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Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs) , which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs) , enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG) , an HIF analog.
Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2) .
Figure 2 HIF-1a in FH⁺ and FH⁻ cells grown in vitro under normoxic (N) and hypoxic (H) conditions
Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7) :4524-32.
-During hypoxia, skeletal muscles have been shown to accumulate succinate and ADP molecules. Which of the following explains this result?

Question

Passage
Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs) , which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs) , enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG) , an HIF analog.

Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2) .

Figure 2 HIF-1a in FH⁺ and FH⁻ cells grown in vitro under normoxic (N) and hypoxic (H) conditions
Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7) :4524-32.
-During hypoxia, skeletal muscles have been shown to accumulate succinate and ADP molecules. Which of the following explains this result?

Quizplus · Accepted Answer

11ecba2d_0dcf_cee8_a3bf_c9267fe6e6ba_MD0008_00 The accumulation of succinate and ADP during hypoxia would occur because anaerobic conditions prevent the normal regeneration of the NAD⁺ and FAD needed for progression of the citric acid cycle. This cycle involves reactions that occur within the mitochondrial matrix and perform aerobic processing (oxidation) of pyruvate to generate CO₂ and ATP, the cell's energy currency. The dehydrogenase enzymes in this cycle transfer hydride ions to an electron acceptor (eg, NAD⁺ or FAD), creating the high-energy electron carriers NADH and FADH₂. NADH and FADH₂ are then shuttled to the electron transport chain (ETC) in the inner mitochondrial membrane where proteins transfer the released electrons to oxygen, the final electron acceptor. NAD⁺ and FAD are also regenerated, and the electron movement between ETC proteins generates a proton motive force that drives ATP synthase to phosphorylate ADP to ATP. During hypoxia, less oxygen is available to accept electrons from the citric acid cycle; therefore, NAD⁺ and FAD are not regenerated in the mitochondria. Consequently, ADP concentrations increase (due to the absent proton motive force) as ATP production is significantly reduced. In addition, there is an accumulation of succinate and other citric acid cycle intermediates that require NAD⁺ or FAD for their oxidation reactions. (Choice B) Succinate dehydrogenase remains fully functional during hypoxia, so its k_cat is unchanged. However, because FAD is a substrate of succinate dehydrogenase, hypoxia-induced depletion of FAD causes a decrease in the reaction rate. (Choice C) Fumarate is not converted back to succinate in ATP-rich conditions. Instead, excess citric acid cycle intermediates such as fumarate are used as amino acid precursor molecules. (Choice D) High concentrations of NADH inhibit the α-ketoglutarate dehydrogenase that generates succinyl-CoA from α-ketoglutarate. As a result, succinyl-CoA production is reduced, not stimulated. Educational objective: The citric acid cycle uses oxidation reactions to convert NAD⁺ and FAD into NADH and FADH₂, respectively. The movement of electrons from these high-energy electron carriers to electron transport chain (ETC) proteins drives phosphorylation of ADP to ATP. Regeneration of NAD⁺ and FAD in the ETC is only possible when oxygen is available to act as a final electron acceptor.

Passage Tumor Hypoxia Is a Marker of Resistance to Chemotherapy and and Radiation