BA, BSc, and MSc Degree Examinations

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1 Examination Candidate Number: Desk Number: BA, BSc, and MSc Degree Examinations Department : BIOLOGY Title of Exam: Metabolism in health and disease - open assessment Marking Scheme: Total marks available for this paper: 80 The marks available for each ques on are indicated on the paper Instructions: Submission deadline: Monday 8 January, 12 noon Work should be submi ed in hard copy to Biology Student Services and via the Yorkshare VLE Answer all ques ons. Each ques on should be answered within the stated line or word limit, using Arial font size 11 or larger. Answers should include relevant literature cita ons only where requested. All ques ons should be answered on this ques on paper. For marker use only: For office use only: Module total as % page 1 of 13

2 LEARNING OUTCOMES (indicate relevant LO(s) for each question) Students studying this module will be able to: Understand the fundamental biochemical principles of cell signalling, membrane transport and glucose and lipid metabolism. Discuss how fundamental metabolic pathways are compromised in human disease and ageing. Evaluate whether particular membrane proteins or signalling pathways are appropriate therapeutic targets for human disease. Acquire, analyse and interpret experimental data to formulate hypotheses about cell signalling and metabolic pathways. Solve metabolism - and signalling - themed problems. Frans Maathuis: 17 marks (remove before release to students) 1. a) Summarise the signalling roles of the reaction products of phospholipase C. (maximum 3 lines, 2 marks) DAG remains in the membrane and is an activator of PKC by moving it from the cytoplasm to the membrane (1 mark). IP 3 binds to IP 3 receptors on the ER membrane to release Ca 2+ (1 mark). c) How might you argue that PLC is involved in Ca 2+ -induced Ca 2+ release? (maximum 3 lines, 1 mark) PLC contains 4 (Ca 2+ -binding) EF hands and is activated by cytoplasmic Ca 2+. Its product IP 3 is a very potent opener of Ca 2+ channels in the ER so overall this amounts to a strong amplification of the Ca 2+ signal. 2. a) Explain why the mitochondrial proton motive force (PMF) could be a target to treat obesity. LO1-4 (maximum 8 lines, 3 marks) The PMF is instrumental in controlling aerobic phosphorylation (i.e. ATP production) and heat production. Metabolic energy that is lost as heat (rather than turned into ATP) essentially constitutes a loss of calories (1 mark). For example, in adipose tissue, natural uncoupler proteins (UCPs) can increase heat production by rapidly oxidising fats and in general, uncoupling greatly accelerates metabolic rate (and hence weight loss) (1 mark). There are also many chemical uncouplers (DNP, FCCP, CCCP) which are ionophores that create a shunt pathway for H + (1 mark). b) Discuss pros and cons of exploiting chemical and endogenous page 2 of 13

3 uncouplers (UCPs) in the treatment of obesity and related diseases such as type II diabetes mellitus. (maximum 10 lines, 4 marks) Though potentially an efficient manner to lose weight, chemical uncouplers like dinitrophenol (DNP) that can be bought online are highly toxic because they do not show any selectivity and affect all tissues. Over dosing leads to hyperthermia, lack of ATP and a host of other symptoms (1 mark) and can be lethal. Research into the regulation (at the tissue or cellular level) of specific UCPs may prove more successful (e.g. Busillio et al 2015 Front Physiol.). For example, among humans, alleles have been identified that carry mutations in UCP isoforms that impact on body mass index. (1 mark). Uncoupling proteins may also have other functions such as protection from ROS, mediation of fatty acids oxidation and export of fatty acids, which are related to obesity/diabetes (1 mark). 3. Figure 1 shows insulin secretion in rat pancreatic β-cells in response to TP (thiopental) or glucose. LO1-5 Figure 1. Insulin secretion in rat pancreatic beta cells. Cells were treated in the presence or absence (control) of 20 mm glucose or 0.3 mm thiopental (TP) and insulin secretion quantified. ** P<0.001 compared to 0 mm glucose. a) What is thiopental and why would you study its effect on insulin secretion? (maximum 5 lines, 2 marks) page 3 of 13

