Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress
Baran A. Ersoy, … , Ipek Alpertunga, David E. Cohen
Baran A. Ersoy, … , Ipek Alpertunga, David E. Cohen
Published November 20, 2017
Citation Information: J Clin Invest. 2018;128(1):141-156. https://doi.org/10.1172/JCI93123.
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Research Article Cell biology Metabolism

Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress

Abstract

The incorporation of excess saturated free fatty acids (SFAs) into membrane phospholipids within the ER promotes ER stress, insulin resistance, and hepatic gluconeogenesis. Thioesterase superfamily member 2 (Them2) is a mitochondria-associated long-chain fatty acyl-CoA thioesterase that is activated upon binding phosphatidylcholine transfer protein (PC-TP). Under fasting conditions, the Them2/PC-TP complex directs saturated fatty acyl-CoA toward β-oxidation. Here, we showed that during either chronic overnutrition or acute induction of ER stress, Them2 and PC-TP play critical roles in trafficking SFAs into the glycerolipid biosynthetic pathway to form saturated phospholipids, which ultimately reduce ER membrane fluidity. The Them2/PC-TP complex activated ER stress pathways by enhancing translocon-mediated efflux of ER calcium. The increased cytosolic calcium, in turn, led to the phosphorylation of calcium/calmodulin-dependent protein kinase II, which promoted both hepatic insulin resistance and gluconeogenesis. These findings delineate a mechanistic link between obesity and insulin resistance and establish the Them2/PC-TP complex as an attractive target for the management of hepatic steatosis and insulin resistance.

Authors

Baran A. Ersoy, Kristal M. Maner-Smith, Yingxia Li, Ipek Alpertunga, David E. Cohen

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Figure 3

Them2 and PC-TP promote efflux of ER calcium into the cytosol.

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Them2 and PC-TP promote efflux of ER calcium into the cytosol. (A) ER st...
(A) ER stress induced by 0.5 μM thapsigargin (Tg) in mouse primary hepatocytes. Immunoblots are representative of 3 independent experiments. (B) ER calcium release into cytosol induced by 2 μM Tg in HEK 293E cells following knockdown of Them2, PC-TP, or scrambled control. Calcium release into cytosol was measured by Fluo4-AM relative fluorescence units (RFU). Inset barplot displays the AUC. *P < 0.025 compared with scrambled. (C) HEK 293E cells were preincubated with the translocon inhibitor anisomycin (200 μM) or vehicle (0.1% v/v DMSO) for 1 hour prior to Tg-induced ER calcium release into the cytosol following knockdown of Them2, PC-TP, or scrambled control. (D) ER calcium release into cytosol induced by 5 μM ionomycin (IO) in HEK 293E cells following knockdown of Them2, PC-TP, or scrambled control. *P < 0.025 compared with scrambled. (E and F) Immunoblot analyses of IP3R and Serca2b following knockdown of Them2 (E), PC-TP (F), or scrambled control in HEK 293E cells. (G and H) mRNA abundance of Ip3r1, Ip3r2, Ip3r3, Serca2a, and Serca2b was determined by qPCR analysis following knockdown of Them2 (G), PC-TP (H), or scrambled control in HEK 293E cells. GAPDH mRNA served as reference. Error bars represent SEM for n = 3. *P < 0.05 compared with scrambled. (I and J) IP3R3 expression was knocked down along with Them2 (I), PC-TP (J), or scrambled control. (K) Tg-induced ER calcium release into cytosol following co-knockdown of IP3R3 in HEK 293E cells. AUC was normalized to scrambled control from B. *P < 0.025 compared with scrambled + IP3R3 siRNA. (L) IP3-mediated ER calcium release into cytosol was induced in HEK 293E cells by 40 μM thrombin. Calcium release curves represent 6–9 independent experiments. Statistical significance was determined by Student’s t test adjusted by Bonferroni correction.