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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Nonalcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation and endoplasmic reticulum (ER) stress. Because fatty acids (FAs) may trigger ER stress, we hypothesized that the absence of adipose triglyceride lipase (ATGL/PNPLA2)–the main enzyme for intracellular lipolysis, releasing FAs, and closest homolog to adiponutrin (PNPLA3) recently implicated in the pathogenesis of NAFLD–protects against hepatic ER stress. Wild-type (WT) and ATGL knockout (KO) mice were challenged with tunicamycin (TM) to induce ER stress. Serum biochemistry, hepatic TG and FA profiles, liver histology, and gene expression for markers of hepatic lipid metabolism, ER stress, and inflammation were explored. Moreover, cell-culture experiments were performed in Hepa1.6 cells after the knockdown of ATGL before FA and TM treatment. TM increased hepatic TG accumulation in ATGL KO, but not in WT, mice. Lipogenesis and β-oxidation were repressed at the gene-expression level (sterol regulatory element-binding transcription factor 1c, fatty acid synthase, acetyl coenzyme A carboxylase 2, and carnitine palmitoyltransferase 1 alpha) in both WT and ATGL KO mice. Genes for very-low-density lipoprotein (VLDL) synthesis (microsomal triglyceride transfer protein and apolipoprotein B) were down-regulated by TM in WT and even more in ATGL KO mice, which displayed strongly reduced serum VLDL cholesterol levels. Notably, ER stress markers glucose-regulated protein, C/EBP homolog protein, spliced X-box-binding protein, endoplasmic-reticulum–localized DnaJ homolog 4, and inflammatory markers Tnfα and iNos were induced exclusively in TM-treated WT, but not ATGL KO, mice. Total hepatic FA profiling revealed a higher palmitic acid/oleic acid (PA/OA) ratio in WT mice, compared to ATGL KO mice, at baseline. Phosphoinositide-3-kinase inhibitor–known to be involved in FA-derived ER stress and blocked by OA–was increased in TM-treated WT mice only. In line with this, in vitro OA protected hepatocytes from TM-induced ER stress. Conclusions: Lack of ATGL may protect from hepatic ER stress through alterations in FA composition. ATGL could constitute a new therapeutic strategy to target ER stress in NAFLD. (HEPATOLOGY 2012;56:270–280 )

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation (i.e., steatosis) and can progress to nonalcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and cancer.1, 2 As a result of the pandemic of obesity and diabetes, NAFLD has become a leading cause of liver disease in the Western world.3 As such, more than 20% of the general population4 and 75% of obese individuals5 suffer from NAFLD. Though adipose tissue has the capacity to deposit excess free fatty acids (FAs) as triglycerides (TGs) in lipid droplets, nonadipocyte cell types, such as hepatocytes, have a more limited capacity for lipid storage. When the FA-buffering capacity of a cell is exceeded, the resultant increase in FA levels can become cytotoxic in a series of events termed lipotoxicity.6 Previous studies have demonstrated hepatic endoplasmic reticulum (ER) stress in several animal models of steatosis7, 8 and human NAFLD patients,9, 10 suggesting that ER stress may be associated with lipotoxicity. In response to ER stress, three main pathways are activated, which, in turn, mediate the unfolded protein response (UPR).11 Pancreatic ER eukaryotic translational initiation factor (eIF)-2α kinase (PERK) and inositol-requiring enzyme (IRE)-1α are transmembrane kinases leading to the phosphorylation of eIF2α, which inhibits the translation and production of X-box-binding protein (XBP)-1 transcription factor by a splicing mechanism. Concomitantly, activating transcription factor (ATF)-6α, a transmembrane transcription factor released by stress, regulates intramembrane proteolysis. Each pathway activates transcriptional regulators of gene expression and contributes to the preservation of cellular integrity during ER stress.12 Constituent genes of the UPR, such as the transcription factor, XBP1,13 and the translational regulator, eIF2α,14 have also been proposed to directly regulate lipid metabolic pathways. ATF6α up-regulates chaperones, such as binding immunoglobulin protein/glucose-regulated protein (BiP/Grp78) and the ER-associated protein degradation (ERAD) machinery, and therefore protects ER function during stress.15-17 Deletion of ATF6α leads to persistent ER stress and fatty liver, providing a further mechanistic link between ER stress and hepatic lipid metabolism.16, 18

Emerging evidence suggests that elevated levels of saturated FAs may contribute to lipotoxicity and subsequently to ER stress.19 Hence, long-chain fatty acids activate ER stress in hepatocytes, and palmitic acid (PA) induces apoptosis.20-22 Moreover stearoyl–coenzyme A (CoA) desaturase 1 (SCD1) knockout (KO) mice on a low-fat diet show more pronounced ER stress.23 In human patients with NAFLD and NASH, free FAs tend to be enriched in PA and oleic acid (OA) while containing less polyunsaturated FAs.24 Monounsaturated FAs are more prone to form TGs and are well tolerated by cells. Furthermore, monounsaturated FAs are able to rescue saturated FA-induced lipotoxicity.6 Both saturated and monounsaturated FAs can derive from TG hydrolysis through intracellular lipases. Accordingly, the TG hydrolytic capacity and, possibly, the selective release of certain FA species from the TG pool may modify induction of, or protection from, ER stress. Adipose tissue triglyceride lipase (ATGL) is responsible for the initial step in TG catabolism, and ATGL deficiency provokes TG accumulation in many organs, including the liver.25, 26 Notably, ATGL (PNPLA2) is the closest homolog to adiponutrin (PNPLA3), which has recently been implicated in the pathogenesis and progression of fatty liver disease.1, 27-29 The present study was therefore designed to investigate the role of ATGL in the regulation of the acute hepatic ER stress response, and we hypothesized that lack of ATGL may protect against hepatic ER stress by reducing levels of free FAs and/or altering hepatic FA composition.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals.

Gender- and age-matched C57/BL6 wild-type (WT) and ATGL KO littermates were intraperitoneally injected with 1 mg/kg body weight of a 0.1-mg/mL suspension of tunicamycin (TM) in saline. Mice were reinjected after 24 hours. At 48 hours after the starting point, mice were killed by cervical dislocation. The experimental protocols were approved by the local animal care and use committees according to criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences (National Institutes of Health publication 86-23, revised 1985). The animals were kindly provided by Rudolf Zechner from the Institute of Molecular Biosciences at Karl-Franzens University (Graz, Austria) and were generated as described previously.26

Feeding Protocols.

Animals were fed a standard rodent chow and were housed in a controlled environment with 12-hour light-dark cycles.

Chemicals.

TM, sodium oleate, and sodium palmitate were from Sigma-Aldrich (Vienna, Austria).

Serum Parameters.

Enzymatic assays were used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholesterol (CHOL), TGs (Roche Diagnostics, Mannheim, Germany), and free FAs (Wako Chemical, Neuss, Germany). Lipoprotein subfractions were determined by quantitative agarose gel electrophoresis (Helena Biosciences, Gateshead, UK).

Liver Histology.

For conventional light microscopy, livers were fixed in 4% neutral buffered formaldehyde solution for 24 hours, embedded in paraffin, and stained with hematoxylin and eosin (H&E) or Sirius Red. Frozen tissue was embedded in Tissue Tec (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands), and sections of 2 μm were stained with Oil Red O.

Immunohistochemistry.

Double immunofluorescence staining for cleaved caspase-3 and cytokeratin 18 was performed as described previously.30

Messenger RNA Analysis and Polymerase Chain Reaction.

RNA isolation, complementary DNA synthesis, and real-time polymerase chain reaction (PCR) were performed as described previously.31 All messenger RNA (mRNA) expression data were normalized to 36b4. Oligonucleotide sequences are available upon request.

Cell-Culture Experiments.

Hepa1.6 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium and knockdown for ATGL was performed by using a lentivirus containing a short hairpin RNA against ATGL, as described in the Supporting Materials and Methods.

Western Blotting.

Hepatic nuclei were extracted with the NE-PER Nuclear and Cytoplasmic Extraction kit from Pierce Biotechnology (Rockford, IL). Srebp1c protein levels were determined using the polyclonal antibodiy Srebp-1(K10), sc-367 (Santa Cruz Biotechnology, Santa Cruz, CA).

Hepatic TG Content.

Total lipids were extracted from frozen liver tissue (100 mg) homogenates with chloroform/methanol/glacial acetic acid (66:33:1, v/v), and phase separation was achieved by the addition of water. Dried lipids were redissolved in 1% (v/v) Triton X-100, and TG content was measured using the reagent for quantitative TG measurement (DiaSys, Holzheim, Germany).

Hepatic Total FA Pool Composition: Sample Preparation and Derivatization.

Mouse liver was homogenized in 0.5 mL of phosphate buffered saline and 0.5 mL of methanol. Each sample was spiked with 375 nmol of a C15:0 FA as an internal standard immediately. Lipids were then extracted according to Bligh and Dyer. Lipid extracts were taken to dryness and resuspended in 1 mL of methanolic NaOH. After 10 minutes at 80°C and 5 minutes on ice, 1 mL of BF3 was added, followed by another 10 minutes at 80°C. FA methyl esters were extracted with 1 mL of saturated NaCl solution and 2 mL of hexane. The hexane phase was taken to dryness and redissolved in 1.5 mL of hexane.

Free FAs: Sample Preparation and Derivatization.

Gas chromatography (GC)/electron impact ionization/mass spectrometry (MS) and GC/negative ion chemical ionization/MS was performed as described in the Supporting Materials and Methods.

Statistical Analysis.

