Passage
Whole grains have been shown to reduce the risk of cardiovascular disorders. A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism. Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell. Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria. Based on the structures of 5-AVAB and L-carnitine (Figure 1) , scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine.
Figure 1 Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours. Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources. The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2) .
Figure 2 Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB.
Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1) :13036.
-A colleague suggested that 5-AVAB may interfere with the electron transport chain directly. Which experiment could provide evidence for the effect (if any) of 5-AVAB on the electron transport chain?

Question

Passage
Whole grains have been shown to reduce the risk of cardiovascular disorders. A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism. Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell. Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria. Based on the structures of 5-AVAB and L-carnitine (Figure 1) , scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine.

Figure 1 Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours. Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources. The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2) .

Figure 2 Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB.
Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1) :13036.
-A colleague suggested that 5-AVAB may interfere with the electron transport chain directly. Which experiment could provide evidence for the effect (if any) of 5-AVAB on the electron transport chain?

Quizplus · Accepted Answer

11ecba2d_0efa_92fa_a3bf_0b65866656c9_MD0008_00 The electron transport chain (ETC) uses electrons from NADH and FADH₂ to reduce oxygen to water, providing the energy needed for ATP synthesis. NADH and FADH₂ are largely produced in the citric acid cycle and several metabolic pathways lead to this cycle, including glycolysis and β-oxidation. The scientists hypothesized that 5-AVAB inhibits fatty acid degradation (β-oxidation) in the mitochondria, and thereby decreases oxygen consumption. They further hypothesized that this inhibition is accomplished by blocking L-carnitine from entering cells, impairing L-carnitine-mediated transport of fatty acids into the mitochondria. Although the data in Figure 2 are consistent with this hypothesis, it is possible that 5-AVAB can also decrease oxygen consumption by directly inhibiting the ETC. To determine whether the ETC is directly affected by 5-AVAB, an alternative source of NADH and FADH₂ must be provided. Pyruvate produces NADH and FADH₂ by conversion to acetyl-CoA. Acetyl-CoA enters the citric acid cycle, which the passage says is not affected by 5-AVAB. If 5-AVAB inhibits the ETC directly, then cells provided with pyruvate will have a higher oxygen consumption rate in the absence of 5-AVAB than in its presence. Conversely, if 5-AVAB does not directly impact the ETC, then cells provided with pyruvate will have the same oxygen consumption rate regardless of whether 5-AVAB is present. Therefore, incubating cells with pyruvate and measuring oxygen consumption in the presence and absence of 5-AVAB would reveal whether 5-AVAB interferes with the ETC directly. (Choice B) Adding a known ETC inhibitor will slow down oxygen consumption, but this slowing will only provide evidence that the inhibitor affects the ETC. It will not provide any evidence about direct interactions between 5-AVAB and the ETC. (Choices C and D) Palmitate and oxygen are consumed by β-oxidation and the ETC, respectively. Altering their concentration or partial pressure will not give any information about the effect of 5-AVAB on ETC activity. Educational objective: The electron carriers needed for the electron transport chain (ETC), NADH and FADH₂, can be provided by several pathways including glycolysis, β-oxidation, and the citric acid cycle. A molecule that affects the ETC directly will block the use of electron carriers regardless of which pathway generates them.

Passage Whole Grains Have Been Shown to Reduce the Risk of of Cardiovascular