Uncovering a sugar tolerance network : SIK3 and Cabut as downstream effectors of Mondo-Mlx

Simple carbohydrates constitute a big part of our everyday diet, as significant consumption increases have occurred in recent decades. This has coincided with a dramatic rise in people suffering from metabolic disorders, such as diabetes and obesity. However, the genetic factors ensuring a healthy response to sugar intake remain poorly understood. To keep blood glucose in a healthy range even upon overnight fasting or following a rich meal, animals need to be able to adapt quickly. In a healthy organism, high sugar intake leads to rapid conversion of excess sugars into stored glycogen and triacylglycerols, while starvation triggers glucose production through gluconeogenesis and glycogen breakdown. To cope with these fluctuating nutritional conditions organisms possess glucose-sensing mechanisms. Such pathways have a key role in monitoring changes in cellular and organismal nutrient status and readjusting animal physiology accordingly to maintain homeostasis. Intracellular sugar metabolites are sensed by the conserved Mondo family transcription factors (TFs)(MondoA and MondoB/ChREBP in mammals, Mondo in Drosophila), which act together with TF Mlx. Together, they control the expression of metabolic target genes by binding to the carbohydrate response elements present in their promoters. The known Mondo-Mlx targets include genes involved in carbohydrate metabolism and biosynthesis of fatty acids. Yet, the physiological output of these transcription factors has remained largely elusive. As these TFs are very well conserved in Drosophila, this model organism has been used in this thesis to understand the physiological role of Mondo-Mlx and their target genes in vivo. We show that Mondo-Mlx interact in the fly and are crucial for dietary sugar tolerance. Loss of either transcription factor leads to impaired growth and lethality upon high sugar diet. We further characterize the mlx mutant phenotype to reveal high circulating glucose, and increased glycogen levels. We uncover transcriptional repressor Cabut as a direct target of Mondo-Mlx. The Cabut promoter is directly bound by Mondo-Mlx in a sugar-dependent manner. Loss of Cabut similarly results in dietary sugar intolerance, pointing to a crucial function in carbohydrate metabolism. Mondo-Mlx through Cabut are essential for repressing the expression of pepck – the rate limiting gene in gluconeogenesis and glyceroneogenesis. A failure to regulate this step results in pepck over-expression, leading to several fold higher levels of circulating glycerol, and pupal lethality of mlx mutants. Moreover, we reveal that Cabut interconnects nutrient sensing with the circadian clock, and contributes to circadian gene expression oscillation. We also identify Salt inducible kinase 3 (SIK3) as a direct transcriptional target of Mondo-Mlx required for dietary sugar tolerance. Moreover, we show that the activity of the rate-limiting enzyme in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), is increased following sugar feeding. The sugar-augmented increase in G6PD activity is achieved through SIK3-dependent activating G6PD phosphorylation. Collectively, Mondo-Mlx-mediated transcriptional upregulation, as well as SIK3-dependent phosphorylation promote G6PD enzyme activity in response to high high sugar diet. The increased G6PD activity is required for elevating NADPH/NADP+ ratio in order to reduce glutathione in high sugar conditions. We determine that upregulating G6PD activity through Mondo-Mlx and SIK3 is essential for redox balance maintenance, and dietary sugar tolerance. In sum, thesis demonstrates that maintaining redox balance, directing the flow of carbon backbones, and regulating the expression of metabolic circadian genes is essential for dietary sugar tolerance. We highlight the central role of Mondo-Mlx in orchestrating the necessary transcriptional response required for safe glucose utilization, and ultimately, for maintaining metabolic homeostasis.Yksinkertaiset hiilihydraatit, sokerit, ovat merkittävä osa päivittäistä ravintoamme ja sokerien kulutus on lisääntynyt huomattavasti viime vuosikymmeninä. Samanaikaisesti metabolisten sairauksien, kuten diabeteksen ja lihavuuden, esiintyvyys on lisääntynyt voimakkaasti. Ymmärrämme kuitenkin edelleen huonosti, miten yksilön geenit vaikuttavat ruuan sokerien aiheuttamiin terveysriskeihin. Eläimet pystyvät nopeasti sopeuttamaan elimistönsä aineenvaihdunnan muuttuviin ravitsemusolosuhteisiin. Näinollen esimerkiksi veren glukoosi pysyy tasapainossa aterian jälkeen. Tämä on mahdollista, koska elimistössä on ns. glukoosinaistintamekanismeja, jotka toimivat hormonaalisesti (esim. insuliini ja glukagoni) tai paikallisesti solujen sisällä. Nämä mekanismit reagoivat elimistön muuttuviin glukoosipitoisuuksiin ja muuttavat eläimen fysiologiaa niin, että aineenvaihdunnan tasapaino palautuu. Solun sisäisen glukoosinaistinnan kulmakiviä ovat Mondo-transkriptiotekijät ChREBP (MondoB) ja MondoA, jotka toimivat yhdessä Mlx transkriptiotekijän kanssa. ChREBP/MondoA-Mlx-kompleksi aktivoituu solunsisäisen glukoosin lisääntyessä ja se säätelee monia aineenvaihduntaan vaikuttavia geenejä. Monet aineenvaihdunnan säätelyyn vaikuttavat mekanismit ovat evoluutiossa hyvin säilyneitä, joten ne ovat samankaltaisia ihmisellä ja yksinkertaisemmilla eläimillä, kuten banaanikärpäsellä (Drosophila melanogaster). Tämä mahdollistaa aineenvaihdunnan säätelyn perusmekanismien tutkimisen käyttäen Drosophilaa tutkimusmallina. Myös Drosophilalla on solunsisäinen glukoosinaistintajärjestelmä, joka on hieman yksinkertaisempi kuin ihmisellä: Drosophilalla on yksi Mondo-transkriptiotekijä ja yksi Mlx. Niiden toimintaa ei ollut juurikaan aiemmin tutkittu. Tässä väitöstyössä osoitimme, että Drosophilan Mondo ja Mlx muodostavat yhdessä samanlaisen kompleksin kuin ihmisellä. Lisäksi havaitsimme, että toiminnallinen Mondo-Mlx kompleksi on välttämätön edellytys Drosophilan kyvylle säilyä hengissä sokeripitoisella ravinnolla. Lisäksi löysimme useita Mondo-Mlx:n säätelemiä tekijöitä, jotka osallistuvat solunsisäiseen glukoosinaistintaan. Yksi näistä on transkriptiotekijä Cabut, joka vaikuttaa solunsisäiseen glukoosiaineenvaihduntaan inhiboimalla gluko- ja glyseroneogeneesiä. Lisäksi Cabut toimii yhdistävänä linkkinä glukoosiaineenvaihdunnan ja vuorokausirytmin säätelyn välillä. Toinen löytämämme Mondo-Mlx:n kohdegeeni on SIK3, joka on aineenvaihduntaa säätelevä kinaasi. Me havaitsimme, että SIK3 säätelee solujen hapetus-pelkistys-tasapainoa, mikä puolestaan on keskeinen säätelykohde eläinten sokerinsietokyvyn kannalta. Yhteenvetona, tässä työssä on löydetty uusia säätelyverkostoja, jotka mahdollistavat eläimen fysiologisen adaptoitumisen sokeripitoiseen ravintoon. Koska väitöstyössä tutkitut säätelijät ovat evoluutiossa hyvin säilyneitä, saavuttamamme tieto saattaa olla tärkeää myös ihmisen aineenvaihduntasairauksien näkökulmasta