Statistical analysis was performed using SPSS V.18.0 (SPSS, Inc., Chicago, IL), using the unpaired Student's t test. Data are reported as means ± standard deviation (SD). Unless otherwise noted, animal numbers were as follows: WT control: n = 5; ATGL KO control: n = 5; WT TM treatment: n = 5; and ATGL KO TM treatment: n = 6. Cell-culture experiments were performed in triplicate. A P value ≤0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

ATGL KO Mice Show Increased Hepatic Lipid Accumulation During TM-Induced ER Stress.

Lipotoxicity is known to induce ER stress in vitro32 and in vivo.8 Therefore, we injected TM to induce ER stress in WT and ATGL KO fed mice, which have a defect in cellular TG catabolism.25, 26, 33 Serum ALT levels were not significantly increased in mice injected with TM; ALP levels were increased, whereas total CHOL, TG, and FA were decreased in both genotypes (Fig. 1A). Although serum parameters suggested that WT and ATGL KO mice challenged with TM have disturbed lipid metabolism, only ATGL KO mice showed hepatic lipid accumulation (Fig. 1B; Supporting Fig. 1A). Notably, the liver/body-weight ratio was not changed by TM treatment in both WT and ATGL KO mice (Supporting Fig. 1B). Liver histology in WT mice was not affected by TM, whereas untreated ATGL KO controls exhibited a moderate lipid infiltration, which was further pronounced by TM, as shown by H&E and Oil Red O staining (Fig. 1B; Supporting Fig. 1A). Biochemical quantification of hepatic lipid content revealed a more than 2-fold increase in hepatic TG accumulation in TM-treated ATGL KO mice, compared to TM-treated WT mice (Fig. 1C). Taken together, our data demonstrate that ATGL KO mice show elevated hepatic TG stores after induction of ER stress.

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Figure 1. ATGL KO mice display increased lipid-droplet accumulation in the liver during TM-induced ER stress. (A) TM injection increased serum ALP in both WT and ATGL KO mice, and AST was increased only in WT TM mice, whereas ALT serum levels were not affected by TM. Serum CHOL, TGs, and free FAs were significantly reduced after TM injection. (B) Hepatic steatosis appeared in ATGL KO mice 48 hours after TM injection, but not in treated WT mice (H&E staining; 20× magnification). (C) After TM injection, livers of ATGL KO mice showed a significant increase of hepatic TG. *Significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; §significant difference from TM-treated WT; P < 0.05.

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Absence of ATGL Protects Against Hepatic ER Stress and Inflammation In Vivo.

To further address the role of ATGL in the hepatic ER stress response, we checked mRNA expression levels of ER stress markers in the presence and absence of ATGL in vivo. When challenged with TM, WT, but not ATGL KO, mice showed markedly increased expression levels of spliced Xbp1 (sXbp1), reflecting ER stress (Fig. 2A). In line with this, mRNA expression of the sXbp1 downstream target, endoplasmic-reticulum–localized DnaJ homolog 4 (ERdJ4), was exclusively elevated in TM-treated WT mice. A similar expression profile was observed for Grp78–a heat shock chaperone located in the lumen of the ER that activates the UPR–and C/EBP homolog protein (Chop) as a downstream target of the integrated stress response (Fig. 2A). Notably, gene expression of inflammatory markers tumor necrosis factor alpha (Tnfα) and inducible nitric oxide synthase (iNos) in response to TM was suppressed exclusively in ATGL KO mice, whereas WT mice displayed increased gene expression (Fig. 2B). mRNA levels of collagen I α I (Col1a1) (an indicator for fibrosis) (Supporting Fig. 2B) and vascular cell adhesion protein-1 (Vcam-1) (Supporting Fig. 2C)–which is not only correlating with inflammation, but also with fibrosis–did not yet reach significant differences 48 hours after TM treatment. B-cell lymphoma 2 (Bcl-2) (an antiapoptosis marker) mRNA expression levels did not differ between untreated and treated WT mice and were even increased in TM-injected ATGL KO mice, compared to respective controls (Supporting Fig. 3B). In line with this, Sirius Red (Supporting Fig. 2A) and cytokeratin 18/caspase 3 double-immune staining revealed no changes (Supporting Fig. 3A). These data demonstrate that lack of ATGL protects mice from hepatic ER stress and the subsequent inflammatory response. Notably, kidneys of TM-treated ATGL KO mice were not protected from ER stress (Supporting Fig. 4A), whereas white adipose tissue (WAT) was not affected by TM injection in both ATGL KO and WT mice (Supporting Fig. 4B), emphasizing the specificity of the findings for liver.

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Figure 2. TM induces hepatic ER stress and hepatic inflammation in WT mice. Real-time PCR was used to assess the expression of (A) ER stress markers sXbp1, ErDj4, Grp78, and Chop and (B) hepatic inflammation markers Tnfα and iNos. In contrast to WT, ATGL KO mice had less ER stress and inflammation. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. *Significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; §significant difference from TM-treated WT; P < 0.05.

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ER Stress Reduces Circulating Very-Low-Density Lipoprotein CHOL, Very-Low-Density Lipoprotein Assembly, Endogenous FA Synthesis, and FA β-Oxidation.

Because the ER plays a central role in very-low-density lipoprotein (VLDL) metabolism, we next investigated whether VLDL and FA metabolism were affected by TM treatment. High-density lipoprotein (HDL) and VLDL CHOL serum levels were drastically reduced in TM-challenged mice (Fig. 3A). In line with the serum data, mRNA expression of microsomal triglyceride transfer protein (Mttp) and apolipoprotein B (ApoB), two key genes involved in VLDL formation, were down-regulated in TM-treated mice (Fig. 3B). Together, these findings suggest that TM treatment impaired VLDL synthesis in both WT and ATGL KO mice.

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Figure 3. TM injection in mice reduces circulating VLDL CHOL and expression of genes involved in VLDL assembly. (A) Both WT and ATGL KO mice have reduced serum levels of total CHOL, HDL, and VLDL CHOL after TM injection. (B) mRNA expression of Mttp and ApoB, key genes involved in VLDL synthesis, were measured by real-time PCR. Independently of the genotype, TM injection reduced Mttp and ApoB gene expression. mRNA expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. (A) *Significant difference from corresponding controls (Ctrl); (B) *significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; §significant difference from TM treated WT; P < 0.05.

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To explore whether differences in ER stress and hepatic steatosis after TM application might be the result of differences in de novo lipogenesis and/or FA β-oxidation, we next assessed hepatic sterol regulatory element-binding transcription factor 1c (Srebp1c) and fatty acid synthase (FasN) mRNA (Fig. 4A) as well as nuclear Srebp1c protein levels (Supporting Fig. 5) as markers for de novo lipogenesis and carnitine palmitoyltransferase 1 alpha (Cpt1α) and acetyl-coenzyme A (CoA) carboxylase 2 (Acc2) mRNA levels (Fig. 4B) as markers for β-oxidation. mRNA expression levels of Srebp1c-the master regulator of de novo lipogenesis-and its downstream target, FasN, were down-regulated after TM injection in WT and ATGL KO mice, with ATGL KO TM-injected mice having slightly higher Srebp1c expression than the WT TM-treated mice (Fig. 4A). In line with the decrease in Srebp1c mRNA levels in mice challenged with TM, the nucleic mature Srebp1c protein expression was also diminished. Both WT and ATGL KO mice challenged with TM showed low mRNA levels for Cpt1α (Fig. 4B), whereas acyl CoA oxidase mRNA levels were not changed in mice challenged with TM (data not shown). Moreover, Acc2 expression (responsible for malonyl-CoA generation potentially inhibiting Cpt1α) was similarly repressed in WT and ATGL KO mice after TM injection. These findings demonstrate that de novo lipogenesis and FA β-oxidation cannot explain the differences in hepatic lipid accumulation and ER stress.

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Figure 4. TM injection in mice reduces de novo lipogenesis and β-oxidation. Real-time PCR was used to measure the expression of (A) markers for de novo lipogenesis (e.g., Srebp1c and FasN) and (B) β-oxidation (Cpt1α and Acc2). Srebp1c, FasN, Cpt1α, and Acc2 gene expression was down-regulated as a result of TM challenging. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. *Significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; §significant difference from TM-treated WT; P < 0.05.

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Next, we explored gene-expression levels of key players involved in hepatic TG synthesis: acylglycerol-3-phosphate O-acyltransferase 9 (Agpat9; also known as Gpat3) and acylglycerol-3-phosphate O-acyltransferase 3 (Agpat3; also known as Lpaat). mRNA expression levels of these genes (Fig. 5) were not increased in WT mice upon TM treatment, whereas TM-treated ATGL KO mice showed a marked increase in the expression of Agpat9 (Gpat3) (8-fold) and Agpat3 (Lpaat) (2.5-fold), compared to untreated ATGL KO mice. Collectively, these findings suggest that an increase in hepatic TG formation in ATGL KO mice challenged with TM may be involved in protection against the induction of ER stress.

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Figure 5. TM injection up-regulates TG formation in ATGL KO TM-challenged mice. Real-time PCR was used to measure the expression of markers for TG formation (e.g., Agpat9/Gpat3 and Agpat3/Lpaat). Acyltransferase gene expressions were increased in ATGL KO, but not in WT, mice injected with TM. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. *Significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; P < 0.05.

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Defective Hepatic FA and TG Metabolism in WT and ATGL KO Mice Under ER Stress.