4 Thiopental is a common anaesthetic, often used to sedate rats, including those used in insulin studies (1 mark). However, the suspicion that TP might have direct effects on insulin production (as shown above) prompted more detailed studies and confirmed that TP is not a good way to sedate rats for insulin related studies (1 mark). b) TP evokes a Ca 2+ signal in pancreatic cells that is abolished by thapsigargin and by heparin. What does this suggest about the origin of the Ca 2+? (maximum 4 lines, 1 mark) Thapsigargin is an inhibitor of the ER Ca-pump while heparin inhibits IP3 receptors. Thus, the Ca 2+ originates from the ER Ca store and is released via IP3 receptors on the ER membrane. c) How does this compare to the Ca 2+ signal evoked by glucose as stimulus? (maximum 3 lines, 2 marks) Glucose causes a depolarisation and subsequent opening of voltage dependent Ca channels in the plasma membrane. Thus, the Ca 2+ comes from outside rather than internal stores. d) If you added TP and glucose together to pancreatic beta cells, approximately what value would you expect for insulin secretion? Explain your answer. (maximum 5 lines, 2 marks) The value could be anywhere between 4.5 and 7.5 because the signal can vary from no change since the downstream Ca 2+ response may already be saturated at these concentrations of glucose (and/or TP) (1 mark), to entirely additive because 2 separate Ca 2+ stores are used. (1 mark) page 4 of 13

5 Daniel Ungar: 37 marks (remove before release to students) 4. The actions of the two hormones insulin and adrenaline generally oppose each other. Therefore the downstream signalling from their receptors is carefully coordinated. The experiments below try to address how this coordination may affect the output of these important hormonal stimuli. Figure 2. Cross-talk of adrenaline and insulin signalling A. Muscle cells were treated with adrenaline (solid line) or adrenaline and an unknown drug X (dashed line) continuously for 20 min. camp production in the cells was monitored over time, and the measured values (normalised to time 0) shown on the graph. The error bars represent standard error of the mean for 3 measurements. B. Muscle cells were treated with adrenaline and/or insulin as indicated for 15 min, and the proportion of cell-surface localised beta-adrenergic receptors determined. The results shown are the average of three experiments with the error bars showing standard error of the mean. a) Using both parts of the figure give a reason for the decline of camp production seen for adrenaline treated cells after 10 min. LO1, 4 (maximum 5 lines, 3 marks) Panel B shows that 15 min of adrenaline treatment causes a decline of βar at the cell surface (2 marks). This timing coincides with the time of camp production decline, so the reduced camp production is almost certainly due to internalisation of the receptor (1 mark). b) What could be the identity of drug X? Explain. LO3, 5 (maximum 7 lines, 5 marks) page 5 of 13

6 The drug inhibits adrenaline signalling early on (1 mark), but then stops internalisation as shown by the stabilised level of camp production (1 mark). Both of these could be explained by the actions of insulin signalling (1 mark), as insulin signalling enhances PDE production that degrades camp (1 mark), but inhibits internalization of βar as seen in panel B (1 mark). So the drug could simply be insulin, but more likely an artificial agonist of insulin signalling. c) Explain whether adrenaline and insulin oppose each other or not in this experiment. What could be the physiological relevance of this? LO1, 4 (maximum 8 lines, 5 marks) Up to 10 min insulin opposes adrenaline s action by reducing camp levels (1 mark), but later insulin promotes adrenaline mediated camp production by opposing bar internalisation (2 marks). A possible explanation could be that a dampened flight response during high blood glucose levels (ie when insulin is high) should not be dampened even further with time, as this would potentially keep blood glucose high and prohibit the full benefits of the flight response (2 marks - other similar explanations accepted) 5. AMPK is a key player in aging, and has been shown to be directly connected to insulin signalling. The relationship between adrenergic signalling and AMPK is less clear though. The following experiments are looking to clarify this in muscle cells. page 6 of 13

7 Figure 3. Influence of adrenaline on AMPK activity AMPK activity in muscle cells treated with adrenaline and the indicated chemicals for 1 h. AMPK activity is expressed relative to untreated muscle cells (labeled no addition ). The error bars represent standard error of the mean for 3 measurements. Asterisks indicate statistically significant differences, compared to untreated control samples: ** for p<0.01, *** for p< a) Based on the data in Figure 3, describe which signalling pathways initiated by adrenergic receptor signalling are or are not involved in regulating AMPK. LO1, 4 (maximum 5 lines, 4 marks) The use of db-camp suggests that the adenylyl cyclase-camp-pka pathway is not involved, as db-camp does not inhibit AMPK (2 marks). EGTA does not block adrenaline's effect, suggesting that the PLC-PKC pathway going through Ca 2+ is not involved either (2 marks). page 7 of 13

8 b) These data do not provide enough evidence to conclude if the effect of adrenaline on AMPK is independent of insulin s influence on AMPK or not. Explain why. LO1, 5 (maximum 7 lines, 4 marks) Rapamycin inhibits mtor, which is the main signal transducer between insulin and AMPK (1 mark). Addition of rapamycin does not alter the inhibitory effect of adrenaline on AMPK (1 mark). However, without further data we cannot tell if rapamycin and adrenaline work in the same pathway and therefore have no additive effects (1 mark), or if rapamycin in this experiment just has no effect because mtor is not turned on to start with (1 mark). c) Suggest further experiments that would extend the currently shown figure and could provide the missing evidence to assess if adrenaline and insulin act independently on AMPK or not. Explain how the experiments provide the missing evidence. LO2, 5 (maximum 7 lines, 4 marks) One experiment would be to treat cells with rapamycin in the absence of adrenaline (1 mark). If this shows a reduction of AMPK activity that would suggest the two work in the same pathway, as their co-application is not additive (1 mark). Adding insulin by itself as well as together with adrenaline (2 marks) would also address this question by assessing if the effects seen after co-administration are additive compared to the two separate ones (2 marks). [more marks in marking scheme to allow for the fact that the rapamycin experiment is not essential if the insulin one is given] d) The aim of aging research is to expand healthspan of individuals. It is established that oxygen consumption increases with age, most likely due to less efficient conversion of electron transport through the electron transport chain into a proton motive force. Design an experiment to test the negative effects of AMPK inhibition on the healthspan of mice by using the oxygen consumption of mitochondria as a proxy for health. Test if the adrenergic and insulin signalling inputs into AMPK inhibition are additive or not. The plan should describe the experimental setup (how/when mice are treated, how mitochondrial respiration is measured and when) as well as some possible results you expect. LO 3,4 (maximum 250 words, 12 marks) Plan (8 marks total) should describe treatments with various page 8 of 13

9 agonists/inhibitors of the adrenergic (e.g. adrenaline, beta blockers) and insulin pathways (e.g. insulin, rapamycin) as well as metformin (AMPK agonist) as a control. Treatments should last weeks or months rather than hours, and several mice need to be used per treatment group. Mitochondria should be isolated at the end of treatment regimes and oxygen consumption measured using a Clarke oxygen electrode. Would be good to see determination of P:O ratios as a measure of intact membrane as well as electron transport efficiency. Expected results (4 marks) should detail how P:O ratio (or other measure of oxygen consumption will inform on health of mitochondria and consequently mice. Then some inference on the effects of the used inhibitors/agonists, e.g. adrenaline could increase insulins aging effects if the two work in parallel, and a beta blocker, as well as rapamycin should partly reverse this, while metformin should block the aging effect of both of them. page 9 of 13

10 Gareth Evans: 26 marks (remove before release to students) 6. Two forms of inherited hypercholesterolemia (HA and HB) were mapped to genes A and B respectively. A comparison of control patients, patients with hypercholesterolemia A (HA) or B (HB) is presented in Table 1. Table 1. Comparison of plasma lipid profile, LDL receptor affinity, prevalence of heart disease and response to statin treatment in patients with hypercholesterolemia A (HA; n=54), hypercholesterolemia B (HB; n=58), and non-carrier family members (n=51). HA HB non-carriers LDL cholesterol (mmol/l) 15.4 ± ± ± 1.1 HDL cholesterol (mmol/l) 0.86 ± ± ± 0.52 Affinity of LDL for LDL receptor (Kd; nm) Incidence of coronary heart disease (CHD) Age <20 Age Age Maximum reduction in LDL cholesterol by statin treatment % 85 % 100 % 0 % 45 % 78 % 0 % 3 % 20 % 30 % 60 % 50 % To investigate the function of genes A and B, researchers generated knockout mouse models and obtained the data presented in Figure 4. page 10 of 13

11 Figure 4. Uptake of radiolabelled lipoproteins in WT, A KO and B KO mice. Mice were injected with 125 I-labelled VLDL (left panel) or LDL (right panel) and at the indicated times, blood was removed and the plasma content of 125 I-labelled lipids was measured. a) Propose molecular roles for genes A and B in lipoprotein metabolism. Use your interpretation of the data in Table 1 and Figure 4 to justify your answer. LO 1,2,4,5 (maximum 15 lines; 8 marks) Gene A is related to the uptake of LDL into the liver/tissues and the data are consistent with it being the LDL-receptor (1 mark). HA patients have a high plasma [LDL] and their LDL receptor has a low affinity for LDL (1 mark). The A KO mouse is unable to take up 125 I-LDL or 125 I-VLDL into the tissues/liver, which both require the LDL-receptor (1 mark). The interpretation for gene B is more complex. HB patients have a normal LDL binding affinity to the LDL receptor but high levels of [LDL], similar to HA (1 mark), and the KO mouse is unable to take up LDL. However, there is a measurable amount of VLDL clearance (1 mark). The most logical interpretation is that B is necessary for LDL uptake, but not VLDL (1 mark). VLDL is known to cluster LDL receptors via ApoB48, unlike, LDL which binds via a single ApoB100 (1 mark). VLDL mediated clustering might help stimulate endocytosis in a defective pathway (1 mark). [This is reminiscent of the phenotype of LDLRAP/ARH, an adapter that regulates LDL endocytosis.] b) Based on your interpretation of the data, explain why hypercholesterolemia B is a less severe disease than hypercholesterolemia A. LO 2,5 (maximum 8 lines; 4 marks) Although the [LDL] plasma concentrations are comparable for HA and HB, there is likely to be a much larger lipid burden in HA due to the lack of VLDL uptake (1 mark). There is a lower [HDL] in HA (HB is comparable to controls), which will reduce cholesterol scavenging and increase the risk of atherosclerosis (2 marks). This explains why the incidence of heart disease is delayed compared to HA. HB is responsive to statins because VLDL uptake provides a viable mechanism to clear endogenous cholesterol (1 mark). c) Speculate as to how hypercholesterolemia A could be treated beyond statin therapy. Support your rationale by citing relevant literature. page 11 of 13

12 LO 2,3,5 (maximum 8 lines; 4 marks) In the absence of LDL clearance, familial hypercholesterolemia is usually treated with LDL apheresis (1 mark). This is akin to dialysis of the blood plasma to remove LDL from the bloodstream (1 mark). The process involves immunodepletion of LDL using an affinity column comprising anti-ldl antibodies (1 mark). Additional mark for citing a relevant study. Other viable alternatives will gain marks, which might include gene therapy, or targeting other processes to prevent atherosclerosis. 7. Orexin A acting via its G protein coupled receptor, OX1-R, stimulates neurons in the hypothalamus to regulate appetite, arousal and energy balance. More recently it has been proposed that there are OX1 receptors in peripheral tissues. Figure 5 presents data from experiments investigating the effect of orexin A on adipocyte function. Figure 5. Effect of orexin A on lipid metabolism in adipocytes. A. Cultured rat adipocytes were treated with 100 nm orexin A (OXA) or OXA + 1 µm U0126. ERK phosphorylation was detected by immunoblot and the band intensity normalised to total ERK. ** P<0.01 vs. control. B. Adipocytes received the same treatments as in A, and after 72 h, triacylglycerol content was quantified. ** P<0.01 vs. control. # P<0.001 OXA vs. OXA + U0126. a) Based on the data in Figure 5, how and why might the OX1 receptor be targeted by pharmaceutical companies? LO 2,3,4,5 (maximum 7 lines; 3 marks) The data show that orexin A enhances triacylglycerol content in page 12 of 13

13 adipocytes (1 mark) and does so via MAPK signalling, as evidenced by the activation of ERK1/2 (by phosphorylation) and the inhibition of TAG accumulation by U0126 (a specific MEK inhibitor; 1 mark). A drug company could design an antagonist to the OX1 receptor to inhibit adipocyte maturation, which could treat obesity (1 mark). [researchers have shown this to be viable and orexin antagonists also reduce food intake]. b) Speculate as to how signalling downstream of the OX1 receptor causes the effects observed in Figure 5B and design an experiment to test your hypothesis. LO 1,2,5 (maximum 12 lines; 7 marks) The metabolic pathway for TAG synthesis was covered in detail in the lectures and so any sensible hypothesis involving ERK regulation of this pathway will gain 3 marks. An example might be the regulation of a rate limiting step, such as the lipin mediated dephosphorylation of phosphatidic acid, necessary for TAG synthesis. 4 marks will be available for a sensible experiment to test the hypothesis. For example, assaying the enzyme/protein in question in the presence and absence of OXA, with other relevant controls. This might involve detecting ERK phosphorylation of the protein and then assaying a readout for the protein s function and/or TAG synthesis. page 13 of 13

1Why lipids cannot be transported in blood alone? 2How we transport Fatty acids and steroid hormones?

1Why lipids cannot be transported in blood alone? 2How we transport Fatty acids and steroid hormones? 3How are dietary lipids transported? 4How lipids synthesized in the liver are transported? 5 Lipoprotien