b) Describe mechanism how AMPK activity is regulated inside the cell.

Transkript

1 Eksamen ERN4210, 2013 Sensorveiledning Oppgave 1 a) Forklar hvordan insulin og IGF-1 aktiverer de to signalveiene som fører til aktivering av PKB/Akt og MAP-kinase/Erk. Forklar videre signalering nedstrøms for PKB og Erk, og forklar de fysiologiske prosesser som blir regulert. - b) Describe mechanism how AMPK activity is regulated inside the cell. - AMPK activity is regulated by the ATP/AMP ratio (10/1) and by phosphorylation on Thr172 - During activation two AMP bind the AMPK γ-subunits and activates the enzyme in an allosteric fashion. - Binding of the second AMP molecule provokes a conformational shift and sensitize AMPK for upstream kinases such as LKB1 and Calcium calmoduline kinases. Phosphorylation by any of these kinases increases AMP-dependent AMPK activity by folds.

2 c) Explain why and how both fat and glucose is taken up by the muscle during physical activity. - Physical activity alters the ATP/AMP ratio inside the cell to lower than 10. More energy substrates are required. AMPK is activated by the reduced ATP/AMP ratio and Thr172 phosphorylation. This leads to 1) increased mobilization of GLUT4 to the muscle membrane by AMPK. No storage of glucose only consumption is secured by inhibition of glycogen synthase and increased glycolytic activity by AMPK activity. 2) Moreover, increased uptake of fat is associated with increased mobilization of FAT/CD36 and inhibition of AcetylcoA carboxylase (ACC) by AMPK followed by reduction of MalonylCoA levels. Reduced MalonylCo, an inhibitor of free fatty acid uptake by the mitochondria, will lead to increased uptake of free fatty acids by the mitochondria and up regulated β-oxidation. As AMPK increases the activity of the transcription factor PGC-1alfa, this will lead to increased expression of genes associated with increased mitochondrial activity, such as genes responsible for β-ox and ox-phos. All together this will favor increased energy production in the form of ATP both from glucose and fat restoring an optimal ATP/ADM ratio during physical activity. Oppgave 2 Hunger and satiety are closely regulated events resulting from communication between the endocrine and nervous system. a) There are two main centers in the brain that receives endocrine and nervous signals for hunger and satiety. What centers are these? - The dorsal vagus complex, nucleus tractus olitarius, in the Pons/Medulla oblongata and hypothalamus. b) When the ventricle is filled with food several hormones, called incretines are released locally from the ventricle and intestines. Mention at least two incretines and describe how they act. - GLP-1(glukoagon-like peptide 1), GIP (glucos-dependent insulinotropic polypeptid), OXM (oxyntomodulin) and PYY (peptide YY). - - By incretines we mean local hormones released to increase the secretion of hormones from e.g. the pancreas.

3 c)when the ventricle is emptied for food you will get a feeling of hunger, in part due to production of the hormone Ghrelin. Describe how Ghrelin stimulates the feeling of hunger in the hypothalamus. Ghrelin regulerer sult via METTHET SULT PKA ARC Appetittstimulerende MCR = melanocortin reseptor AgRP = Agoutibeslektet protein POMC = pro-opiomelanocortin CART = cocaine-amphetamine-regulated transcript NPY = neuropeptid Y Oppgave 3 a) Describe the molecular structure of lipid droplets (LDs), including the classes of molecules and mention a few examples of proteins often found on LDs. -Hydrophobic lipids in the core (TG, CE, retinoic esters, and more), surrounded by a single layer of phospholipids (phosphatidylserine, phosphatidylcholine, spingolmyelin). Attached to the surface you will find a number of different proteins. Examples of proteins: perilipins, lipases, CE- and TG- metabolism enzymes. b) Where in the cell is the synthesis and growth of lipid droplets believed to occur? -Synthesis is believed to occur in the ER. A large number of enzymes important for synthesis of the neutral core lipids (CE or TAG) locate to the ER membrane. Growth of LDs may also occur at the LD surface, with synthesis of phospholipids (PLs) and core lipids directly on the droplet. LDs might also enlarge by fusion of existing LDs (SNAP/SNARE mediated fusion).

4 c) Describe the molecular regulation of adipose lipolysis. - I basal tilstand finnes perilipin1 i ikke-fosforylert tilstand på LD-overflaten. Den C- terminale delen av Perilipin1 binder til seg cgi-58/abhd5. Det er trolig slik at denne interaksjonen hemmer aktiviteten til cgi-58. Perilipin beskytter innholdet i kjernen fra lipolyse. Ved initiering av lipolyse aktiveres PKA grunnet økt camp nivå. PKA fosforylerer hormone sensitive lipase (HSL) og perilipin1 (perilipin1 har potensielt 6 fosforyleringsseter). CGI-58 frigjøres fra perilipin 1 og binder til adipose triglyderide lipase (ATGL). CGI-58 fungerer som en koaktivator av ATGL som katalyserer nedbrytning av TG til DG. Fosforylering i den N-terminale delen av perilipin1 rekrutterer HSL som katalyserer nedbrytning av DG til MG. Disse molekylære forandringene gir >100-gangers forskjell i lipolytisk hastighet mellom basal og stimulert lipolyse. Følgende kan tenkes nevnt av A-kandidater : Fosforylering av sete 6 (Ser 517, C-terminalen) er nødvendig for at cgi58 skal frigjøres fra perilipin1. Fosforylering av sete 5 (Ser492) er nødvendig for fragmentering av LDer. Gir mer LD overflate, noe som er antatt å øke lipolytisk hastighet. Forsforylering i N-terminalen er viktig (men ikke essensielt) for rekrutering og binding av HSL til perilipin1.

5 Oppgave 4 The hexosamine biosynthetic pathway (HBP) is a nutrient responsive pathway in cells. Make a schematic drawing of this pathway including the enzymes and nutrients involved. A) What is the final product in this pathway and what is it used for? GFAT: L-glutamine:D-fructose-6-phosphate amidotransferase Emeg32: glucosamine-6-phosphate (GlcN6P) acetyltransferase O-GlcNAc signaling: OGT: O-linked GlcNAc transferase (GlcNAc: N-acetylglucosamine), OGA: O-GlcNAcase Synthesis of glycoproteins, proteoglycans, glycolipids: Transferases in ER-Golgi -Final product hexosamin biosynthetic pathway: UDP-GlcNAc is a nucleotide sugar and a coenzyme in metabolism. It is used by glycosyltransferases to transfer N-acetylglucosamine (GlcNAc) residues N-linked on proteins and lipids and extensively involved in intracellular signaling as a substrate for O-linked N-actetylglucosamine transferases (OGTs) in a wide range of species. b)hva er protein O-GlcNAcylering? -O-linked GlcNAcylation is a dynamic, transient posttranslational modification that occurs on many different proteins in the cytoplasm and in the nucleus affecting protein stability, protein-protein interactions, enzyme activity, transcription, translation, cell signalling, apoptosis, cell shape. OGT is the enzyme that confers O-GlcNAc modification of nucleocytoplasmic proteins and this enzyme is a unique transferase that transferes one single

6 GlcNAc molecule on the amino acids serine and threonine O-linked (linked to OH-group on R side chain); O-linked GlcNAc. This is more similar to reversible phosphorylation on ser /thr than classical glycosylation in ER/Golgi. Here, there are other ER-loclised transferases that confer O-linked GalNac and N-linked GlcNAc (NB! Not O-linked GlcNAc!) to proteins in ER/Golgi and more than one sugar molecule (from glycoproteins/glycolipids with a few chains of sugar-molecules to large proteoglycans with long GAG chains (glycosaminoglycans, unbranched polysaccarides-may be further sulphated eg heparin/heparan sulphate). Classical glycosylation in the secretory pathway is not dynamic and reversible as O-GlcNAc in cytosol/nucleus (and mithocondria). Thus, the proteins are constituvely modified and the proteins are either secreted or localised to membranes (ER, Golgi, plasmamembrane) c)biologiske konsekvenser av O-GlcNAc på proteiner: - O-GlcNacylation/cycling on proteins is an essential mechanism in cells important for proper stress responses and cell cycle control. Pathologies associated with dysregulated protein O- GlcNacylation: insulin resistence/diabetes, neurodegenerative disease (eg Alzheimer s disease), cardiomyopathy, cancer, endothelial dysfunction; retinopathy, neuropathy. d) Brusk inneholder en rekke makromolekyler, som bl.a. hyaluronsyre og proteoglykanet aggrekan. Disse har viktige funksjoner for støtdemping ved belastning av knærne. Dårlig leddfunksjon sees ved bla. revmatisk sykdom. Gi din vurdering av bruk av glukosamin eller kombinasjonen glukosamin/kondroitin som behandling for denne type lidelser. Sensorveiledning Svein Olav Kolset: Glukosamin er byggestein i hyaluronsyre. Kondroitin sulfat inneholder galaktosamin. Tilført glukosamin (oftest i form av glukosamin sulfat) eller glukosamin/kondroitin må omdannes til UDP-glukosamin og UDP-galaktosamin. Syntese av hyaluronsyre er avhenging av tilførsel av UDP-glukosamin og UDP-glukuronsyre. Syntese av kondroitin sulfat er avhenging av dannelse av UDP-galaktosamin UDP-glukuronsyre. Tilførte tilskudd må diffundere fra blodbanen til kondrocyttene, som ansees som lite sannsynlig og er ikke dokumentert. Mulige positive effekter kan være sekundære via vekstfaktorer som sitter bundet i preparatene. Mulige effekter hittil mye basert på metoder som er diskutable og mange er subjektive. Behov for mer objektive parametre. Sensorveiledning Line Grønning-Wang: Glucosamin kan også aktivere hexosaminveien nedstrøms for det hastighetsbegrensende trinnet GFAT som illustrert over. På denne måten dannes UDP-GlcNAc som er substrat for proteoglykansyntese, men også for O-GlcNAc signalering via OGT. Vi vet at økt UDP-GlcNAc fører til økt O-GlcNAcylering på proteiner og dette er vist å være involvert i insuinresistens og diabetiske komplikasjoner. Mao, kan det være betenkelig å gi glukosamin til diabetes pasienter (og individer med glukose intoleranse). Det kan også vurderes om ikke-diabetikere bør unngå glukosamin supplement ved at de teoretisk sett kan utvikle insulin-resistens. ( Begge momenter over kan diskuteres. Svein Olav har snakket om glukosamin supplement i sine forelesninger, jeg har ikke snakket spesielt om det, men mener en god kandidat kan ressonere seg

7 frem til at glukosamin aktiverer O-GlcNAc signalering og via dette muligens insulin resistens (at glukosamin kan aktivere HBP, er forelest). Oppgave 5 a) What is a nuclear receptor (NR) and what influences the activity of a NR? Ligand (eg fatty acids, oxysterols) and/or post translational modifications (eg acetylation, phosphorylation, O-GlcNAcylation) downstream of nutrient/stress/hormonal/cytokine induced signaling pathways mediating association with coactivator or corepressor proteins. Nuclear receptor gene expression up-regulated during adipocyte differentiation (PPARgamma, LXRalpha), specific expression in different tissues (eg LXRa: adipose tissue, intestine, kidney, liver). b) What are the known natural ligands for LXRs, PPARs and FXR? -LXRs: Oxysterols (oxygenated derivatives of cholesterol)- PPARs: fatty acids (and eicosanoids. PPARγ is activated by the prostaglandin PGJ 2. PPARα is activated by leukotriene B 4 ). FXR: bile acids (including Chenodeoxycholic acid)

8 c) Briefly describe the pathway of hepatic de novo lipogenesis and the role of LXR in this pathway -Absence of LXRs suppresses DNL in the liver (but stimulates DNL in adipose tissue, demonstrating opposite roles of LXR in DNL regulation in these two tissues). LMG-W har også forelest om hvordan glukose metabolisme/signalering via OGT/O-GlcNAc (ikke bare oxysteroler og insulin-mulig synergi-effekter) kan regulere LXR aktivitet og nedstrøms opp-regulering av SREBP1c, ChREBP og lipogene målgeners mrna.