although trait analyses have become more important in community ecology, trait-environment correlations have rarely been studied along successional gradients. We asked which environmental variables had the strongest impact on intraspecific and interspecific trait variation in the community and which traits were most responsive to the environment. We established a series of plots in a secondary forest in the Chinese subtropics, stratified by successional stages that were defined by the time elapsed since the last logging activities. On a total of 27 plots all woody plants were recorded and a set of individuals of every species was analysed for leaf traits, resulting in a trait matrix of 26 leaf traits for 122 species. A Fourth Corner Analysis revealed that the mean values of many leaf traits were tightly related to the successional gradient. Most shifts in traits followed the leaf economics spectrum with decreasing specific leaf area and leaf nutrient contents with successional time. Beside succession, few additional environmental variables resulted in significant trait relationships, such as soil moisture and soil C and N content as well as topographical variables. Not all traits were related to the leaf economics spectrum, and thus, to the successional gradient, such as stomata size and density. By comparing different permutation models in the Fourth Corner Analysis, we found that the trait-environment link was based more on the association of species with the environment than of the communities with species traits. The strong species-environment association was brought about by a clear gradient in species composition along the succession series, while communities were not well differentiated in mean trait composition. In contrast, intraspecific trait variation did not show close environmental relationships. The study confirmed the role of environmental trait filtering in subtropical forests, with traits associated with the leaf economics spectrum being the most responsive ones

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field