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Liver Endoplasmic Reticulum Stress and Hepatosteatosis

Larry H Bernstein, MD, FCAP

 

1. Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice.

Fuchs CD, Claudel T, Kumari P, Haemmerle G, et al.
LabExpMol Hepatology, Medical Univ of Graz, Austria.
Hepatology. 2012 Jul;56(1):270-80.   http://dx.doi.org/10.1002/hep.25601. Epub 2012 May 29.

Nonalcoholic fatty liver disease (NAFLD) is characterized by

• triglyceride (TG) accumulation and
• endoplasmic reticulum (ER) stress.

Fatty acids (FAs) may trigger ER stress, therefore,

•  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-

• could protect against hepatic ER stress.

Wild-type (WT) and ATGL knockout (KO) mice

•  were challenged with tunicamycin (TM) to induce ER stress.

Markers of hepatic

•  lipid metabolism,
• ER stress, and
• inflammation were explored
  • for gene expression by
  •  serum biochemistry,
  • hepatic TG and FA profiles,
  • liver histology,
  • 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.

ER stress markers were induced exclusively in TM-treated WT, but not ATGL KO, mice:

•  glucose-regulated protein,
• C/EBP homolog protein,
• spliced X-box-binding protein,
• endoplasmic-reticulum-localized DnaJ homolog 4, and
• inflammatory markers Tnfα and iNos.

Total hepatic FA profiling revealed a higher palmitic acid/oleic acid (PA/OA) ratio in WT mice.
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. 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.
PMID: 22271167 Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92.
http://dx.doi.org/10.1111/j.1463-1326.2010.01275.x.

2. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.
Ferré P, Foufelle F. INSERM, and Université Pierre et Marie Curie-Paris, Paris, France.    PMID: 21029304

Excessive availability of plasma fatty acids and lipid synthesis from glucose (lipogenesis) are important determinants of steatosis.
Lipogenesis is an insulin- and glucose-dependent process that is under the control of specific transcription factors,

•  sterol regulatory element binding protein 1c (SREBP-1c), activated by
• insulin and carbohydrate response element binding protein (ChREBP) activated by glucose.

Insulin induces the maturation of SREBP-1c in the endoplasmic reticulum (ER).

• SREBP-1c in turn activates glycolytic gene expression,
  • allowing glucose metabolism, and
  • lipogenic genes in conjunction with ChREBP.

Lipogenesis activation in the liver of obese markedly insulin-resistant steatotic rodents is then paradoxical.
It appears the activation of SREBP-1c and thus of lipogenesis is

•  secondary in the steatotic liver to an ER stress.

The ER stress activates the

•  cleavage of SREBP-1c independent of insulin,
• explaining the paradoxical stimulation of lipogenesis
• in an insulin-resistant liver.

Inhibition of the ER stress in obese rodents

•  decreases SREBP-1c activation and lipogenesis and
• improves markedly hepatic steatosis and insulin sensitivity.
• ER is thus worth considering as a potential therapeutic target for steatosis and metabolic syndrome.

3. SREBP-1c transcription factor and lipid homeostasis: clinical perspective
Ferré P, Foufelle F
Inserm, Centre de Recherches Biomédicales des Cordeliers, Paris, France.
Horm Res. 2007;68(2):72-82. Epub 2007 Mar 5. PMID:17344645

Insulin has long-term effects on glucose and lipid metabolism through its control on the expression of specific genes.
In insulin sensitive tissues and particularly in the liver,

•  the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) transduces the insulin signal, which is
• synthetized as a precursor in the membranes of the endoplasmic reticulum
• which requires post-translational modification to yield its transcriptionally active nuclear form.

Insulin activates the transcription and the proteolytic maturation of SREBP-1c, which induces the

•  expression of a family of genes
• involved in glucose utilization and fatty acid synthesis and
• can be considered as a thrifty gene.

Since a high lipid availability is

•  deleterious for insulin sensitivity and secretion,
• a role for SREBP-1c in dyslipidaemia and type 2 diabetes
• has been considered in genetic studies.

SREBP-1c could also participate in

•  hepatic steatosis observed in humans
• related to alcohol consumption and
• hyperhomocysteinemia
• concomitant with a ER-stress and
• insulin-independent SREBP-1c activation.

4. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c
Ferré P, Foufelle F
INSERM, Centre de Recherches des Cordeliers and Université Pierre et Marie Curie-Paris, Paris, France.
Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92. PMID: 21029304
http://dx.doiorg/10.1111/j.1463-1326.2010.01275.x.

