PEPCK. The Regulation of Eukaryotic Gene Expression. Why choose PEPCK? PEPCK. PEPCK overexpression in muscle. The Supermouse.

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1 PEPK The Regulation of Eukaryotic Gene Expression..using the example of PEPK This is an acronym for an enzyme PhosphoEnol Pyruvate arboxykinase This enzyme is NLY regulated by gene expression! No allosteric activators, covalent modification etc No activation by camp, inhibition by insulin etc PEPK The enzyme is expressed in liver, kidney, adipose tissue and to a lesser extent in muscle It is a key enzyme in gluconeogenesis (the synthesis of new glucose, usually from lactate, pyruvate or alanine) and glyceroneogenesis (the synthesis of glycerol, usually from lactate, pyruvate or alanine) Why choose PEPK? It is an enzyme. Why would this be good? It is not post-translationally regulated. Why would this be good? A number of hormones influence gene expression in different tissues. PEPK overexpression in muscle The youtube video is of a mouse with PEPK overexpressed in muscle only. This mouse hit the popular press in 2007 and put ase Western Reserve University in leveland hio on the map! Earl Sutherland, the discoverer of camp also hailed from ase Western. The Supermouse. Eats 60% more food than wild type mice Weighs 40% less than wild type mice an run for >4 h until exhaustion whereas the control littermates stop after only 10 min as 2 3 fold less adipose tissue 1

2 PEPK overexpression in muscle This mouse was leaner than wild type mice, ran for longer and lived longer! They were also more aggressive. The overexpression had switched the muscle fuel usage to fatty acids with little lactate production. PEPK overexpression in adipose tissue A less famous cousin mouse has the PEPK enzyme overexpressed in adipose tissue. The results couldn t be further from supermouse! PEPK overexpression in fat cells PEPK overexpression in adipose tissue These mice are obese although metabolically healthy (as measured by glucose tolerance and insulin sensitivity) until you put them on a high fat diet. Then you see insulin resistance and diabetes emerging. PEPK overexpression in liver Leads to altered glucose tolerance Insulin resistance, NIDDM Increased gluconeogenesis causes increased hepatic glucose production which is released into the blood stream This caused increased insulin secretion but ultimately insulin resistance. PEPK Knock out in liver Surprisingly these mice can maintain blood glucose under starvation conditions They develop liver steatosis (fatty livers) probably because of impaired oxidation of fatty acids A total PEPK knock out in all tissues is lethal mice die within days of birth. 2

3 Why the dramatically different outcome for the mouse when PEPK is overexpressed in different tissues? It is after all the same enzyme catalysing the same reaction. 2 xaloacetate The reaction! GTP GDP 2 2 P 3 Phosphoenol pyruvate Where does it fit in? Glyceroneogenesis Gluconeogenesis Glucose Glycolysis 2 GDP GTP Phosphoenol pyruvate PEP 2 PEPcarboxykinase 2 2 P 3 NAD Alanine NAD+ PEP AA Pyruvate NAD NAD+ 3 LD 3 2 oxaloacetate AA Pyruvate arboxylase 3 Pyruvate LD 3 Lactate Glyceroneogenesis PEPK gene Fatty acids 3 S-oA PPAR Glucocorticoid camp Triglycerides 2P 3 2 Glycerol 3-P PEP 2P 3 2 Dihydroxyacetone phosphate (DAP) 2P 3 Glyceraldehyde 3-P PPARRE IRE Insulin -400 GRE TRE Thyroid -300 RE -100 Promoter and regulatory region TATA 3

