The Regulation of Eukaryotic Gene Expression using the

The Regulation of Eukaryotic Gene Expression. . using the example of PEPCK

PEPCK • This is an acronym for an enzyme • Phospho. Enol Pyruvate Carboxy. Kinase • This enzyme is ONLY regulated by gene expression! • No allosteric activators, covalent modification etc • No activation by c. AMP, inhibition by insulin etc

PEPCK • 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 PEPCK? • 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.

PEPCK overexpression in muscle • • The youtube video http: //www. youtube. com/watch? v=4 PXC_mctsg. Y is of a mouse with PEPCK overexpressed in muscle only. This mouse hit the popular press in 2007 and put Case Western Reserve University in Cleveland Ohio on the map! • Earl Sutherland, the discoverer of c. AMP also hailed from Case Western.

The Supermouse…. • Eats 60% more food than wild type mice • Weighs 40% less than wild type mice • Can run for >4 h until exhaustion whereas the control littermates stop after only 10 min • Has 2 – 3 fold less adipose tissue

PEPCK 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.

PEPCK overexpression in adipose tissue • A less famous cousin mouse has the PEPCK enzyme overexpressed in adipose tissue. • The results couldn’t be further from supermouse!

PEPCK overexpression in fat cells

PEPCK 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.

PEPCK 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.

PEPCK 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 PEPCK knock out in all tissues is lethal…mice die within days of birth.

Why the dramatically different outcome for the mouse when PEPCK is overexpressed in different tissues? It is after all the same enzyme catalysing the same reaction.

The reaction! GTP Oxaloacetate GDP Phosphoenol pyruvate

Where does it fit in? Glucose Gluconeogenesis Glycolysis PEP OAA Pyruvate LDH

Glyceroneogenesis PEPcarboxykinase

Glyceroneogenesis PEP

PEPCK gene PPAR response element PPARRE -1000 c. AMP response element Glucocorticoid response element IRE GRE -400 Insulin response element TRE CRE -300 -100 Thyroid response element Promoter and regulatory region TATA

PEPCK regulation in liver • PEPCK activity is highest in liver during starvation • Glucocorticoids such as cortisol and glucagon both activate the expression of the PEPCK gene in liver • The glucocorticoids are steroid hormones whereas glucagon is a peptide hormone

Activating PEPCK activity in liver during starvation • Let’s consider the glucocorticoid response first. • Cortisol is the active glucocorticoid hormone. • Pharmaceutical analogues are cortisone (converted to cortisol by a dehydrogenase) and the synthetic analogues prednisone and dexamethasone • Often administered for their immunosuppressive properties

Activating PEPCK activity in liver during starvation • Cortisol is produced and released by the adrenal gland…. it travels through the circulation and can pass through the cell plasma membrane (unlike peptide hormones) • Once inside the cell it binds to a cytosolic receptor in specific cells

Activating PEPCK activity in liver during starvation • 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 response element (a sequence of DNA upstream of a number of genes including PEPCK)

Activating PEPCK activity in liver during starvation • The binding of this complex greatly enhances the frequency of initiation of the basal transcription apparatus (RNA pol II with all the bits). • Other protein factors (coactivators) also bind. These factors reside in the nucleus of liver cells and are known as hepatic nuclear factors (HNFs).

Activating PEPCK activity in liver during starvation • It is thought that both the cortisol: receptor complex and one or more of the HNFs need to be bound for effective enhancement. • This is important for the tissue specific nature of the PEPCK up-regulation.

PEPCK gene PPAR response element PPARRE -1000 c. AMP response element Glucocorticoid response element IRE GRE -400 Insulin response element TRE CRE -300 -100 Thyroid response element Promoter and regulatory region TATA

blood cortisol Cortisol receptor cytoplasm HNFs nucleus NLS NLS RNA pol II TATA Cortisol binds to its receptor, exposing the NLS

Differing response to glucocorticoids in different tissues • While cortisol up regulates PEPCK transcription in the liver. • It down regulates PEPCK in adipose tissue. • The same gene (single copy in the genome) with the same promoter and regulatory regions! How is this possible?

PEPCK 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 HNFs bound. • With different adipocyte specific nuclear factors you can get the reverse result.

