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. 2017 Feb;1(1):23-35.
doi: 10.1002/hep4.1012. Epub 2016 Nov 11.

Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver

Colleen N Feriod  1   2 , Andre Gustavo Oliveira  3 , Mateus T Guerra  3 , Lily Nguyen  2 , Kisha Mitchell Richards  4 , Michael J Jurczak  5 , Hai-Bin Ruan  6   7 , Joao Paulo Camporez  5 , Xiaoyong Yang  1   6   7 , Gerald I Shulman  1   5   8 , Anton M Bennett  2   7 , Michael H Nathanson  3 , Barbara E Ehrlich  1   2
Affiliations

Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver

Colleen N Feriod et al. Hepatol Commun. 2017 Feb.

Abstract

Fatty liver is the most common type of liver disease, affecting nearly one third of the US population and more than half a billion people worldwide. Abnormalities in ER calcium handling and mitochondrial function each have been implicated in abnormal lipid droplet formation. Here we show that the type 1 isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R1) specifically links ER calcium release to mitochondrial calcium signaling and lipid droplet formation in hepatocytes. Moreover, liver-specific InsP3R1 knockout mice have impaired mitochondrial calcium signaling, decreased hepatic triglycerides, reduced lipid droplet formation and are resistant to development of fatty liver. Patients with non-alcoholic steatohepatitis, the most malignant form of fatty liver, have increased hepatic expression of InsP3R1 and the extent of ER-mitochondrial co-localization correlates with the degree of steatosis in human liver biopsies.

Conclusion: InsP3R1 plays a central role in lipid droplet formation in hepatocytes and the data suggest that it is involved in the development of human fatty liver disease.

Keywords: Calcium; diabetes; endoplasmic reticulum; hepatitis; steatosis.

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Figures

Figure 1
Figure 1
Loss of InsP3R1 in hepatocytes leads to reduced lipid accumulation in vitro and in vivo. (A) Body composition measured by 1H‐magnetic resonance spectroscopy after 8‐9 weeks of HFD feeding (n = 6‐9). (B) Hematoxylin and eosin staining of formalin‐fixed sections of the left liver lobe after 10 weeks of HFD feeding (representative image; n = 6). Scale bars = 50 μm. Quantification of the steatotic area relative to total liver area is shown in the histogram. (C) Serum triglycerides from overnight‐fasted mice (n = 8). (D) Liver triglycerides in overnight‐fasted animals (n = 8). (E) Cellular uptake of 250 μM oleic acid measured at 0, 2, 4, 8, and 16 hours by way of quantification of Bodipy fluorescence, normalized to baseline (n = 40 cells, n = 5 animals). (F) Relative mRNA expression of lipogenic and beta‐oxidation genes in WT and InsP3R1 LSKO livers after 10 weeks of HFD feeding (n = 5). Statistical significance was determined using a Student t test. All error bars represent the SEM. *P < 0.05. ***P < 0.001.
Figure 2
Figure 2
Hepatocytes lacking InsP3R1 have impaired mitochondrial Ca2+ signals. (A,B) Freshly isolated hepatocytes from WT C57/Bl6 mice, InsP3R1 LSKO, and InsP3R2 whole body KO mice were maintained in collagen sandwich cultures and transfected with a genetically encoded cytosolic calcium indicator, R‐GECO (A), and a mitochondrial matrix‐targeted calcium indicator, Inverse PeriCam (B). Cells were stimulated with 10 μM ATP at 50 seconds. Whole cell fluorescence was recorded over time. Representative traces for each group and summarized results from n = 12 cells, n = 2‐4 coverslips per group. Histograms show average amplitude of ATP‐induced Ca2+ signals. (C) Representative traces of cytosolic (light gray) and mitochondrial (dark gray) Ca2+ signals after stimulation with 5 μM phenylephrine (n = 3). Statistical significance was determined using a Student t test. All error bars represent the SEM. **P < 0.01.
Figure 3
Figure 3
ER–mitochondria association is maintained in InsP3R1 LSKO hepatocytes. (A) Representative EM micrograph of a WT mouse hepatocyte in which regions of close contact between the ER membranes (green) and mitochondria (magenta) are magnified. (B) Quantification of total mitochondrial length located within 40 nm of ER membranes shows no significant difference between WT (30.86 ± 4.31%; n = 20 fields) and InsP3R1 LSKO (34.59 ± 3.82%; n = 20 fields) hepatocytes. P = 0.52.
Figure 4
Figure 4
InsP3R1 LSKO mice have normal rates of glucose production and insulin sensitivity after HFD feeding. (A,B) Plasma glucose levels (A) and glucose infusion rates (B) during an HE clamp (n = 10‐12). (C) Basal endogenous glucose production measured under fasting conditions before the start of the clamp (n = 10‐12). (D‐F) Glucose production measured during the clamp (D), glucose uptake (E), and circulating free fatty acids (FFA) (F). Statistical significance was determined using a Student t test. All error bars represent the SEM.
Figure 5
Figure 5
HFD hepatocytes have enhanced mitochondrial Ca2+ signals and increased ER–mitochondrial colocalization. (A) ATP‐induced mitochondrial Ca2+ signals were measured in hepatocytes isolated from mice chronically fed an HFD for 6 months and their chow control littermates (n = 10 cells from three mice). The histogram shows the peak amplitude of mitochondrial Ca2+ signals at 150 seconds. (B) Labeling of ER (PDI, green) and mitochondria (Mitotracker Red, red) indicates an increase in the interaction of both organelles in HFD hepatocytes; colocalization test of immunostaining images (n = 3). Statistical significance was determined using a Student t test. All error bars represent the SEM. *P < 0.05.
Figure 6
Figure 6
Increased association of ER and mitochondria in human fatty liver disease. (A) Representative images of hematoxylin and eosin staining of a normal, simple steatosis, and NASH human liver biopsy. Regions of lipid accumulation (negative image) are present in both simple steatosis and NASH. In addition to steatosis, NASH specimens also display inflammatory infiltrate and fibrosis. Scale bar = 20 μm. (B) Representative image of a histologically normal human liver biopsy specimen stained with markers for an ER protein (PDI, green) and a mitochondrial protein (Tom‐22, red). Colocalized pixels are highlighted in white. The inset shows a magnified area within the field. Scale bar = 20 μm. (C) Mander's colocalization coefficients of mitochondria to ER shows that the fraction of mitochondria associated with ER is increased in both simple steatosis (n = 5 patients) and NASH (n = 4) in comparison with normal liver (n = 5) and that the coefficient for biopsies from NASH patients is significantly greater than that for patients with simple steatosis. Coefficient values are based on 25 separate determinations in each biopsy specimen. (D) InsP3R1 mRNA expression in human liver specimens is significantly increased in NASH patients compared with control and simple steatosis (n = 6 biopsies per condition). Statistical significance was determined by way of analysis of variance (C) or by using a Student t test (D). All error bars represent the SEM. *P < 0.05. ***P < 0.001.
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