Initial B Cell Activation Induces Metabolic Reprogramming and Mitochondrial Remodeling.

B lymphocytes provide adaptive immunity by generating antigen-specific antibodies and supporting the activation of T cells. Little is known about how global metabolism supports naive B cell activation to enable an effective immune response. By coupling RNA sequencing (RNA-seq) data with glucose isotopomer tracing, we show that stimulated B cells increase programs for oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and nucleotide biosynthesis, but not glycolysis. Isotopomer tracing uncovered increases in TCA cycle intermediates with almost no contribution from glucose. Instead, glucose mainly supported the biosynthesis of ribonucleotides. Glucose restriction did not affect B cell functions, yet the inhibition of OXPHOS or glutamine restriction markedly impaired B cell growth and differentiation. Increased OXPHOS prompted studies of mitochondrial dynamics, which revealed extensive mitochondria remodeling during activation. Our results show how B cell metabolism adapts with stimulation and reveals unexpected details for carbon utilization and mitochondrial dynamics at the start of a humoral immune response

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

Author Posting. © American Society for Biochemistry and Molecular Biology, 2004. This article is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 279 (2004): 42954-42963, doi:10.1074/jbc.M405040200.ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36–70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1–35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable {alpha}-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106–715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.This work was supported by National Institutes of Health Grants R01HL071629, P41RR001395, and P01HL032262

Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Author Correction: LKB1 loss links serine metabolism to DNA methylation and tumorigenesis

Erratum for: LKB1 loss links serine metabolism to DNA methylation and tumorigenesis. [Nature. 2016

A Phylogenomic Assessment of Processes Underpinning Convergent Evolution in Open-Habitat Chats.

Insights into the processes underpinning convergent evolution advance our understanding of the contributions of ancestral, introgressed, and novel genetic variation to phenotypic evolution. Phylogenomic analyses characterizing genome-wide gene tree heterogeneity can provide first clues about the extent of ILS and of introgression and thereby into the potential of these processes or (in their absence) the need to invoke novel mutations to underpin convergent evolution. Here, we were interested in understanding the processes involved in convergent evolution in open-habitat chats (wheatears of the genus Oenanthe and their relatives). To this end, based on whole-genome resequencing data from 50 taxa of 44 species, we established the species tree, characterized gene tree heterogeneity, and investigated the footprints of ILS and introgression within the latter. The species tree corroborates the pattern of abundant convergent evolution, especially in wheatears. The high levels of gene tree heterogeneity in wheatears are explained by ILS alone only for 30% of internal branches. For multiple branches with high gene tree heterogeneity, D-statistics and phylogenetic networks identified footprints of introgression. Finally, long branches without extensive ILS between clades sporting similar phenotypes provide suggestive evidence for a role of novel mutations in the evolution of these phenotypes. Together, our results suggest that convergent evolution in open-habitat chats involved diverse processes and highlight that phenotypic diversification is often complex and best depicted as a network of interacting lineages

Carbohydrate absorption by blackcap warblers (Sylvia atricapilla) changes during migratory refuelling stopovers

Passerine birds migrating long distances arrive at stopover sites to refuel having lost as much as 50% of their initial body mass (mb), including significant losses to digestive organs that may serve as a reservoir of protein catabolised for fuel during flight. Birds newly arrived at a stopover show slow or no mb gain during the initial 2–3 days of a stopover, which suggests that energy assimilation may be limited by reduced digestive organs. Measurements of migrants and captive birds subjected to simulated migratory fasts have shown reductions in intestine mass, morphological changes to the mucosal epithelium, and reductions in food intake and assimilation rate upon initial refeeding. We found that blackcaps (Sylvia atricapilla, Linnaeus) newly arrived at a migratory stopover after crossing the Sahara and Sinai deserts had significantly increased paracellular nutrient absorption (non-carrier mediated uptake occurring across tight junctions between enterocytes) that may provide partial compensation for reduced digestive capacity resulting from changes to intestinal tissues. Indeed, newly arrived birds also had a slightly reduced capacity for absorption of a glucose analogue (3-O-methyl-D-glucose) transported simultaneously by both carrier-mediated and non-mediated mechanisms. Increased paracellular absorption coupled with extended digesta retention time may thus allow migratory blackcaps to maintain high digestive efficiency during initial stages of refuelling while digestive organs are rebuilt.C.R. Tracy, T.J. McWhorter, M.S. Wojciechowski, B. Pinshow and W.H. Karaso

Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BADS. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BADS, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BADS. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes

Structures and functions of mitochondrial ABC transporters

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters

Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity

AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy