Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish - PubMed (original) (raw)

Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish

Prakash C Thakur et al. Hepatology. 2011 Aug.

Abstract

Hepatic steatosis is the initial stage of nonalcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipthi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver, and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5 days postfertilization. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of endoplasmic reticulum stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, and xbp1, are selectively up-regulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis.

Conclusion: cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis, and evaluation of novel therapeutics of NAFLD.

Copyright © 2011 American Association for the Study of Liver Diseases.

PubMed Disclaimer

Figures

Figure 1

Figure 1. hi559 larvae exhibit defects in liver development

(A) 5-dpf larval morphology**,** wild-type on top, mutant below. (B) hi559 larvae show globular liver (yellow) and smaller intestine (red). (C) Globular liver is apparent by Cy3-SA labeling. Lateral view of ISH at 5-dpf showing expression of liver-specific markers sepp1b (D), cp (E), and fabp10a (F). L, liver, ib; intestinal bulb; gb, gas-bladder; y, yolk.

Figure 2

Figure 2. Disruption of cdipt expression and PtdIns synthesis in hi559 larvae

(A) The retroviral insertion (triangle) was mapped to the first intron of the cdipt gene. White boxes indicate exons, grey boxes introns. (B) cdipt expression is disurpted in hi559 larvae (RT-PCR amplification of three different regions, indicated in A). (C-E) Developmental expression pattern of cdipt in wild-type embryos. Note the absence of cdipt expression in hi559 larvae (wild-type, F, left; mutant, F, right). Intestine (arrow), liver (arrowhead). (F) PtdIns synthesis is disrupted in hi559 larvae. The bar chart represents values from three biological replicates.

Figure 3

Figure 3. Histopathological abnormalities of hi559 liver

(A) H&E-stained sagittal sections of 5-dpf wild-type and hi559 liver. Mutant liver architecture is abnormal with vacuolar appearance and smaller sinuses (asterisk). (B) Toluedene staining of semi-thin transverse sections reveals large, vacuolated hepatocytes with marginalized nuclei (arrow) in hi559 liver. (C) Whole mount ORO staining of 5-dpf larvae shows fatty liver (dotted line) in the mutant. (D) ORO staining of frozen tissue sections of 5-dpf larvae reveals substantial steatosis in hi559 hepatocytes. Arrows indicate lipid droplets (red), arrowheads nuclei. ib, intestinal bulb; in, intestine; L, liver; y, yolk; wt, wild-type. Scale bars: A, 20 μM; B, 50 μM; D, 20 μm.

Figure 4

Figure 4. NAFLD progression in hi559 liver

H&E sections of wild-type liver at 6-dpf (A), and stages of NAFLD progression in hi559 liver at 5-dpf (B), 5.5-dpf (C), and 6-dpf (D). At early stage of NAFLD at 5-dpf, foci of microvesicular (arrow) and macrovesicular (arrowhead) steatosis are evident with no apparent apoptosis. At 5.5-dpf, most of the hepatocytes exhibit macrovesicular steatosis with a few apoptotic hepatocytes (arrow, the dotted line area magnified in the inset). At 6-dpf, hepatocellular ballooning (arrow), often with perinuclear hyaline inclusions (arrowhead), and necrotic foci (dotted line) are apparent. (E) Bar-charts showing percentages of embryos with hepatic steatosis at 5-dpf. (F) Bar-charts showing percentages of apoptotic hepatocytes in 6-dpf liver. Data are representative of 5 biological replicates. Scale bars: 20 μM.

Figure 5

Figure 5. Deregulation of genes involved in ERSR

GSEA enrichment plots and expression profile (shown by heat-map) of genes involved in the ERSR/UPR pathway (A-B) and integrated stress-response pathway (C-D). Running enrichment score (ES, top) and signal-to-noise ratio (bottom) used for ranking genes (positive: upregulated in mutant; negative: downregulated in mutant) are shown in the GSEA plots. Vertical bars (middle) indicate the position of genes in the ERSR/UPR within the sorted microarray data, showing enrichment among genes upregulated in hi559. Normalized ES (NES) and nominal _p_-value, below.

Figure 6

Figure 6. Preferential upregulation of ERSR/UPR genes in the liver of hi559 embryos

ISH of ERSR markers in wild-type (top) and hi559 embryos (bottom) at 5-dpf. The respective gene symbols are indicated in each panel. Note the elevated expression in the hi559 liver (arrow).

Figure 7

Figure 7. Ultrastructural pathology of hepatocytes

(A-D) Electron-micrographic comparison of wild-type and hi559 hepatocytes. Cytoplasmic clearing (asterisk, B), ER luminal swelling (triangle, D), and abnormal mitochondria are frequently observed in hi559 hepatocytes. (E) hi559 hepatocyte shows mitochondrial damage and excessive ER luminal vacuolization. The ER lumens are filled with granular materials. (F) The hi559 hepatocytes often contain membrane-bound lipids (lipolysosomes, arrow) of variable electron density. (G) Autophagosome-like structures (arrow) containing reticular materials are noticed within hi559 hepatocytes. (H) Presence of a macrophage (arrow) adjacent to necrotic hepatocytes. mc, mitochondria; n, nucleus; er: endoplasmic reticulum. wt, wild-type; mut, hi559 mutant. Scale bars: A-B & F, 2 μm_; C-E_ & G,-H, 500 nm.

Figure 8

Figure 8. Tunicamycin-induced ER stress causes fatty liver in wild-type larvae

(A) Elevated expression of hspa5 in hi559 liver at 4-dpf by ISH (arrow). (B) Chronic exposure of 1 μM tunicamycin from 3.5-dpf to 5.5-dpf causes globular darkish liver (yellow outline) similar to hi559. (C) hspa5 expression is ubiquitously elevated, including liver (arrow) in tunicamycin-treated larvae. (D) ORO staining shows presence of fatty liver (yellow outline) in tunicamycin-treated larvae. (E) Bar-charts indicating percentage of larvae with hepatic steatosis at 5.5-dpf in DMSO and tunicamycin (TM) treated groups. Data are representative of 3 clutches (n=55).

Similar articles

Cited by

References

    1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med. 2002;346:1221–31. - PubMed
    1. Brunt EM. Pathology of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 7:195–203. - PubMed
    1. Stuckenholz C, Ulanch PE, Bahary N. From guts to brains: using zebrafish genetics to understand the innards of organogenesis. Curr Top Dev Biol. 2005;65:47–82. - PubMed
    1. Wallace KN, Pack M. Unique and conserved aspects of gut development in zebrafish. Dev Biol. 2003;255:12–29. - PubMed
    1. Stuckenholz C, Lu L, Thakur P, Kaminski N, Bahary N. FACS-Assisted Microarray Profiling Implicates Novel Genes and Pathways in Zebrafish Gastrointestinal Tract Development. Gastroenterology. 2009 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources