Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

Natural variants suppress mutations in hundreds of essential genes.

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, and lethal in another. The extent to which mutant phenotypes are malleable, the architecture of modifiers and the identities of causal genes remain largely unknown. Here, we measure the fitness effects of ~ 1,100 temperature-sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds and map the modifiers for 19 combinations. Altogether, fitness defects for 149 of the 580 tested genes (26%) could be suppressed by genetic variation in at least one yeast strain. Suppression was generally driven by gain-of-function of a single, strong modifier gene, and involved both genes encoding complex or pathway partners suppressing specific temperature-sensitive alleles, as well as general modifiers altering the effect of many alleles. The emerging frequency of suppression and range of possible mechanisms suggest that a substantial fraction of monogenic diseases could be managed by modulating other gene products.

SUR1, newly expressed in astrocytes, mediates neuropathic pain in a mouse model of peripheral nerve injury.

BACKGROUND: Neuropathic pain following peripheral nerve injury (PNI) is linked to neuroinflammation in the spinal cord marked by astrocyte activation and upregulation of interleukin 6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 1 (CXCL1), with inhibition of each individually being beneficial in pain models. METHODS: Wild type (WT) mice and mice with global or pGfap-cre- or pGFAP-cre/ERT2-driven Abcc8/SUR1 deletion or global Trpm4 deletion underwent unilateral sciatic nerve cuffing. WT mice received prophylactic (starting on post-operative day [pod]-0) or therapeutic (starting on pod-21) administration of the SUR1 antagonist, glibenclamide (10 µg IP) daily. We measured mechanical and thermal sensitivity using von Frey filaments and an automated Hargreaves method. Spinal cord tissues were evaluated for SUR1-TRPM4, IL-6, CCL2 and CXCL1. RESULTS: Sciatic nerve cuffing in WT mice resulted in pain behaviors (mechanical allodynia, thermal hyperalgesia) and newly upregulated SUR1-TRPM4 in dorsal horn astrocytes. Global and pGfap-cre-driven Abcc8 deletion and global Trpm4 deletion prevented development of pain behaviors. In mice with Abcc8 deletion regulated by pGFAP-cre/ERT2, after pain behaviors were established, delayed silencing of Abcc8 by tamoxifen resulted in gradual improvement over the next 14 days. After PNI, leakage of the blood-spinal barrier allowed entry of glibenclamide into the affected dorsal horn. Daily repeated administration of glibenclamide, both prophylactically and after allodynia was established, prevented or reduced allodynia. The salutary effects of glibenclamide on pain behaviors correlated with reduced expression of IL-6, CCL2 and CXCL1 by dorsal horn astrocytes. CONCLUSION: SUR1-TRPM4 may represent a novel non-addicting target for neuropathic pain.

ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels.

Neuroendocrine-type ATP-sensitive K+ (KATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)4, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K+-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower KD for [3H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.

Salutary effects of glibenclamide during the chronic phase of murine experimental autoimmune encephalomyelitis.

BACKGROUND: In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial. METHODS: EAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35-55. Glibenclamide (10 µg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET. RESULTS: Administering glibenclamide beginning 24 days after MOG35-55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2. CONCLUSIONS: SUR1-TRPM4 may be a druggable target for disease modification in MS.

Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae.

BACKGROUND: Transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding RNAs (lncRNAs). However, only a few lncRNAs have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. Even less lncRNAs have been annotated as essential hence implying that the majority may be functionally redundant. Therefore, the function of lncRNAs could be illuminated through systematic analysis of their synthetic genetic interactions (GIs). RESULTS: Here, we employ synthetic genetic array (SGA) in Saccharomyces cerevisiae to identify GIs between long intergenic non-coding RNAs (lincRNAs) and protein-coding genes. We first validate this approach by demonstrating that the telomerase RNA TLC1 displays a GI network that corresponds to its well-described function in telomere length maintenance. We subsequently performed SGA screens on a set of uncharacterised lincRNAs and uncover their connection to diverse cellular processes. One of these lincRNAs, SUT457, exhibits a GI profile associating it to telomere organisation and we consistently demonstrate that SUT457 is required for telomeric overhang homeostasis through an Exo1-dependent pathway. Furthermore, the GI profile of SUT457 is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. Accordingly, we show that ectopic expression of this lincRNA suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for SUT457 in telomere biology. CONCLUSIONS: Overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncRNAs in yeast and other complex organisms.

The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia.

BACKGROUND: Harmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca(2+)-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca(2+) influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2. METHODS: We studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8-/- and Trpm4-/- mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation. RESULTS: In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca(2+)]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca(2+)]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca(2+)/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation. CONCLUSIONS: Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca(2+)-sensitive gene expression, including Nos2.

Silencing of Abcc8 or inhibition of newly upregulated Sur1-Trpm4 reduce inflammation and disease progression in experimental autoimmune encephalomyelitis.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression. METHODS: EAE was induced in wild-type (WT) and Abcc8-/- mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage. RESULTS: Sur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8-/-/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes. CONCLUSIONS: Sur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.

Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

Reinterpreting the action of ATP analogs on K(ATP) channels.

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting ß-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(ß,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(ß,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2.

K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic ß-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, ß-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.

Quantitative analysis of fitness and genetic interactions in yeast on a genome scale.

Global quantitative analysis of genetic interactions is a powerful approach for deciphering the roles of genes and mapping functional relationships among pathways. Using colony size as a proxy for fitness, we developed a method for measuring fitness-based genetic interactions from high-density arrays of yeast double mutants generated by synthetic genetic array (SGA) analysis. We identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate single- and double-mutant fitness measurements, which rival the accuracy of other high-resolution studies. We applied the SGA score to examine the relationship between physical and genetic interaction networks, and we found that positive genetic interactions connect across functionally distinct protein complexes revealing a network of genetic suppression among loss-of-function alleles.

Comparative evaluation of no dye assistance, indocyanine green and triamcinolone acetonide for internal limiting membrane peeling during macular hole surgery.

PURPOSE: To evaluate the anatomical closure rate and visual outcome in patients undergoing pars plana vitrectomy with internal limiting membrane peeling without dye enhancement and with indocyanine green or triamcinolone acetonide assistance. METHODS: This is a retrospective, comparative interventional case series. The electronic charts of 435 eyes of 415 patients with idiopathic macular holes who underwent macula hole surgery were reviewed from January 2003 to April 2010. The patients were assigned to 3 groups: no dye assistance (Group 1), indocyanine green-assisted (Group 2), and triamcinolone acetonide-assisted internal limiting membrane peel (Group 3). The data were recorded for 3-, 6-, and 12-month follow-up visits. Main outcome measures were postoperative visual acuity and macular hole closure. RESULTS: The overall macular hole closure rate with a single surgery was 94.7% (108 of 114). In Group 1: 91.4% (191 of 209), in Group 2: 94.1% (112 of 119), and in Group 3: 95.9% (93 of 97) achieved closure. Closure rate for holes that were ≤400 µm in diameter was 98.8% compared with >400 µm, which was 90.4% (P = 0.001). There was a statistically significant (P < 0.001) improvement from preoperative visual acuity to postoperative visual acuity in all groups across all time points. There was no statistically significant visual acuity difference among three groups. At 12-month follow-up, 77.7% in Group 1, 66.1% in Group 2, and 81.3% in Group 3 achieved 20/50 visual acuity or better. CONCLUSION: The study shows that good anatomical and functional results can be achieved with no dye and with indocyanine green or triamcinolone acetonide dye assistance for internal limiting membrane peeling during macular hole surgery. Overall, visual acuity levels did not differ among groups, although subanalysis of the results suggests subtle indocyanine green toxicity.

Cation flux through SUR1-TRPM4 and NCX1 in astrocyte endfeet induces water influx through AQP4 and brain swelling after ischemic stroke.

Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na+/Ca2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na+ through SUR1-TRPM4 induced Ca2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca2+. This increase in Ca2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.

Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8.

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

Neonatal diabetes mellitus.

An explosion of work over the last decade has produced insight into the multiple hereditary causes of a nonimmunological form of diabetes diagnosed most frequently within the first 6 months of life. These studies are providing increased understanding of genes involved in the entire chain of steps that control glucose homeostasis. Neonatal diabetes is now understood to arise from mutations in genes that play critical roles in the development of the pancreas, of beta-cell apoptosis and insulin processing, as well as the regulation of insulin release. For the basic researcher, this work is providing novel tools to explore fundamental molecular and cellular processes. For the clinician, these studies underscore the need to identify the genetic cause underlying each case. It is increasingly clear that the prognosis, therapeutic approach, and genetic counseling a physician provides must be tailored to a specific gene in order to provide the best medical care.

Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis.

Increased glucose production is a hallmark of type 2 diabetes and alterations in lipid metabolism have a causative role in its pathophysiology. Here we postulate that physiological increments in plasma fatty acids can be sensed within the hypothalamus and that this sensing is required to balance their direct stimulatory action on hepatic gluconeogenesis. In the presence of physiologically-relevant increases in the levels of plasma fatty acids, negating their central action on hepatic glucose fluxes through (i) inhibition of the hypothalamic esterification of fatty acids, (ii) genetic deletion (Sur1-deficient mice) of hypothalamic K(ATP) channels or pharmacological blockade (K(ATP) blocker) of their activation by fatty acids, or (iii) surgical resection of the hepatic branch of the vagus nerve led to a marked increase in liver glucose production. These findings indicate that a physiological elevation in circulating lipids can be sensed within the hypothalamus and that a defect in hypothalamic lipid sensing disrupts glucose homeostasis.

Clinical and image-guided chorioretinal findings in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency.

The purpose of this study is to report clinical, optical coherence tomography (OCT), and fluorescein angiogram/indocyanine green angiography (FA/ICG) findings in patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) enzyme deficiency in two siblings. A 13-year-old girl and her 14-year-old brother presented with progressive decrease in central vision. Clinically, there were blond-looking fundi, diffuse retinal pigment epithelial (RPE) disruption/atrophy in the macula and peripheral retina with choriocapillaris atrophy in both of them. OCT showed RPE irregularity and diffuse disruption of the RPE layer. FA/ICG imaging demonstrated transmitted choroidal fluorescence secondary to diffuse RPE atrophy with no evidence of leakage. Electroretinogram and electrooculogram findings were suggestive of primary abnormality of pigment epithelium. The boy died of cardiac/respiratory illness, whereas his sister is alive at the last follow-up. Abnormal chorioretinal findings in LCHAD patients should be carefully followed. Regular follow-up is recommended to monitor the ocular and systemic status.

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG.

Transfer RNA variants increase the frequency of mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, to frequencies approaching 10% in yeast and bacteria. Cells cope with these variants by having multiple copies of each tRNA isodecoder and through pathways that deal with proteotoxic stress. In this study, we define the genetic interactions of the gene encoding tRNASerUGG,G26A, which mistranslates serine at proline codons. Using a collection of yeast temperature-sensitive alleles, we identify negative synthetic genetic interactions between the mistranslating tRNA and 109 alleles representing 91 genes, with nearly half of the genes having roles in RNA processing or protein folding and turnover. By regulating tRNA expression, we then compare the strength of the negative genetic interaction for a subset of identified alleles under differing amounts of mistranslation. The frequency of mistranslation correlated with the impact on cell growth for all strains analyzed; however, there were notable differences in the extent of the synthetic interaction at different frequencies of mistranslation depending on the genetic background. For many of the strains, the extent of the negative interaction with tRNASerUGG,G26A was proportional to the frequency of mistranslation or only observed at intermediate or high frequencies. For others, the synthetic interaction was approximately equivalent at all frequencies of mistranslation. As humans contain similar mistranslating tRNAs, these results are important when analyzing the impact of tRNA variants on disease, where both the individual's genetic background and the expression of the mistranslating tRNA variant need to be considered.

Hypothalamic K(ATP) channels control hepatic glucose production.

Obesity is the driving force behind the worldwide increase in the prevalence of type 2 diabetes mellitus. Hyperglycaemia is a hallmark of diabetes and is largely due to increased hepatic gluconeogenesis. The medial hypothalamus is a major integrator of nutritional and hormonal signals, which play pivotal roles not only in the regulation of energy balance but also in the modulation of liver glucose output. Bidirectional changes in hypothalamic insulin signalling therefore result in parallel changes in both energy balance and glucose metabolism. Here we show that activation of ATP-sensitive potassium (K(ATP)) channels in the mediobasal hypothalamus is sufficient to lower blood glucose levels through inhibition of hepatic gluconeogenesis. Finally, the infusion of a K(ATP) blocker within the mediobasal hypothalamus, or the surgical resection of the hepatic branch of the vagus nerve, negates the effects of central insulin and halves the effects of systemic insulin on hepatic glucose production. Consistent with these results, mice lacking the SUR1 subunit of the K(ATP) channel are resistant to the inhibitory action of insulin on gluconeogenesis. These findings suggest that activation of hypothalamic K(ATP) channels normally restrains hepatic gluconeogenesis, and that any alteration within this central nervous system/liver circuit can contribute to diabetic hyperglycaemia.