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. 2017 Nov;67(5):1009-1017.
doi: 10.1016/j.jhep.2017.05.023. Epub 2017 Jul 17.

High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver

Emily S Wires  1 , Kathleen A Trychta  1 , Susanne Bäck  1 , Agnieszka Sulima  2 , Kenner C Rice  2 , Brandon K Harvey  3
Affiliations

High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver

Emily S Wires et al. J Hepatol. 2017 Nov.

Abstract

Background & aims: Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis.

Methods: A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity.

Results: Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration.

Conclusions: Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.

Keywords: Cafeteria diet; Dantrolene; Endogenous SERCaMP; Endoplasmic reticulum; Endoplasmic reticulum calcium; Gaussia luciferase; High fat diet; Palmitate; SERCA2b; SERCaMP.

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Conflict of interest statement

Authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1. GLuc-SERCaMP is responsive to ER calcium perturbations
(A) Schematic of viral-mediated delivery of GLuc-SERCaMP to the liver and expected outcomes from challenges that alter ER calcium homeostasis in the liver. (B) Temporal profile of GLuc-SERCaMP activity in plasma over time with two thapsigargin injections (1mg/kg i.p.) (mean ± SEM, n=4 rats, *p<0.05, **p<0.01, 1-way ANOVA, Dunnett’s multiple comparison test). (C) SH-SY5Y-GLuc-SERCaMP or SH-SY5Y-GLuc-No Tag stable cell lines were treated with palmitate (0–600μM), and luminescence in the cell media was read 24hrs post-treatment (mean ± SEM, n=6 wells/treatment/cell line, ****p<0.0001, 2-way ANOVA, Tukey’s multiple comparison test, 0μM vs other concentrations).
Fig. 2
Fig. 2. Cafeteria diet induces GLuc-SERCaMP response
(A) Effect of dietary intake on plasma levels of GLuc-SERCaMP. Rats intrahepatically expressing GLuc-SERCaMP were allowed ad lib access to a cafeteria diet plus chow (green circles) or restricted access to standard chow (blue triangles). Rats allowed ab lib access to cafeteria diet plus chow showed increased release of GLuc-SERCaMP (mean ± SEM, n=7 rats/cafeteria group, n= 4 rats/restricted chow group, *p<0.05, **p<0.01, 2-way ANOVA, Sidak’s multiple comparison test). (B) Caloric intake of ad lib cafeteria diet plus chow versus restricted chow. (C, D) Fat intake tracks with changes in plasma GLuc-SERCaMP (percent of calories from fat calculated as percentage of total caloric intake based off nutritional labels). (E) Increased fat intake (calculated based on average per day on menu item) correlates with increase in GLuc-SERCaMP release in cafeteria diet-fed rats (Pearson’s correlation, r= 0.3217, p= 0.0101, n= 6 rats).
Fig. 3
Fig. 3. High fat pellets induce GLuc-SERCaMP and endogenous SERCaMP response
(A) Schematic of experimental paradigm color coded to match subsequent panels. (B) Ad lib access to high fat pellets increases GLuc-SERCaMP release (mean ± SEM, n=6 rats/group, *p<0.05, **p<0.01, ***p<0.001, 2-way ANOVA, Sidak’s multiple comparison test). Color change indicates access change (dark blue= ad lib access, light blue= restricted access). Symbols and line pattern (closed circles and solid line versus open triangles and dotted line) represent same group of animals throughout experiment. (C) Control pellet intake has no effect on GLuc-SERCaMP release regardless of food access (mean ± SEM, n=6 rats/group, n.s. p=0.2760, 2-way ANOVA, Sidak’s multiple comparison test). Color change indicates access change (dark red= ad lib access, pink= restricted access). (D) Switch from restricted high fat pellet to ad lib access increases endogenous SERCaMP release (mean ± SEM, n=6 rats/group, *p<0.05, 1-way ANOVA, Dunnett’s multiple comparison test). (E) Switch from restricted control pellet to ad lib access increases endogenous SERCaMP release (mean ± SEM, n=6 rats/group, *p<0.05, 1-way ANOVA, Dunnett’s multiple comparison test). (F) Caloric intake over the course of the study in rats allowed ad lib and restricted access to high fat pellets and control pellets. Color, symbol, and line key same as previous panels. (G) Sum of caloric intake among feeding cohorts. Rats with ad lib access to high fat pellets consumed more calories than rats with ad lib access to control pellets (mean ± SEM, n=6 rats/group, *p=0.0127, **p=0.0016, unpaired t-test with Welch’s correction). (H) Rats with ad lib access to high fat pellets consumed less food than rats with ad lib access to control pellets (mean ± SEM, n=6 rats/group, **p=0.0066, n.s. p=0.1588, Unpaired t-test with Welch’s correction).
Fig. 4
Fig. 4. Ad lib access to high fat pellets alters liver morphology, function and calcium ATPase activity
(A) Representative images of left lateral lobe harvested from rats allowed ad lib or restricted access to high fat pellets. (B) Weights of left lateral lobe harvested from rats allowed ad lib or restricted access to high fat pellets (mean ± SEM, n=6 rats/group, t-test, P<0.0001, t=13.52, df=10). (C) Table of peripheral markers of liver inflammation and damage (mean ± SEM, n=6 rats/group, t-test, *p<0.05, **p<0.01). (D, E) Western blot analysis of SERCA2b from liver microsomes (15 μg). No significant changes in the expression levels of SERCA 2b (mean ± SEM, n=6 rats/group, t-test, p=0.2375). (F) Ca2+ ATPase activity reflecting SERCA activity in rats allowed ad lib access to high fat pellets (mean ± SEM, n=6 rats/group, t-test, *p=0.0308).
Fig. 5
Fig. 5. Dantrolene attenuates GLuc-SERCaMP and endogenous SERCaMP
(A, B) Dantrolene decreases GLuc-SERCaMP and endogenous SERCaMP release during ad lib access to high fat pellets. Rats were administered dantrolene (15mg/kg) or vehicle for 7 days during diet (mean ± SEM, n=6 rats/group, *p<0.05, 2-way ANOVA, Sidak’s multiple comparison test). (C) Decreased food consumption among dantrolene injected rats (mean ± SEM, n=6 rats/group, ****p<0.0001, 2-way ANOVA). (D) Dantrolene decreases BiP mRNA levels in liver tissue (data expressed as fold change (2^-ΔΔCt) relative to vehicle-treated rats (mean ± upper and lower limits (2ΔΔCt±SD), n=6 rats/group, multiple t-test, Holm-Sidak method (dantrolene versus vehicle) transformed from ΔCt±SD, *p=0.01).
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