Integrating adipocyte insulin signaling and metabolism in the multi-omics era.

Insulin stimulates glucose uptake into adipocytes via mTORC2/AKT signaling and GLUT4 translocation and directs glucose carbons into glycolysis, glycerol for TAG synthesis, and de novo lipogenesis. Adipocyte insulin resistance is an early indicator of type 2 diabetes in obesity, a worldwide health crisis. Thus, understanding the interplay between insulin signaling and central carbon metabolism pathways that maintains adipocyte function, blood glucose levels, and metabolic homeostasis is critical. While classically viewed through the lens of individual enzyme-substrate interactions, advances in mass spectrometry are beginning to illuminate adipocyte signaling and metabolic networks on an unprecedented scale, yet this is just the tip of the iceberg. Here, we review how 'omics approaches help to elucidate adipocyte insulin action in cellular time and space.

Author Correction: mTORC2-AKT signaling to ATP-citrate lyase drives brown adipogenesis and de novo lipogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

mTORC2-AKT signaling to ATP-citrate lyase drives brown adipogenesis and de novo lipogenesis.

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.

Modeling Urban Hydrology and Green Infrastructure Using the AGWA Urban Tool and the KINEROS2 Model.

Urban hydrology and green infrastructure (GI) can be modeled using the Automated Geospatial Watershed Assessment (AGWA) Urban tool and the Kinematic Runoff and Erosion (KINEROS2) model. The KINEROS2 model provides an urban modeling element with nine overland flow components that can be used to represent various land cover types commonly found in the built environment while treating runoff-runon and infiltration processes in a physically based manner. The AGWA Urban tool utilizes a Geographic Information System (GIS) framework to prepare parameters required for KINEROS2, executes the model, and imports results for visualization in the GIS. The AGWA Urban tool was validated on a residential subdivision in Arizona, USA, using 47 rainfall events (June 2005 to September 2006) to compare observed runoff volumes and peak flow rates with simulated results. Comparison of simulated and observed runoff volumes resulted in a slope of 1.00 for the regression equation with an R2 value of 0.80. Comparison of observed and simulated peak flows had a slope of 1.12 with an R 2 value of 0.83. A roof runoff analysis was simulated for 787 events, from January 2006 through December 2015, to analyze the water availability from roof runoff capture. Simulation results indicated a 15% capture of the average monthly rainfall volume on the watershed. Additionally, rainwater captured from roofs has the potential to provide for up to 70% of the domestic annual per capita water use in this region. Five different scenarios (S1 - base, S2 - with retention basins, S3 - with permeable driveways, S4 - with rainwater harvesting cisterns, and S5 - all GI practices from S2, S3, and S4) were simulated over the same period to compare the effectiveness of GI implementation at the parcel level on runoff and peak flows at the watershed outlet. Simulation results indicate a higher runoff volume reduction for S2 (53.41 m3 average capacity, average 30% reduction) as compared to S3 (average 14% reduction), or S4 (3.78 m3 capacity, average 6% reduction). Analysis of peak flows reveal larger peak flow reduction for S2. S3 showed more reduction of smaller peak flows as compared to S4.

Spatial patterns of grassland­shrubland state transitions: a 74­year record on grazed and protected areas.

Tree and shrub abundance has increased in many grasslands causing changes in ecosystem carbon and nitrogen pools that are related to patterns of woody plant distribution. However, with regard to spatial patterns of shrub proliferation, little is known about how they are influenced by grazing or the extent to which they are influenced by intraspecific interactions. We addressed these questions by quantifying changes in the spatial distribution of Prosopis velutina (mesquite) shrubs over 74 years on grazed and protected grasslands. Livestock are effective agents of mesquite dispersal and mesquite plants have lateral roots extending well beyond the canopy. We therefore hypothesized that mesquite distributions would be random on grazed areas mainly due to cattle dispersion and clustered on protected areas due to decreased dispersal and interspecific interference with grasses; and that clustered or random distributions at early stages of encroachment would give way to regular distributions as stands matured and density-dependent interactions intensified. Assessments in 1932, 1948, and 2006 supported the first hypothesis, but we found no support for the second. In fact, clustering intensified with time on the protected area and the pattern remained random on the grazed site. Although shrub density increased on both areas between 1932 and 2006, we saw no progression toward a regular distribution indicative of density-dependent interactions. We propose that processes related to seed dispersal, grass­shrub seedling interactions, and hydrological constraints on shrub size interact to determine vegetation structure in grassland-to-shrubland state changes with implications for ecosystem function and management.

