Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan 2;128(1):141-156.
doi: 10.1172/JCI93123. Epub 2017 Nov 20.

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

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

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

Baran A Ersoy et al. J Clin Invest. .

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.

Keywords: Calcium; Cell Biology; Cell stress; Diabetes; Metabolism.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Them2 and PC-TP regulate ER stress in mice.
(A) The livers of Them2–/– and Pctp–/– mice are protected against ER stress due to high-fat feeding. Liver homogenates from high-fat-fed Them2+/+ (n = 8), Them2–/– (n = 9), Pctp+/+ (n = 5), and Pctp–/– (n = 5) mice were subjected to immunoblot analyses, and bands were quantified by densitometry and normalized to β-actin. Error bars represent SEM. *P < 0.05 compared with Them2–/– or Pctp+/+, ‡P = 0.057 compared with Pctp+/+. (B and C) Them2 and PC-TP modulate the resolution of hepatic ER stress following refeeding. Liver homogenates (n = 3 per genotype) were subjected to immunoblot analyses, and bands were quantified by densitometry. (D) The influence of refeeding on the interactions between PC-TP and Them2 was determined by coimmunoprecipitation in the liver lysates that were harvested from Pctp+/+ (n = 3) mice, and bands were quantified by densitometry. (E–N) Them2 or PC-TP expression enables tunicamycin-induced liver injury. Eight-week-old chow-fed mice were injected i.p. with tunicamycin (0.25 mg/kg body weight) or vehicle (DMSO, 0.25% v/v) for 2 consecutive days. (E and F) Body weights (n = 3 per genotype) following tunicamycin injections (arrows). (G) Livers were harvested and imaged 6 hours following food restriction. Liver sections were subjected to hematoxylin and eosin (H&E) staining. Microscope images (×60) are representatives of n = 3. (H–M) Livers were analyzed for the concentrations of triglycerides (TG) (H and K), NEFA (I and L), and total cholesterol (J and M). (N) ER stress markers were detected in the liver lysates by immunoblot analyses. Error bars represent SEM. *P < 0.05 compared with WT. Statistical significance was determined by Student’s t test.
Figure 2
Figure 2. Them2–/– and Pctp–/– mouse primary hepatocytes are protected against induction of ER stress by tunicamycin and palmitic acid.
(A and B) ER stress was induced in mouse primary hepatocytes that were harvested from Them2+/+ and Them2–/– mice (A) and Pctp+/+ and Pctp–/– mice (B) by treatment of cells with tunicamycin (1 μg/ml) for 5 hours. (C and D) ER stress was induced in mouse primary hepatocytes harvested from Them2+/+ and Them2–/– mice (C) and Pctp+/+ and Pctp–/– mice (D) by treatment of cells with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Rescue of SFA-induced ER stress was performed by treatment of cells with the monounsaturated NEFA oleic acid (0.5 mM) in the presence of palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Hepatocytes were serum-starved overnight before treatments. Immunoblots are representative of 3 independent experiments.
Figure 3
Figure 3. Them2 and PC-TP promote efflux of ER calcium into the cytosol.
(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.
Figure 4
Figure 4. Them2 and PC-TP enable palmitic acid–induced calcium efflux from the ER.
(A–C) Serum-starved HEK 293E cells were treated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours following knockdown of scrambled control (black/gray) (A), Them2 (red/pink) (B), or PC-TP (blue/aqua) (C). Calcium release into the cytosol was measured as a function of the fluorescence intensity of the cytosolic calcium indicator Fluo4-AM following the induction of ER calcium release by thapsigargin (top panels; Tg, 2 μM) or ionomycin (bottom panels; IO, 5 μM). (D) Steady-state cytosolic calcium levels in HEK 293E cells following treatment with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Cells were treated with Them2, PC-TP, or scrambled control siRNA for 48 hours and serum-starved overnight before palmitic acid treatment. *P < 0.025 vs. scrambled. Time-dependent calcium release curves and cytosolic calcium measurements represent 6–9 independent experiments. Error bars represent SEM. Statistical significance was determined by Student’s t test adjusted by Bonferroni correction.
Figure 5
Figure 5. Them2 and PC-TP decrease ER membrane fluidity.
