“…Fourth, reduction of pyruvate to lactate by LDH is not the only reaction by which NAD+ is regenerated. Recent studies have identified how serine pathway modulation, polyunsaturated fatty acid synthesis, and the glycerol-phosphate shunt all contribute to NADH turnover [57][58][59] . Future work remains to assess these mechanisms in PDA cells in vivo and if they compensate for an impaired MAS.…”
The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and consequently nutrient and oxygen access and drug delivery. Concurrently, recent work also demonstrates that the TME directly provides metabolites that facilitate cancer cell survival, growth, and therapeutic resistance. Redox imbalance is another restraint on cellular proliferation, yet it is unknow how the TME contributes to the maintenance redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle (MAS), disturbs redox homeostasis and halts proliferation of PDA cells in vitro. Surprisingly, we found GOT2 knockdown has no effect on in vivo tumor growth. We demonstrate that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts (CAF). More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Blocking pyruvate uptake through genetic or pharmacologic inhibition of monocarboxylate transporter 1 (MCT1) abrogated pyruvate-mediated restoration of redox homeostasis. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.
“…Fourth, reduction of pyruvate to lactate by LDH is not the only reaction by which NAD+ is regenerated. Recent studies have identified how serine pathway modulation, polyunsaturated fatty acid synthesis, and the glycerol-phosphate shunt all contribute to NADH turnover [57][58][59] . Future work remains to assess these mechanisms in PDA cells in vivo and if they compensate for an impaired MAS.…”
The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and consequently nutrient and oxygen access and drug delivery. Concurrently, recent work also demonstrates that the TME directly provides metabolites that facilitate cancer cell survival, growth, and therapeutic resistance. Redox imbalance is another restraint on cellular proliferation, yet it is unknow how the TME contributes to the maintenance redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle (MAS), disturbs redox homeostasis and halts proliferation of PDA cells in vitro. Surprisingly, we found GOT2 knockdown has no effect on in vivo tumor growth. We demonstrate that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts (CAF). More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Blocking pyruvate uptake through genetic or pharmacologic inhibition of monocarboxylate transporter 1 (MCT1) abrogated pyruvate-mediated restoration of redox homeostasis. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.
“…The activity of the other major redox shuttle (glycerol-3-phosphate shuttle, GPS) in brain has been long questioned. Its activity in brain is strikingly low [ 34 ]. For a long time, the two enzymatic constituents of GPS (cytosolic and mitochondrial glycerol 3-phosphate dehydrogenases, cGPDH and mGPDH) were not found to co-localize in the same cell type, a fact that would be required for the GPS to be functional.…”
Section: Agc1/slc25a12/aralar-mas Pathway In Brain Metabolismmentioning
AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named “early infantile epileptic encephalopathy 39” (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.
“…ETC dysfunction or hypoxia increases the NADH/NAD + ratio to inhibit NADH-generating reactions and cause metabolic derangements. Elevated NADH/NAD + ratio inhibits cell proliferation in vitro , limits tumor growth in vivo , and underlies mitochondrial disease pathogenesis ( Liu et al., 2021 ; McElroy et al., 2020 ). Thus, measurement of the elevated NADH/NAD + ratio and the related metabolic derangements is necessary to understand the metabolic consequences under ETC dysfunction.…”
Section: Before You Beginmentioning
confidence: 99%
“…Here we describe a protocol to systematically analyze glucose metabolism under ETC inhibition. The deuterium of [4- 2 H]-glucose can be transferred to NADH in the glycolysis pathway and is used to trace electron transfer of glycolytic NADH ( Figure 1 B); the deuterium of [3- 2 H]-glucose can be transferred to NADPH in the oxidative pentose phosphate pathway (PPP) and is used to trace electron transfer of oxidative PPP ( Figure 1 C) ( Lewis et al., 2014 ; Liu et al., 2021 ).We also use [U- 13 C]-glucose to trace glucose carbon. Figure 1 Schematic of glucose metabolism and deuterium glucose tracing (A) Schematic of glucose metabolism.…”
Section: Before You Beginmentioning
confidence: 99%
“…These protocols comprehensively analyze the metabolic derangements resulting from increased NADH/NAD + ratio in in vitro and in vivo models. For complete details on the use and execution of this profile, please refer to Liu et al. (2021) .…”
Summary
Mitochondrial electron transport chain (ETC) dysfunction elevates the NADH/NAD
+
ratio to cause metabolic derangements. Here we describe a protocol to measure the NADH/NAD
+
ratio and analyze the rewiring of glucose metabolism using [4-
2
H]-glucose, [3-
2
H]-glucose, and [U-
13
C]-glucose in ETC-inhibited human cancer cells. We also describe a protocol to analyze the NADH/NAD
+
ratio-sensitive metabolites in mouse plasma and mouse liver following phenformin treatment. These protocols comprehensively analyze the metabolic derangements resulting from increased NADH/NAD
+
ratio in
in vitro
and
in vivo
models.
For complete details on the use and execution of this profile, please refer to
Liu et al. (2021)
.
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