RIPGIS-NET: a GIS tool for riparian groundwater evapotranspiration in MODFLOW.

RIPGIS-NET, an Environmental System Research Institute (ESRI's) ArcGIS 9.2/9.3 custom application, was developed to derive parameters and visualize results of spatially explicit riparian groundwater evapotranspiration (ETg), evapotranspiration from saturated zone, in groundwater flow models for ecohydrology, riparian ecosystem management, and stream restoration. Specifically RIPGIS-NET works with riparian evapotranspiration (RIP-ET), a modeling package that works with the MODFLOW groundwater flow model. RIP-ET improves ETg simulations by using a set of eco-physiologically based ETg curves for plant functional subgroups (PFSGs), and separates ground evaporation and plant transpiration processes from the water table. The RIPGIS-NET program was developed in Visual Basic 2005, .NET framework 2.0, and runs in ArcMap 9.2 and 9.3 applications. RIPGIS-NET, a pre- and post-processor for RIP-ET, incorporates spatial variability of riparian vegetation and land surface elevation into ETg estimation in MODFLOW groundwater models. RIPGIS-NET derives RIP-ET input parameters including PFSG evapotranspiration curve parameters, fractional coverage areas of each PFSG in a MODFLOW cell, and average surface elevation per riparian vegetation polygon using a digital elevation model. RIPGIS-NET also provides visualization tools for modelers to create head maps, depth to water table (DTWT) maps, and plot DTWT for a PFSG in a polygon in the Geographic Information System based on MODFLOW simulation results.

Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy.

Small molecule inhibitors that selectively target cancer cells and not normal cells would be valuable anti-cancer therapeutics. The mammalian target of rapamycin complex 2 (mTORC2) is emerging as a promising candidate target for such an inhibitor. Recent studies in cancer biology indicate that mTORC2 activity is essential for the transformation and vitality of a number of cancer cell types, but in many normal cells, mTORC2 activity is less essential. These studies are intensifying interest in developing inhibitors that specifically target mTORC2. However, there are many open questions regarding the function and regulation of mTORC2 and its function in both normal and cancer cells. Here, we summarize exciting new research into the biology of mTORC2 signaling and highlight the current state and future prospects for mTOR-targeted therapy.

Tracking acid mine-drainage in Southeast Arizona using GIS and sediment delivery models.

This study investigates the application of models traditionally used to estimate erosion and sediment deposition to assess the potential risk of water quality impairment resulting from metal-bearing materials related to mining and mineralization. An integrated watershed analysis using Geographic Information Systems (GIS) based tools was undertaken to examine erosion and sediment transport characteristics within the watersheds. Estimates of stream deposits of sediment from mine tailings were related to the chemistry of surface water to assess the effectiveness of the methodology to assess the risk of acid mine-drainage being dispersed downstream of abandoned tailings and waste rock piles. A watershed analysis was preformed in the Patagonia Mountains in southeastern Arizona which has seen substantial mining and where recent water quality samples have reported acidic surface waters. This research demonstrates an improvement of the ability to predict streams that are likely to have severely degraded water quality as a result of past mining activities.

Interaction between the noncatalytic region of Sid1p kinase and Cdc14p is required for full catalytic activity and localization of Sid1p.

Sid1p is a group II p21-activated kinase/germinal center kinase family member that is part of a signaling network required for cytokinesis in fission yeast. Germinal center kinases are characterized by well conserved amino-terminal catalytic domains followed by less conserved carboxyl termini. The carboxyl termini among group I germinal center kinases are moderately conserved and thought to be regulatory regions. Little is known about the carboxyl termini of group II family members. Sid1p has been shown to bind the novel protein Cdc14p; however, the functional significance of this interaction is unknown. Here we report that the carboxyl terminus of Sid1p is an essential regulatory region. Our results indicate that this region contains the binding domain for Cdc14p, and this association is required for full Sid1p catalytic activity as well as intracellular localization. Furthermore, overexpression of the carboxyl terminus of Sid1p alone compromises the signaling of cytokinesis. We conclude that Cdc14p positively regulates the Sid1p kinase by binding the noncatalytic carboxyl-terminal region of the protein.

