Oxidized Low-Density Lipoprotein Accumulation in Macrophages Impairs Lipopolysaccharide-Induced Activation of AKT2, ATP Citrate Lyase, Acetyl-Coenzyme A Production, and Inflammatory Gene H3K27 Acetylation.

The accumulation of lipid and the formation of macrophage foam cells is a hallmark of atherosclerosis, a chronic inflammatory disease. To better understand the role of macrophage lipid accumulation in inflammation during atherogenesis, we studied early molecular events that follow the accumulation of oxidized low-density lipoprotein (oxLDL) in cultured mouse macrophages. We previously showed that oxLDL accumulation downregulates the inflammatory response in conjunction with downregulation of late-phase glycolysis. In this study, we show that within hours after LPS stimulation, macrophages with accumulated oxLDL maintain early-phase glycolysis but selectively downregulate activation of AKT2, one of three AKT isoforms. The inhibition of AKT2 activation reduced LPS-induced ATP citrate lyase activation, acetyl-CoA production, and acetylation of histone 3 lysine 27 (H3K27ac) in certain inflammatory gene promoters. In contrast to oxLDL, multiple early LPS-induced signaling pathways were inhibited in macrophages with accumulated cholesterol, including TBK1, AKT1, AKT2, MAPK, and NF-κB, and early-phase glycolysis. The selective inhibition of LPS-induced AKT2 activation was dependent on the generation of mitochondrial oxygen radicals during the accumulation of oxLDL in macrophages prior to LPS stimulation. This is consistent with increased oxidative phosphorylation, fatty acid synthesis, and oxidation pathways found by comparative transcriptomic analyses of oxLDL-loaded versus control macrophages. Our study shows a functional connection between oxLDL accumulation, inactivation of AKT2, and the inhibition of certain inflammatory genes through epigenetic changes that occur soon after LPS stimulation, independent of early-phase glycolysis.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Targeting Apollo-NADP+ to Image NADPH Generation in Pancreatic Beta-Cell Organelles.

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

TRANSPORTER OF IBA1 Links Auxin and Cytokinin to Influence Root Architecture.

Developmental processes that control root system architecture are critical for soil exploration by plants, allowing for uptake of water and nutrients. Conversion of the auxin precursor indole-3-butyric acid (IBA) to active auxin (indole-3-acetic acid; IAA) modulates lateral root formation. However, mechanisms governing IBA-to-IAA conversion have yet to be elucidated. We identified TRANSPORTER OF IBA1 (TOB1) as a vacuolar IBA transporter that limits lateral root formation. Moreover, TOB1, which is transcriptionally regulated by the phytohormone cytokinin, is necessary for the ability of cytokinin to exert inhibitory effects on lateral root production. The increased production of lateral roots in tob1 mutants, TOB1 transport of IBA into the vacuole, and cytokinin-regulated TOB1 expression provide a mechanism linking cytokinin signaling and IBA contribution to the auxin pool to tune root system architecture.

Three-Dimensional Time-Lapse Analysis Reveals Multiscale Relationships in Maize Root Systems with Contrasting Architectures.

Understanding how an organism's phenotypic traits are conditioned by genetic and environmental variation is a central goal of biology. Root systems are one of the most important but poorly understood aspects of plants, largely due to the three-dimensional (3D), dynamic, and multiscale phenotyping challenge they pose. A critical gap in our knowledge is how root systems build in complexity from a single primary root to a network of thousands of roots that collectively compete for ephemeral, heterogeneous soil resources. We used time-lapse 3D imaging and mathematical modeling to assess root system architectures (RSAs) of two maize (Zea mays) inbred genotypes and their hybrid as they grew in complexity from a few to many roots. Genetically driven differences in root branching zone size and lateral branching densities along a single root, combined with differences in peak growth rate and the relative allocation of carbon resources to new versus existing roots, manifest as sharply distinct global RSAs over time. The 3D imaging of mature field-grown root crowns showed that several genetic differences in seedling architectures could persist throughout development and across environments. This approach connects individual and system-wide scales of root growth dynamics, which could eventually be used to predict genetic variation for complex RSAs and their functions.

Whole-Plant Manual and Image-Based Phenotyping in Controlled Environments.

Phenotypic measurements and images of crops grown under controlled-environment conditions can be analyzed to compare plant growth and other phenotypes from diverse varieties. Those demonstrating the most favorable phenotypic traits can then be used for crop improvement strategies. This article details a protocol for image-based root and shoot phenotyping of plants grown in the greenhouse to compare traits among different varieties. Diverse maize lines were grown in the greenhouse in large 8-gallon treepots in a clay granule substrate. Replicates of each line were harvested at 4 weeks, 6 weeks, and 8 weeks after planting to capture developmental information. Whole-plant phenotypes include biomass accumulation, ontogeny, architecture, and photosynthetic efficiency of leaves. Image analysis was used to measure leaf surface area and tassel size and to extract shape variance information from complex 3D root architectures. Notably, this framework is extensible to any number of above- or below-ground phenotypes, both morphological and physiological. © 2017 by John Wiley & Sons, Inc.

Evidence against equimolarity of large repeat arrangements and a predominant master circle structure of the mitochondrial genome from a monkeyflower (Mimulus guttatus) lineage with cryptic CMS.

Despite intense investigation for over 25 years, the in vivo structure of plant mitochondrial genomes remains uncertain. Mapping studies and genome sequencing generally produce large circular chromosomes, whereas electrophoretic and microscopic studies typically reveal linear and multibranched molecules. To more fully assess the structure of plant mitochondrial genomes, the complete sequence of the monkeyflower (Mimulus guttatus DC. line IM62) mitochondrial DNA was constructed from a large (35 kb) paired-end shotgun sequencing library to a high depth of coverage (~30×). The complete genome maps as a 525,671 bp circular molecule and exhibits a fairly conventional set of features including 62 genes (encoding 35 proteins, 24 transfer RNAs, and 3 ribosomal RNAs), 22 introns, 3 large repeats (2.7, 9.6, and 29 kb), and 96 small repeats (40-293 bp). Most paired-end reads (71%) mapped to the consensus sequence at the expected distance and orientation across the entire genome, validating the accuracy of assembly. Another 10% of reads provided clear evidence of alternative genomic conformations due to apparent rearrangements across large repeats. Quantitative assessment of these repeat-spanning read pairs revealed that all large repeat arrangements are present at appreciable frequencies in vivo, although not always in equimolar amounts. The observed stoichiometric differences for some arrangements are inconsistent with a predominant master circular structure for the mitochondrial genome of M. guttatus IM62. Finally, because IM62 contains a cryptic cytoplasmic male sterility (CMS) system, an in silico search for potential CMS genes was undertaken. The three chimeric open reading frames (ORFs) identified in this study, in addition to the previously identified ORFs upstream of the nad6 gene, are the most likely CMS candidate genes in this line.