Because TM-injected mice exhibited selective fat accumulation in ATGL KO (but not WT) livers, we next addressed the effect of TM treatment on serum and hepatic FA species and their potential role in ER stress induction or protection by measuring free serum as well as total and free hepatic FA composition in nonfasted mice (Supporting Fig. 6; Fig. 6A; Supporting Table 1). Interestingly, TM treatment resulted in an increase of total hepatic PA (16:0) and OA (18:1n9) levels in both WT and ATGL KO mice. However, only untreated WT mice showed higher amounts of total PA related to OA at the baseline (Fig. 6B). In contrast, ATGL KO mice exhibited higher levels of OA before and after TM injection, reflected by a lower PA/OA ratio (as shown in Fig. 6B). In line with the changes in PA/OA ratios, Scd1-the enzyme responsible for FA desaturation-was down-regulated under TM treatment (Fig. 6C), indicating that TM-treated WT mice are not able to convert potentially lipotoxic PA into nontoxic-or even protective-OA; in contrast, ATGL KO mice exposed to TM might have been protected by their higher basal amount of OA from PA-induced ER stress.

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Figure 6. Defective hepatic FA and TG metabolism in WT and ATGL KO mice under ER stress conditions. (A and B) Total hepatic FA composition was measured from nonfasted mice by MS, as described in Materials and Methods. (A) Total OA (18:1n9) was significantly higher in ATGL KO mice and increased further after TM injection. (B) WT mice have more total PA (16:0) than OA at the baseline, as shown by the PA/OA ratio. (C and D) Real-time PCR was used to measure the expression of Scd1 and Pik3ip1. Scd1 expression was reduced after TM injection, but slightly more in ATGL KO mice. Pik3ip1 levels were increased in WT TM-injected mice only. mRNA expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. (A) $Significant difference from PA levels of the same group; (A, C, and D) *significant difference from untreated WT controls (Ctrl); #significant difference from ATGL KO Ctrl; §significant difference from TM-treated WT; (B) $significant differences from OA/PA ratio in WT controls (Ctrl); P < 0.05.

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In line with our hypothesis, phosphoinositide-3-kinase inhibitor 1 (Pik3ip1) mRNA was up-regulated in WT, but not in ATGL KO, mice subjected to TM (Fig. 6D). Pik3ip1 expression is induced by PA in vitro34 and plays an essential role in PA-induced ER stress. Taken together, these data suggest that saturated FAs, as with PA, are able to induce hepatic ER stress through activation of the Pi3K inhibitor in WT mice. Conversely, high hepatic OA contents may counteract Pik3ip1 activation and therefore prevent ER stress induction in ATGL KO mice.

OA Protects Against TM-Induced Hepatic ER Stress.

To further test whether the absence of ATGL has a direct protective function against TM-induced ER stress, we knocked down ATGL in mouse hepatocytes (Hepa1.6 ATGL KD) by 50% (Fig. 7A) and subsequently treated them with TM (Fig. 7B). No significant differences in mRNA expression levels of ER stress markers Grp78, Chop (data not shown), sXbp1, and ErDj4 were observed under baseline conditions (Fig. 7B). ATGL knockdown markedly attenuated the induction of ER stress markers in response to TM (Fig. 7B). To test whether monounsaturated OA (accumulating in vivo in ATGL-deficient mice) is able to protect Hepa1.6 ATGL KD cells against TM- and/or PA-induced ER stress, we cotreated these cells with OA, PA, and TM (Fig. 7C). Cells treated with PA and TM showed increased expression levels of ER stress markers Chop, sXbp1, and ErDj4, compared to untreated cells, whereas ER stress-marker expression levels in cells treated with OA did not differ from controls (Fig. 7C). Notably, cells treated with similar amounts of OA and PA did not show an increase in mRNA expression levels of ER stress markers after TM exposure (Fig. 7C), further demonstrating the protective role of monounsaturated OA against PA-induced lipotoxicity and ER stress. Collectively, these data demonstrate that increased cellular concentrations of nonesterified OA in hepatocytes are able to protect from TM-induced hepatic ER stress by interfering with Pik3ip1 expression.

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Figure 7. OA protects against TM-induced hepatic ER stress. mRNA expression levels of markers for ER stress were assessed by real-time PCR in Hepa1.6 cells and Hepa1.6 cells knocked down for ATGL (Hepa1.6 ATGL KD). (A) ATGL mRNA expression levels were repressed in Hepa1.6 ATGL KD cells by 50%. (B) Hepa1.6 cells and Hepa1.6 ATGL KD were treated with TM for 7 hours. Cells exposed to TM showed induction in sXbp1 and ErDj4 mRNA expression levels, but to a greater extent in Hepa1.6 cells, compared to Hepa1.6 ATGL KD cells. (C) Hepa1.6 ATGL KD were treated with OA and/or PA for 24 hours and cotreated with TM for 7 hours. Cells exposed to OA and PA with and without TM did not show an increase in mRNA expression levels of ER stress markers, compared to PA- or TM-treated cells. mRNA expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged Hepa1.6 cells. (A and B) *Significant difference from Hepa1.6 control cells (Ctrl); #significant difference from Hepa1.6 ATGL KD control cells (Ctrl); §significant difference from TM-treated Hepa1.6 cells; (C) *significant difference from Hepa1.6 ATGL KD control cells (Ctrl); #significant difference from TM-treated Hepa1.6 ATGL KD cells; $significant difference from PA-treated Hepa1.6 ATGL KD cells; P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we explored the effect of ATGL (PNPLA2) in the development of acute hepatic ER stress. The antibiotic, TM, is a widely used experimental tool to induce ER stress by inhibition of GlcNAc phosphotransferase, the main enzyme in the first step of glycoprotein synthesis, resulting in the induction of the UPR. Notably, the absence of ATGL in KO mice in vivo and silencing of ATGL in hepatocytes in vitro protected from TM-induced hepatic ER stress and inflammation through alterations of potentially lipotoxic FA profiles and subsequent downstream modulation of Pik3ip1 (Fig. 8).