Activating PEPCK activity in liver during starvation • 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 Gcoupled protein receptor)

Activating PEPCK activity in liver during starvation • 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 c. AMP activates Protein Kinase A moves to the nucleus phosphorylates transcription factors (CREBs)

Activating PEPCK activity in liver during starvation • The phosphorylated CREBs then bind to the CRE (c. AMP response element) site on the DNA • effective enhancement of PEPCK transcription (amongst other genes you need up regulated in starvation)

PEPCK gene PPAR response element PPARRE -1000 c. AMP response element Glucocorticoid response element IRE GRE -400 Insulin response element TRE CRE -300 -100 Thyroid response element Promoter and regulatory region TATA

glucagon Blood Liver cytoplasm Glucagon receptor GDP Adenylyl cyclase G protein R C Nucleus R C Protein kinase A

Glucagon binds to receptor Blood Liver cytoplasm Adenylyl cyclase GTP GDP R C Nucleus R C Protein kinase A

Adenylyl cyclase Glucagon binds to receptor Blood Liver cytoplasm GTP R C P P CREB Nucleus ATP c. AMP R C CREB R R P CREB C C

PEPCK down regulation by Insulin What we know…. . • Insulin inhibits the basal PEPCK transcription apparatus • Insulin antagonizes the induction of PEPCK expression by glucagon or glucocorticoids

PEPCK 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 PEPCK. • It would be nice to say that an intermediate produced by insulin signalling phosphorylated a transcription factor which binds to the IRE…. BUT I CAN’T

Summary: Transcriptional Regulation of PEPCK • Use the liver in starvation as the context • PEPCK 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 PEPCK Cortisol, a steroid hormone, up-regulates PEPCK Cortisol 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 PEPCK • Glucagon, a peptide hormone up-regulates PEPCK • Glucagon can’t enter the cell binds to G -coupled protein receptor activates adenylyl cyclase c. AMP↑ binds to Protein kinase A R subunits dissociate from C subunits enter nucleus phosphorylate CREB dimerise and bind to CRE

Post transcriptional regulation of PEPCK • Glucocorticoids and c. AMP also stabilise the PEPCK m. RNA in the liver cytoplasm. • Insulin destabilises it. • m. RNA stability contributes significantly to the overall up or down regulation of gene expression. • PEPCK is normally very unstable. • m. RNA stability is measured by its half life.

Why would it be advantageous for an m. RNA sequence like PEPCK to be unstable? • If PEPCK is only regulated by gene expression it is difficult to down regulate the sequence at the level of synthesis if the m. RNA persists in the cytoplasm. • This also applies to the Trp operon enzymes

cytoplasm AAAAA 3’ 5’ Me. G Translation Processed mature m. RNA Nucleus 5’ Me. G Processing Transcription DNA AAAAA 3’ Primary transcript

PEPCK m. RNA stability • A sequence at the 3’ UTR of PEPCK m. RNA has been identified which “destabilises” the m. RNA. • If that sequence is inserted into the 3’UTR of other more stable m. RNAs, such as globin, the half life reduces significantly. • We are yet to determine how c. AMP or cortisol stabilises this m. RNA.

PEPCK gene expression in adipose tissue • Another response element 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 PPARg, the other is PPAR d • liver has PPARa and PPARg

cytoplasm PPARg Nucleus PPARg RXR PPARg activates the transcription of genes involved with adipogenesis and fat storage

Pharmaceutical applications • A new group of insulin sensitizers, the thiazolidinediones (TZDs) act on PPARg. • The most commonly prescribed are Rosiglitozone and Piogliterzone • These artificial ligands for PPARg. • We don’t even know the natural ligand for PPARg although the favoured candidates are fatty acids and their derivatives, in particular polyunsaturated fatty acids.

TZDs cytoplasm PPARg Nucleus PPARg RXR TZDs are artificial ligands for PPARg. These are used as insulin sensitising agents.

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 • Obesity 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 PEPCK mouse) • They have adipocytes that can contain the FFAs

Fat mice who are metabolically healthy

Pharmaceutical applications: TZDs • act to up-regulate PEPCK 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 PEPCK activity will improve the fat storage in the adipocyte.

Obesity: other areas • As well as elevated FFAs obese adipose tissue is often characterised by macrophage infiltration. • Obesity is now considered to be a low grade, chronic inflammatory condition. • The inflammatory response may account for the cardiovascular and diabetic symptoms associated with most sufferers.

Obesity • There is a strong link between nutrient sensing and pathogen sensing in an organism • There has been very strong selection for – strong immune response – The ability to process and store energy – In times of chronic nutrient overload the immune response may become overly sensitive

Obesity: 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.

Obesity: other areas • Anti-TNF alpha treatments such as infliximab (often prescribed for rheumatoid arthritis and other inflammatory diseases) and even salicylic acid derivatives are being trialled. • Metformin, the most commonly prescribed insulin sensitising drug, suppresses gluconeogenesis by inhibiting the expression of PEPCK and G 6 Pase

For the final exam…. • ELMA will NOT be examined • Material from the labs after the ELMA will be examined: – Beta galactosidase induction (gene expression) – Protein purification

For the final exam…. The BCHM 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 email address. This material will also appear in the exam.