NADPH and glutathione redox link TCA cycle activity to endoplasmic reticulum stress

Abstract

1 2 Endoplasmic reticulum (ER) stress is associated with dysregulated metabolism, but little is 3 known about how the ER responds to metabolic activity. Here, working primarily in mouse 4 hepatocytes, we show that decreasing the availability of substrate for the TCA cycle diminished 5 NADPH production and attenuated ER stress in a manner that depended on glutathione 6 oxidation. ER stress was also alleviated by impairing either TCA-dependent NADPH production 7 or Glutathione Reductase. Conversely, stimulating TCA activity favored NADPH production, 8 glutathione reduction, and ER stress. Validating these findings, we show that deletion of the 9 mitochondrial pyruvate carrier, which is known to decrease TCA cycle activity and protect the 10 liver from diet-induced injury, also diminished NADPH, elevated glutathione oxidation, and 11 alleviated ER stress. These results provide independent genetic evidence that mitochondrial 12 oxidative metabolism is linked to ER homeostasis. Our results demonstrate a novel pathway of 13 communication between mitochondria and the ER, through relay of redox metabolites. 14 15 16

Interference with the HNF4-dependent gene regulatory network diminishes ER stress in hepatocytes

“…7D, E). Having previously demonstrated that pharmacological inhibition of PDKs causes ER stress (16), and here showing the same with knockdown (Fig. 7F, G), this represents an appealing-but as yet unproven-pathway linking HNF4a to ER homeostasis.…”

“…The other liver-expressed isoform of PDK, Pdk2, was unaffected in both conditions (Figure S4A, B). We have previously demonstrated that attenuation of TCA cycle flux protects hepatocytes from ER stress (16). Consistent with that idea, combined knockdown of Pdk2/4 (Figure S4C) was sufficient to cause ER stress, as seen by upregulation of UPR target genes (Figure 7F, S4D) and splicing of Xbp1 (Figure 7G).…”

“…Pyruvate Dehydrogenase exerts a strong influence on TCA cycle activity by controlling the availability of Acetyl-CoA for condensation with oxaloacetate, and inhibition of PDK stimulates TCA cycle flux (51,52). In our previous work, we showed that production of NADPH by the TCA cycle favors glutathione reduction, leaving the ER relatively hypo-oxidized (16). Since ER homeostasis is known to be exquisitely sensitive to reducing conditions in the organelle (53), upregulation of PDK4 (along with PDK2, the most highly expressed hepatic PDK isozymes ( 54)) could thereby alleviate ER stress through the suppression of TCA cycle activity.…”

“…Protein was isolated by homogenization of samples in RIPA buffer, and RNA by homogenization in Trizol (ThermoFisher), and qRT-PCR (including piloting of primer sets to confirm efficiency and specificity), immunoblot, and Xbp1 splicing assays were as described (16,65). qRT-PCR Ct values were normalized against the average of two…”

“…One of the processes suppressed by the UPR in the liver is fatty acid oxidation, and we have shown that fatty acid oxidation stresses ER protein processing capacity by promoting hypooxidizing conditions within ER lumen. This effect occurs through the production of NADPH and the consequent reduction of glutathione that occur during TCA cycle flux (16,17).…”