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast.

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.

Early reduction in insulin-like growth factor gene expression in diabetic nerve.

Diabetic neuropathy is a common and disabling complication of diabetes mellitus whose pathogenesis remains unknown. Insulin-like growth factors (IGFs) have been recently implicated in the development and maintenance of the peripheral nervous system, and circulating IGF levels are decreased in experimental and clinical diabetes. Therefore, we tested the hypothesis that IGF gene expression is reduced in peripheral nerves early after the onset of diabetes. Sciatic nerves from nondiabetic and streptozotocin-treated rats were removed 5-7 days after the induction of diabetes. RNA was isolated and analyzed by Northern and slot blots. IGF-I mRNA content was significantly decreased per milligram wet weight nerve (P < 0.025) as well as per poly(A)+ RNA (P < 0.01) in diabetic vs nondiabetic nerves. Likewise, the amount of IGF-II mRNA was significantly decreased per milligram wet weight nerve (P < 0.01) as well as per poly(A)+ RNA (P < 0.005). These effects were selective because histone 3.3 mRNA content, as well as poly(A)+ mRNA content, per milligram nerve were unchanged. Insulin treatment partially prevented this decline in IGF-I and IGF-II mRNA levels. The diminished IGF mRNA content is one of the earliest biochemical abnormalities to be observed in the diabetic nerve, supporting the hypothesis that a reduction in IGF activity in diabetic nerves precedes and contributes to the development of neuropathy.

Reduced insulin-like growth factor-I mRNA content in liver, adrenal glands and spinal cord of diabetic rats.

Circulating insulin-like growth factor I (IGF-I) concentrations are known to be reduced in experimental and clinical diabetes mellitus. The IGF-I mRNA content was measured in several tissues of rats treated with streptozotocin to determine whether a correlation with neuropathy could be found. IGF-I mRNA content was sharply reduced relative to total and poly(A)+ RNA in diabetic liver and adrenal glands. In contrast, histone 3.3 mRNA content was not significantly reduced relative to poly(A)+ RNA in liver, and alpha-tubulin mRNA content instead was increased in adrenal glands, showing that the decline in IGF-I mRNAs in these tissues was selective. In addition, spinal cord IGF-I mRNA content was significantly reduced per tissue, total RNA, and poly(A)+ RNA after 1 and 2 weeks of diabetes. This was correlated with a concurrent and significant decrease in conduction velocity in both spinal cord and peripheral nerves in a separate study. The decline in liver and spinal cord IGF-I mRNA was not due to streptozotocin toxicity, because it was significantly opposed by insulin which was continuously infused beginning the day after diabetes induction. These results, when taken together with those of others, indicate that the reduction in IGF-I mRNA content may be widespread among diabetic tissues, and might contribute in part to certain syndromes of diabetes, such as neuropathy.

[Analysis of the behavior of the coach in relation to violence in minor league hockey]. / Analyse des comportements de l'entraîneur par rapport à la violence au hockey mineur.

The purpose of this study was to verify if, during games, the behavior of ice hockey coaches at the bantam level tends to incite players to use roughness and to infringe upon the rules of the game, as the Néron report (1977) states. The video recording of 27 games using a split-screen technique made it possible to view simultaneously the players in action as well as the coaches' behavior. Analysis of the videotapes revealed that the coaches (n = 11) at the bantam level often exhort their players to put more intensity in their physical contacts (legal body checking), but they more often encouraged them to control themselves and avoid penalties. In general, the coaches displayed very little behavior that encouraged violent actions from the players.

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue.

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.

ATP- and coenzyme A-dependent fatty acid incorporation into proteins of cell-free extracts from mouse tissues.

The incorporation of tritiated fatty acids into proteins has been studied in cell-free extracts from mouse tissues. Incubation of heart extracts with [3H]tetradecanoic or [3H]palmitic acid in the presence of ATP and CoA resulted in the time-dependent and selective labeling of proteins (Mr = 60,000, 47,000, 42,000, 31,000, 16,000, and 13,000) which could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Two polypeptides (Mr = 47,000 and 42,000) reached a maximum in fatty acid incorporation very rapidly and were mainly localized in the membrane subcellular fractions of the extract. These proteins underwent transient labeling with [3H] tetradecanoyl-CoA, the maximum incorporation being obtained within 1 min. The fatty acid-labeled proteins from tissue extracts had the same properties as other proteins known to be acylated in intact cells, i.e. the acyl moiety was resistant to delipidation with organic solvents but could be hydrolyzed by treatment with neutral hydroxylamine. Screening of different tissues showed that extracts from liver and kidney also catalyze the ATP- and CoA-dependent formation of a similar group of fatty acid-acylated proteins. The results provide evidence for a group of proteins in mammalian tissues which selectively incorporate fatty acids in vitro and should be of value for further studies on the biosynthesis of acylated proteins.

mRNA secondary structure as a determinant in cap recognition and initiation complex formation. ATP-Mg2+ independent cross-linking of cap binding proteins to m7I-capped inosine-substituted reovirus mRNA.

Polypeptides of Mr = 50,000 and 80,000 in rabbit reticulocyte initiation factor preparations can be specifically cross-linked to the oxidized t' cap structure of native reovirus mRNA in an ATP-Mg2+-dependent manner (Sonenberg, N., Guertin, D., Cleveland, D., and Trachsel, H. (1981) Cell 27, 563-572). However, specific cross-linking of these polypeptides can occur in the absence of ATP-Mg2+ when m7I-capped inosine substituted mRNA, which contains less secondary structure than native reovirus mRNA, is used. We also found, using wheat germ extract, that inhibition of initiation complex formation by high salt concentrations is directly related to the degree of secondary structure of the mRNA. Binding of ribosomes to bromouridine-substituted reovirus mRNA is severely inhibited at high K+ concentrations, while binding to inosine-substituted mRNA is only slightly inhibited and binding of native reovirus mRNA is inhibited to an intermediate degree. The results are consistent with the hypothesis that cap recognition factors mediate an ATP-dependent melting of secondary structures involving 5' proximal sequences to the initiation codon in order to facilitate binding of ribosomes during translation initiation.

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells.

Extracts from poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, we demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosaic virus 4 RNA, which is most probably devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture.

The present results demonstrate that Chinese hamster embryo cell populations in culture can be adapted to grow in the presence of chloramphenicol. It is shown that tryptose phosphate broth and uridine, one of its components, prevent the growth inhibitory effect of the drug. Study of some respiratory parameters (cytochrome c oxidase, cytochrome spectra, oxygen consumption) indicated that neither the broth nor uridine prevented the inhibitory effect of chloramphenicol on mitoribosomal protein synthesis. The cells grew with mitochondria devoid of a functional respiratory chain. Auxotrophy for pyrimidines appeared to result from the absence of dihydroorotate dehydrogenase, a respiratory chain-linked enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis. These and other results suggest that the synthesis of orotic acid may be considered as one of the main contributions of mitochondria to the growth of animal cells in culture.

Probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins.

A monoclonal antibody directed against cap-binding proteins was used to elucidate the possible mechanism by which cap-binding proteins function in initiation of eucaryotic translation. The monoclonal antibody preparation employed in this study exhibited a marked differential effect in inhibiting the translation of folded, capped eucaryotic-mRNAs to a far greater extent than naturally uncapped mRNAs or native capped mRNAs that do not possess extensive 5' end secondary structure. These findings were consistent with the effects of the antibody on initiation complex formation with three different types of reovirus mRNA: native reovirus mRNA; inosine-substituted reovirus mRNA, which has a relaxed secondary structure; and bromouridine-substituted reovirus mRNA, in which base pairing is enhanced relative to regular reovirus mRNA. The extent that translation initiation complex formation was inhibited by the monoclonal antibody directly correlated to the degree of secondary structure present in the mRNA. Binding of bromouridine-substituted reovirus mRNA to ribosomes was inhibited to the greatest extent, while binding of inosine-substituted reovirus mRNA was not inhibited at all in the reticulocyte lysate system or was slightly inhibited in a wheat-germ system. These results support the hypothesis that cap-binding proteins are involved in unwinding of the 5' terminal, secondary structure of many eucaryotic mRNAs, thus facilitating the attachment of ribosomes to mRNA.