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Figure 8. Proposed mechanism for the protection of TM-induced hepatic ER stress of ATGL KO mice, in contrast to ER stress in treated WT mice. Monounsaturated FAs, such as OA, are efficiently incorporated into TG, therefore protecting from ER stress. In contrast to OA, unsaturated FAs, such as PA, are not easily stored as TG, and free PA leads to ER stress. The first step of TG formation is catalyzed by Gpat3/Agpat9, which converts glycerol-3-phosphate (G3P) into lysophosphatidic acid (LPA). LPA is then converted into phosphatidic acid (PhA) by Lpaat/Agpat3, and subsequently TGs are formed. In ATGL KO mice under ER stress conditions, OA-rich TGs were formed. In WT mice under TM, TG formation was prevented by high PA levels, therefore inducing Pik3ip1 that, in turn, increased ER stress, leading to activation of heat shock chaperones (e.g., Grp78) and integrated ER stress response (e.g., sXbp1). Moreover, WT mice challenged with TM showed increased inflammation. Both WT and ATGL KO mice treated with TM mice lowered FA synthesis and decreased β-oxidation and VLDL synthesis (open arrows indicate transcriptional up-regulation).

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Serum markers for liver damage (e.g., ALT, AST, and ALP) and lipid parameters (e.g., CHOL, TG, and FA) were comparable in WT and ATGL KO mice upon TM treatment (Fig. 1A). However, hepatic inflammation markers were significantly increased in WT mice challenged with TM, compared to ATGL KO TM mice (Figs. 2B and 8), indicating a role for ATGL in protection from potentially harmful consequences of ER stress in response to TM.

mRNA expression levels for markers of de novo lipogenesis (Fig. 4A) and β-oxidation (Fig. 4B) were down-regulated in both genotypes challenged with TM. Repression of genes involved in FA synthesis was in line with decreased nuclear Srebp1c protein levels in mice exposed to TM (Supporting Fig. 5) and a pronounced reduction of Mttp and ApoB mRNA expression levels (Fig. 3B). Furthermore, we could show that key players in hepatic TG formation, such as Agpat9 (Gpat3) and Agpat3 (Lpaat), were up-regulated exclusively in ATGL KO TM-challenged mice (Fig. 5), suggesting that TG formation could have a protective role against hepatic ER stress (Fig. 8). Increased accumulation of hepatic lipids in ATGL KO mice 48 hours after TM injection is consistent with this hypothesis. Through FA profiling, we could further demonstrate that ATGL KO mice had higher levels of total hepatic OA-an “antilipotoxic” monounsaturated FA-independent of TM treatment, whereas untreated as well as treated WT mice contained more total hepatic PA than OA (Fig. 6A,B). Moreover, the high serum levels of free PA that were observed in the WT TM-challenged mice (Supporting Fig. 6) are consistent with higher hepatic PA levels in these mice. Listenberger et al.6 and our in vitro studies (Fig. 7) showed that (at least an equal concentration of) OA (related to PA concentration) is able to protect against PA-induced toxicity. Together, these factors suggest that the higher concentration of total OA in the ATGL KO mice, compared to total PA concentration, could be able to rescue these mice from PA-induced hepatic ER stress. In addition, the low levels of free hepatic LA (Supporting Table 1), which has a proinflammatory effect, in ATGL KO TM mice, compared to treated WT mice, are further in line with protection against inflammation, as reflected by reduced levels of respective mRNA markers (e.g., Tnfα and iNOS; Fig. 2B).

The increase in OA after TM injection in ATGL KO mice (Fig. 6A) was unexpected, because Scd1, the central enzyme in PA, and stearate desaturation to monounsaturated FA,35 was down-regulated during ER stress (Fig. 6C). OA is the preferential substrate for glycerol esterification, TG synthesis, and lipid-droplet formation in the liver. Therefore, liver OA accumulation could be a consequence of ATGL deficiency.36 This concept is supported by the low PA/OA ratio found in ATGL KO mice at baseline (Fig. 6B). ATGL may be specific for the release of certain FA species, including OA.36 We propose that cellular OA concentrations are determined by a cycle of TG hydrolysis and reesterification to TG, and that ATGL is required to release OA from the TG pool. Future studies will have to address the preference of ATGL for various FAs during hydrolysis.