4 PEPK regulation in liver PEPK activity is highest in liver during starvation Glucocorticoids such as cortisol and glucagon both activate the expression of the PEPK gene in liver The glucocorticoids are steroid hormones whereas glucagon is a peptide hormone Activating PEPK activity in liver Let s consider the glucocorticoid first. ortisol is the active glucocorticoid hormone. Pharmaceutical analogues are cortisone (converted to cortisol by a dehydrogenase) and the synthetic analogues prednisone and dexamethasone ften administered for their immunosuppressive properties Activating PEPK activity in liver ortisol is produced and released by the adrenal gland.it travels through the circulation and can pass through the cell plasma membrane (unlike peptide hormones) nce inside the cell it binds to a cytosolic receptor in specific cells Activating PEPK activity in liver The formation of the cortisol:receptor complex exposes a nuclear localisation signal The complex moves to the nucleus It binds as a dimer to the glucocorticoid (a sequence of DNA upstream of a number of genes including PEPK) Activating PEPK activity in liver The binding of this complex greatly enhances the frequency of initiation of the basal transcription apparatus (RNA pol II with all the bits). ther protein factors (coactivators) also bind. These factors reside in the nucleus of liver cells and are known as hepatic nuclear factors (NFs). Activating PEPK activity in liver It is thought that both the cortisol:receptor complex and one or more of the NFs need to be bound for effective enhancement. This is important for the tissue specific nature of the PEPK up-regulation. 4

5 PEPK gene blood cortisol PPAR Glucocorticoid camp ortisol receptor PPARRE IRE GRE TRE RE TATA Insulin -400 Thyroid nucleus NLS NLS NFs RNA pol II TATA NLS ortisol binds to its receptor, exposing the NLS Promoter and regulatory region Differing to glucocorticoids in different tissues While cortisol up regulates PEPK transcription in the liver. It down regulates PEPK in adipose tissue. The same gene (single copy in the genome) with the same promoter and regulatory regions! ow is this possible? PEPK down regulation by cortisol in adipose tissue We are not sure! The accepted logic at present is that for effective up regulation in the liver you need both the cortisol:receptor dimer and some NFs bound. With different adipocyte specific nuclear factors you can get the reverse result. Activating PEPK activity in liver During starvation glucagon is secreted by the alpha cells of the pancreas (it is synthesised there) Glucagon is a peptide hormone which cannot cross the plasma membrane It binds to a cell surface receptor (a G- coupled protein receptor) Activating PEPK activity in liver The binding of glucagon to this receptor causes a conformational change, associations of subunits and ultimately the activation adenylyl cyclase. This causes an increase in camp activates Protein Kinase A moves to the nucleus phosphorylates transcription factors (REBs) 5

6 Activating PEPK activity in liver The phosphorylated REBs then bind to the RE (camp ) site on the DNA effective enhancement of PEPK transcription (amongst other genes you need up regulated in starvation) PPAR PPARRE IRE Insulin Glucocorticoid -400 GRE PEPK gene TRE Thyroid -300 camp RE -100 TATA Promoter and regulatory region Blood glucagon Blood Glucagon binds to receptor Liver Glucagon receptor GDP G protein Adenylyl cyclase Liver GTP GDP Adenylyl cyclase R R R R Protein kinase A Protein kinase A Blood Glucagon binds to receptor Adenylyl cyclase PEPK down regulation by Insulin Liver P REB REB P REB P GTP ATP camp R R R R What we know.. Insulin inhibits the basal PEPK transcription apparatus Insulin antagonizes the induction of PEPK expression by glucagon or glucocorticoids REB 6

7 PEPK down regulation by Insulin It is thought that intermediates in the insulin signalling pathway are involved. In spite of all we know about insulin we still don t know exactly how insulin inhibits the transcription of PEPK. It would be nice to say that an intermediate produced by insulin signalling phosphorylated a transcription factor which binds to the IRE. BUT I AN T Summary: Transcriptional Regulation of PEPK Use the liver in starvation as the context PEPK needs to be up-regulated to make glucose (GLNG) to maintain blood glucose and thus to supply the brain with fuel In adipose tissue it has the role of making glycerol for the packaging of fatty acids to triglycerides Summary: Transcriptional Regulation of PEPK ortisol, a steroid hormone, up-regulates PEPK ortisol can enter the cell (because it is hydrophobic enough) where it binds to a cytosolic receptor NLS unmasked enters nucleus dimerises binds to GRE Summary: Transcriptional Regulation of PEPK Glucagon, a peptide hormone upregulates PEPK Glucagon can t enter the cell binds to G-coupled protein receptor activates adenylyl cyclase camp binds to Protein kinase A R subunits dissociate from subunits subunits enter nucleus phosphorylate REB dimerise and bind to RE Post transcriptional regulation of PEPK Glucocorticoids and camp also stabilise the PEPK mrna in the liver. Insulin destabilises it. mrna stability contributes significantly to the overall up or down regulation of gene expression. PEPK is normally very unstable. mrna stability is measured by its half life. Why would it be advantageous for an mrna sequence like PEPK to be unstable? If PEPK is only regulated by gene expression it is difficult to down regulate the sequence at the level of synthesis if the mrna persists in the. This also applies to the Trp operon enzymes 7