(A) ER membrane fluidity as measured by the excimer-to-monomer ratio of pyrenedecanoic acid (PDA). ER microsomes were purified from the livers of chow- or high-fat diet–fed Them2+/+ (n = 4) and Them2–/– (n = 4) mice following 6-hour food restriction. Error bars represent SEM. *P < 0.05 vs. Them2+/+ mice. (B) ER membrane fluidity of HEK 293E cells as a function of excimer-to-monomer ratio of PDA. Cells were treated with siRNA against Them2, PC-TP, or scrambled control, serum-starved overnight, and incubated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours prior to purification of ER fractions. Error bars represent SEM for the triplicate. *P < 0.025 vs. vehicle. (C) ER membrane fluidity of mouse primary hepatocytes as determined by the inverse correlation of diphenylhexatriene (DPH) polarization anisotropy whereby decreased DPH polarization indicates increased membrane fluidity. Cells were serum-starved overnight and treated with palmitic acid (0.5 mM) (right) or vehicle (4.8 mM BSA) (left) for 6 hours. Error bars represent SEM for the triplicate. Statistical significance was determined by Student’s t test adjusted by Bonferroni correction.
Figure 6
Figure 6. Them2 and PC-TP traffic saturated fatty acids into the ER and reduce fatty acyl chain unsaturation of ER membrane phospholipids in the setting of high-fat diet.
(A and B) Influence of Them2 (A) and PC-TP (B) on the subcellular distribution of palmitic acid (PA) and oleic acid (OA). Mouse primary hepatocytes were treated with [9,10-3H]palmitic acid (500 μM, 10 μCi/mmol) or [9,10-3H]oleic acid (500 μM, 10 μCi/mmol) for 1 hour. Radioisotope distributions to mitochondria (Mito), ER, and cytosol (Cyto) were normalized for subcellular fraction protein amounts, as well as total cellular uptake. (C–H) Four-week-old mice were fed chow or high-fat diet for 8 weeks, and livers were harvested following 6 hours of food restriction. Lipids were extracted from purified ER microsomes and subjected to mass spectrometry analysis. (C and D) Hepatic ER membrane PC (C) and PE (D) fatty acyl chain composition for Them2+/+ (n = 4) and Them2–/– (n = 4) mice. (E) Phospholipid molecular species from C and D were divided into 2 groups by fatty acyl chain saturation: ≤3 double bonds (left) or ≥4 double bonds (right). (F and G) Hepatic ER membrane PC (F) and PE (G) fatty acyl chain composition for Pctp+/+ (n = 4) and Pctp–/– (n = 4) mice. (H) Phospholipid molecular species from F and G were divided into 2 groups as in C. Error bars represent SEM. *P < 0.05 vs. WT mice. Statistical significance was determined by Student’s t test.
Figure 7
Figure 7. Them2 and PC-TP regulate the activity of CaMKII.
(A and B) Influence of Them2 or PC-TP knockdown on CaMKII activation was determined in HEK 293E cells treated with Bapta-AM (5 nM) (A), anisomycin (200 μM) (B), or vehicle for 1 hour. Immunoblots represent 3 independent experiments. (C) Reduced activation of CaMKII in livers of Them2–/– mice. Liver homogenates from 12-week-old high-fat diet–fed Them2+/+ (n = 4) and Them2–/– (n = 5) mice were subjected to immunoblot analyses, and bands were quantified by densitometry and normalized to β-actin as control. Error bars represent SEM. *P < 0.05 vs. Them2+/+. Statistical significance was determined by Student’s t test. (D) Postulated mechanism by which Them2 and PC-TP regulate hepatic glucose homeostasis. In the setting of overnutrition, Them2 and PC-TP are proposed to facilitate the incorporation of SFAs into the ER membrane phospholipid composition, which induces loss of ER calcium into the cytosol via translocons. Increased cytosolic calcium accumulation activates CaMKII, which in turn promotes insulin resistance and enhances hepatic glucose production. Efflux of ER calcium promotes ER stress, which is associated with insulin resistance and de novo lipogenesis.
See this image and copyright information in PMC

References

    1. Hu FB, van Dam RM, Liu S. Diet and risk of Type II diabetes: the role of types of fat and carbohydrate. Diabetologia. 2001;44(7):805–817. doi: 10.1007/s001250100547. - DOI - PubMed
    1. Feskens EJ, Kromhout D. Habitual dietary intake and glucose tolerance in euglycaemic men: the Zutphen Study. Int J Epidemiol. 1990;19(4):953–959. doi: 10.1093/ije/19.4.953. - DOI - PubMed
    1. Vessby B, et al. Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: the KANWU Study. Diabetologia. 2001;44(3):312–319. doi: 10.1007/s001250051620. - DOI - PubMed
    1. Borradaile NM, Han X, Harp JD, Gale SE, Ory DS, Schaffer JE. Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death. J Lipid Res. 2006;47(12):2726–2737. doi: 10.1194/jlr.M600299-JLR200. - DOI - PubMed
    1. Rong X, et al. LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition. Cell Metab. 2013;18(5):685–697. doi: 10.1016/j.cmet.2013.10.002. - DOI - PMC - PubMed

Publication types

MeSH terms