Because our mouse model systemically lacks ATGL, it is difficult to differentiate from the in vivo findings whether ATGL deficiency in WAT or liver or both provided the protection against TM-induced hepatic ER stress. On the one hand, lack of ATGL in WAT reduces the FA flux from WAT to the liver,25 therefore lowering the amount of FA entering the liver. On the other hand, lack of ATGL in the liver reduces hepatic TG hydrolysis and, subsequently, the release of FA from the hepatic TG pool, thereby affecting FA levels in the liver. Tissue-specific ATGL KO mice will clarify this issue in vivo. Because such mice were not yet available at the time of our study, we addressed this question by ATGL knockdown in hepatocytes before treatment with OA or PA and TM in vitro. This set of experiments clearly showed that knockdown of ATGL and OA protected from ER stress in vitro (Fig. 7C). Therefore, it is tempting to speculate that ATGL deficiency in the WAT may protect the liver from ER stress by changing the balance between lipotoxic PA and protective OA taken up by the liver.

OA has been shown to prevent PA-induced ER stress6 through down-regulation of Pik3ip1.34 Our data indicate that the baseline amount of OA in ATGL KO mice may have a protective function against PA-derived ER stress. Thus, it is tempting to speculate that the first FA that enters the-nontoxic-TG has to be a monounsaturated FA that is, that monounsaturated FAs are the preferential substrate for Agpat9 (Gpat3), whereas Agpat3 (Lpaat) would favor saturated FAs (Fig. 8). The low free hepatic PA levels that we found in WT TM mice (Supporting Table 1) further supports the assumption that under conditions of low concentrations of monounsaturated FAs for TG synthesis, saturated FAs are not able to enter the TG and undergo the synthesis of lipid components that are supposed to be toxic.

The effect of FA-induced ER stress on lipogenesis is controversial. Though some studies demonstrated that ER stress can activate SREBPs, which are transcription factors involved in the activation of the lipogenic gene machinery,37, 38 other studies showed that ER stress down-regulates de novo lipogenesis as a result of the negative regulation of C/EBP.18 Notably, in our study, WT and ATGL KO mice had reduced mRNA and protein expression levels of Srebp1c, the master regulator of de novo lipogenesis after TM injection (Fig. 4A; Supporting Fig. 5). Interestingly, although Srebp1c mRNA was significantly down-regulated in TM-treated WT mice, compared to treated ATGL KO mice, we observed a significantly higher mRNA expression of its downstream targets, FasN and Scd1, in TM-injected WT, compared to ATGL KO, mice, suggesting that (at the mRNA level) another factor different from Srebp1c may activate the expression of genes involved in de novo lipogenesis under ER stress conditions. Recently, Lee et al.13 suggested that Xbp1 may also have a role in the regulation of de novo lipogenesis. Therefore, the higher levels of FasN and Scd1 expression in TM-treated WT mice, compared to TM-challenged ATGL KO mice, could be the result of increased sXbp1 expression.

Puri et al.39 found no activation of the ATF4/CHOP/GADD34 pathway, despite an increase in the phosphorylation of eIF-2α in NAFLD and NASH patients, indicating potential differences between our acute ER stress model and the pathogenesis of NAFLD. It appears that during progression to NASH, the ER transmembrane proteins, PERK and inositol-requiring enzyme 1-alpha, are already activated, whereas their downstream targets are still repressed. This could indicate that the integrated stress response and subsequent ER stress may be a rather late event in the progression to more advance fibrosis or cirrhosis. However, our in vitro data (Fig. 7) support that hepatic FA composition may play a major role in the development of hepatic ER stress. It is tempting to speculate that hepatocytes are able to cope with increased FA uptake, as long as balance between OA and PA is maintained, because such high levels of toxic PA could result in the up-regulation of downstream targets of the UPR, such as sXBP1, ERdJ4, and Chop.

PNPLA3 (the closest homolog to ATGL/PNPLA2) has recently been implicated in NAFLD in humans, because a missense mutation [I148M] is associated with increased steatosis and progression to NASH and fibrosis.1, 27-29 However, PNPLA3 KO mice do not show altered susceptibility to steatosis,40 which could be the result of species-specific differences in the regulation and function of PNPLA3.41 Dubuquoy et al.42 showed that Srebp1c directly regulates PNPLA3 in mice. We observed a repression of Srebp1c mRNA by TM and a trend for reduced PNPLA3 levels in TM-treated WT mice, whereas PNPLA3 expression remained preserved in ATGL KO mice after TM challenge (Supporting Fig. 7), suggesting a potential role for PNPLA3 in the rescue from ER stress in ATGL KO mice. It is tempting to speculate that under conditions with high amounts of OA-the potentially favored FA for TG formation-PNPLA3 could function as an acyltransferase,43 thus facilitating the TG formation protecting from lipotoxicity.44 Moreover, ATGL (PNPLA2) variants could also play a role for the progression of NAFLD in humans, possibly through modulation of ER stress.