Mondo/ChREBP-Mlx-Regulated Transcriptional Network Is Essential for Dietary Sugar Tolerance in Drosophila

Sugars are important nutrients for many animals, but are also proposed to contribute to overnutrition-derived metabolic diseases in humans. Understanding the genetic factors governing dietary sugar tolerance therefore has profound biological and medical significance. Paralogous Mondo transcription factors ChREBP and MondoA, with their common binding partner Mlx, are key sensors of intracellular glucose flux in mammals. Here we report analysis of the in vivo function of Drosophila melanogaster Mlx and its binding partner Mondo (ChREBP) in respect to tolerance to dietary sugars. Larvae lacking mlx or having reduced mondo expression show strikingly reduced survival on a diet with moderate or high levels of sucrose, glucose, and fructose. mlx null mutants display widespread changes in lipid and phospholipid profiles, signs of amino acid catabolism, as well as strongly elevated circulating glucose levels. Systematic loss-of-function analysis of Mlx target genes reveals that circulating glucose levels and dietary sugar tolerance can be genetically uncoupled: Krüppel-like transcription factor Cabut and carbonyl detoxifying enzyme Aldehyde dehydrogenase type III are essential for dietary sugar tolerance, but display no influence on circulating glucose levels. On the other hand, Phosphofructokinase 2, a regulator of the glycolysis pathway, is needed for both dietary sugar tolerance and maintenance of circulating glucose homeostasis. Furthermore, we show evidence that fatty acid synthesis, which is a highly conserved Mondo-Mlx-regulated process, does not promote dietary sugar tolerance. In contrast, survival of larvae with reduced fatty acid synthase expression is sugar-dependent. Our data demonstrate that the transcriptional network regulated by Mondo-Mlx is a critical determinant of the healthful dietary spectrum allowing Drosophila to exploit sugar-rich nutrient sources.Peer reviewe

Mondo-Mlx Mediates Organismal Sugar Sensing through the Gli-Similar Transcription Factor Sugarbabe

The ChREBP/Mondo-Mlx transcription factors are activated by sugars and are essential for sugar tolerance. They promote the conversion of sugars to lipids, but beyond this, their physiological roles are insufficiently understood. Here, we demonstrate that in an organism-wide setting in Drosophila, Mondo-Mlx controls the majority of sugar-regulated genes involved in nutrient digestion and transport as well as carbohydrate, amino acid, and lipid metabolism. Furthermore, human orthologs of the Mondo-Mlx targets display enrichment among gene variants associated with high circulating triglycerides. In addition to direct regulation of metabolic genes, Mondo-Mlx maintains metabolic homeostasis through downstream effectors, including the Activin ligand Dawdle and the Gli-similar transcription factor Sugarbabe. Sugarbabe controls a subset of Mondo-Mlx-dependent processes, including de novo lipogenesis and fatty acid desaturation. In sum, Mondo-Mlx is a master regulator of other sugar-responsive pathways essential for adaptation to a high-sugar diet.Peer reviewe

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.Peer reviewe

Hierarchical Nanostructuring of Porous Silicon with Electrochemical and Regenerative Electroless Etching

Hierarchically nanostructured silicon was produced by regenerative electroless etching (ReEtching) of Si powder made from pulverized anodized porous silicon. This material is characterized by ∼15 nm mesopores, into the walls of which tortuous 2–4 nm pores have been introduced. The walls are sufficiently narrow that they support quantum-confined crystallites that are photoluminescent. With suitable parameters, the ReEtching process also provides control over the emission color of the photoluminescence. Ball milling and hydrosilylation of this powder with undecylenic acid produces nanoparticles with hydrodynamic diameter of ∼220 nm that exhibit robust and bright luminescence that can be excited with either one ultraviolet/visible photon or two near-infrared photons. The long-lived, robust visible photoluminescence of these chemically passivated porous silicon nanoparticles is well-suited for bioimaging and theranostic applications