8 5 MeG AAAAAAAAA 3 Translation Processed mature mrna 5 MeG AAAAAAAAA 3 Processing Primary transcript Transcription DNA PEPK mrna stability A sequence at the 3 UTR of PEPK mrna has been identified which destabilises the mrna. If that sequence is inserted into the 3 UTR of other more stable mrnas, such as globin, the half life reduces significantly. We are yet to determine how camp or cortisol stabilises this mrna. PEPK gene expression in adipose tissue Another becomes significant, the PPARRE Peroxisomal Proliferator Activator Receptor (PPAR) Response Element There in fact 4 PPARs; one of the ones of interest to adipocytes is PPARγ, the other is PPAR δ liver has PPARα and PPARγ PPARγ RXR PPARγ RXR PPARγ activates the transcription of genes involved with adipogenesis and fat storage Pharmaceutical applications A new group of insulin sensitizers, the thiazolidinediones (TZDs) act on PPARγ. The most commonly prescribed are Rosiglitozone and Piogliterzone These are artificial ligands for PPARγ. We don t even know the natural ligand for PPARγ although the favoured candidates are fatty acids and their derivatives, in particular polyunsaturated fatty acids. TZDs PPARγ RXR PPARγ RXR TZDs are artificial ligands for PPARγ. These are used as insulin sensitising agents. 8

9 Pharmaceutical applications They work to sensitize the body to insulin in an interesting way. Insulin resistance is thought, in part to be brought on by elevated free fatty acids (FFA) in the serum interfering with insulin signalling. Elevated FFAs are commonly associated with obesity which gives one of the putative links between obesity and insulin resistance. Pharmaceutical applications besity is characterised by lots of large adipocytes which become leaky, hence losing weight is one of the most effective ways of enhancing insulin sensitivity. There are some mice that, although fat are metabolically healthy (remember the PEPK mouse) They have adipocytes that can contain the FFAs Fat mice who are metabolically healthy Pharmaceutical applications: TZDs act to up-regulate PEPK synthesis in adipocytes, thus increase glyceroneogenesis more repackaging of FFAs in the adipocyte less FFAs in serum Stimulate adipogenesis (differentiation of new fat cells from fibroblasts) thus increasing the storage for FFAs and again lowering FFAs in serum. Implications of TZD treatment The patient may actually put on weight as adipogenesis is stimulated BUT the fat cells will be able to contain the FFAs and stop the release into the bloodstream. The increase in PEPK activity will improve the fat storage in the adipocyte. besity: other areas As well as elevated FFAs obese adipose tissue is often characterised by macrophage infiltration. besity is now considered to be a low grade, chronic inflammatory condition. The inflammatory may account for the cardiovascular and diabetic symptoms associated with most sufferers. 9

10 besity There is a strong link between nutrient sensing and pathogen sensing in an organism There has been very strong selection for strong immune The ability to process and store energy In times of chronic nutrient overload the immune may become overly sensitive besity: other areas Some recent treatments for type-2 diabetes associated with obesity involve treating patients with anti-inflammatory drugs to reduce the inflammatory effects and so lessen the type 2 diabetic symptoms. For the final exam. ELMA will NT be examined Material from the labs after the ELMA will be examined: Beta galactosidase induction (gene expression) Protein purification For the final exam. The BM contribution All material covered in my lectures and Gareth s lectures will be examined. I will place some reading material on the web and send it to your usyd address. This material will also appear in the exam. 10