In summary, our data established that WT mice exposed to ER stress are not able to form TG, as a result of low hepatic OA and high PA levels, which furthermore efficiently promotes Pik3ip1 expression and thereby increases ER stress. Conversely, we show that an enrichment of OA in the hepatic TG pool of ATGL KO mice prevents against TM-induced hepatic ER stress. Accordingly, ATGL-mediated TG hydrolysis may constitute a novel target in the treatment of ER stress, which is typically present in patients suffering from NAFLD and NASH.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Bianca Maurer from the Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine; Lusik Balayan, Christina Haas, and Sabine Paulitsch from the Clinical Institute of Medical and Chemical Laboratory Diagnostics, and Astrid Knopf from the Core Facility for Mass Spectrometry, Center for Medical Research (ZMF), all at the Medical University of Graz (Graz, Austria), as well as Nicole Bachhofner from the Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna (Vienna, Austria) for their excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25601_sm_SuppFig1.tif10228KSupplementary Figure 1: Hepatic lipid accumulation in ATGL KO TM injected mice. (A) Liver/bodyweight ratio is not changed in WT and ATGL KO mice after Tunicamycin (TM) injection, compared to their respective control groups. (B) Oil red O staining (10x magnification) showed a marked accumulation of lipids in the liver of ATGL KO TM injected mice. § indicates a significant difference from TM treated WT; P<0.05.
HEP_25601_sm_SuppFig2.tif10271KSupplementary Figure 2: 48h TM injection did not induce fibrosis. (A) Sirius red staining was performed to assess fibrosis and (B) real time PCR was used to measure the expression of Col1a1 and Vcam-1 as markers for fibrosis. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. No differences in Sirius red stainings and mRNA markers of fibrosis were seen among the groups.
HEP_25601_sm_SuppFig3.tif6830KSupplementary Figure 3: 48h TM injection did not induce apoptosis. (A) Cytokeratin 18 caspase 3 double immunostaining to assess apoptosis was negative among the groups while positive control (liver section of a mouse injected with the Fas agonist Jo2) display activated caspase 3 (red dots). (B) Real time PCR was used to measure expression of Bcl-2 as marker for apoptosis. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. Note up-regulation of the anti-apoptotic marker Bcl-2 in ATGL KO mice upon MCD treatment. # indicates a significant difference from ATGL KO Ctrl; P<0.05.
HEP_25601_sm_SuppFig4.tif324KSupplementary Figure 4: Induction of ER stress markers in kidney but not in white adipose tissue. Real time PCR was used to measure the mRNA expression of markers for ER stress (sXbp1, ERdJ4) in (A) kidney and (B) white adipose tissue (WAT). Tunicamycin (TM) treatment led to increase in mRNA expression of both, sXBP1 and ERdJ4 in kidney, but not in WAT. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. * indicates a significant difference from untreated WT controls (Ctrl); # indicates a significant difference from ATGL KO Ctrl; P<0,05. (n(WT Ctrl) = 4; n(ATGL KO Ctrl) = 4; n(WT TM) = 4; n(ATGL KO TM) = 3).
HEP_25601_sm_SuppFig5.tif508KSupplementary Figure 5: Nuclear hepatic Srebp1c protein levels. Nuclear protein expression levels of Srebp1c were assessed by western blot. Densitometry data are expressed as n-fold change related to unchallenged WT controls. * indicates a significant difference from untreated WT controls (Ctrl); # indicates a significant difference from ATGL KO Ctrl; P<0.05.
HEP_25601_sm_SuppFig6.tif299KSupplementary Figure 6: Free Fatty acid composition in serum. Free serum fatty acid composition was measured from non-fasted mice by mass spectrometry as described in M&M. Free serum palmitic acid (PA; 16:0) levels were significantly increased in WT tunicamycin (TM) treated mice, whereas ATGL KO TM mice displayed no differences in PA levels compared to untreated ATGL KO mice. Free serum oleic acid (OA; 18:1n9) was decreased due to TM treatment independent of the genotype. $ indicates a significant difference from PA levels in the same group; * indicates a significant difference from untreated WT controls (Ctrl); # indicates a significant difference from ATGL KO Ctrl; § indicates a significant difference from TM treated WT; P<0.05.
HEP_25601_sm_SuppFig7.tif189KSupplementary Figure 7: PNPLA3 mRNA expression is preserved in TM treated ATGL KO mice. mRNA expression of PNPLA3 was assessed by real time PCR. While in WT mice TM injection tended to a decrease of PNPLA3 mRNA levels, ATGL KO mice showed a preserved PNPLA3 expression after TM exposure. Expression values were normalized against 36b4 levels and are shown relative to expression level in unchallenged WT animals. § indicates a significant difference from TM treated WT; P<0.05.
HEP_25601_sm_SuppTab1.doc33KSupplementary Table 1: Hepatic free fatty acid composition in tunicamycin-treated animals in the presence and absence of ATGL
HEP_25601_sm_SuppInfo.docx15KSupporting Materials and Methods

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