Lipogenesis in liver steatosis is

•  an insulin- and glucose-dependent process
• under the control of specific transcription factors,
• sterol regulatory element binding protein 1c (SREBP-1c),
• activated by insulin and carbohydrate response element binding protein (ChREBP)

Insulin induces the maturation of SREBP-1c in the endoplasmic reticulum (ER).
SREBP-1c in turn activates glycolytic gene expression, allowing –

•  glucose metabolism in conjunction with ChREBP.

activation of SREBP-1c and lipogenesis is secondary in the steatotic liver to ER stress, which

•  activates the cleavage of SREBP-1c independent of insulin,
• explaining the stimulation of lipogenesis in an insulin-resistant liver.
• Inhibition of the ER stress in obese rodents decreases SREBP-1c activation and improves
• hepatic steatosis and insulin sensitivity.

ER is thus a new partner in steatosis and metabolic syndrome

5. Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model
Jin-Sook Leea, Ze Zhenga, R Mendeza, Seung-Wook Hac, et al.
Wayne State University SOM, Detroit, MI
Toxicology Letters 20 May 2012; 211(1):29–38      http://dx.doi.org/10.1016/j.toxlet.2012.02.017

Endoplasmic reticulum (ER) stress refers to a condition of

•  accumulation of unfolded or misfolded proteins in the ER lumen, which is known to
• activate an intracellular stress signaling termed
• Unfolded Protein Response (UPR).

A number of pharmacologic reagents or pathophysiologic stimuli

•  can induce ER stress and activation of the UPR signaling,
• leading to alteration of cell physiology that is
• associated with the initiation and progression of a variety of diseases.

Non-alcoholic steatohepatitis (NASH), characterized by hepatic steatosis and inflammation, has been considered the
precursor or the hepatic manifestation of metabolic disease. In this study, we delineated the

• toxic effect and molecular basis
• by which pharmacologic ER stress,
• induced by a bacterial nucleoside antibiotic tunicamycin (TM),
• promotes NASH in an animal model.

Mice of C57BL/6J strain background were challenged with pharmacologic ER stress by intraperitoneal injection of TM. Upon TM injection,

•  mice exhibited a quick NASH state characterized by
• hepatic steatosis and inflammation.

TM-treated mice exhibited an increase in –

•  hepatic triglycerides (TG) and a –
• decrease in plasma lipids, including
• plasma TG,
• plasma cholesterol,
• high-density lipoprotein (HDL), and
• low-density lipoprotein (LDL),

In response to TM challenge,

•  cleavage of sterol responsive binding protein (SREBP)-1a and SREBP-1c,
•  the key trans-activators for lipid and sterol biosynthesis,
• was dramatically increased in the liver.

Consistent with the hepatic steatosis phenotype, expression of

•  some key regulators and enzymes in de novo lipogenesis and lipid droplet formation was up-regulated,
• while expression of those involved in lipolysis and fatty acid oxidation was down-regulated
• in the liver of mice challenged with TM.

TM treatment also increased phosphorylation of NF-κB inhibitors (IκB),

•  leading to the activation of NF-κB-mediated inflammatory pathway in the liver.

Our study not only confirmed that pharmacologic ER stress is a strong “hit” that triggers NASH, but also demonstrated

•  crucial molecular links between ER stress,
• lipid metabolism, and
• inflammation in the liver in vivo.

Highlights
► Pharmacologic ER stress induced by tunicamycin (TM) induces a quick NASH state in vivo.
► TM leads to dramatic increase in cleavage of sterol regulatory element-binding protein in the liver.
► TM up-regulates lipogenic genes, but down-regulates the genes in lipolysis and FA oxidation.
► TM activates NF-κB and expression of genes encoding pro-inflammatory cytokines in the liver.
Abbreviations
ER, endoplasmic reticulum; TM, tunicamycin; NASH, non-alcoholic steatohepatitis; NAFLD,
non-alcoholic fatty liver disease; TG, triglycerides; SREBP, sterol responsive binding protein;
NF-κB, activation of nuclear factor-kappa B; IκB, NF-κB inhibitor
Keywords: ER stress; Non-alcoholic steatohepatitis; Tunicamycin; Lipid metabolism; Hepatic inflammation
Figures and tables from this article:

Fig. 1. TM challenge alters lipid profiles and causes hepatic steatosis in mice. (A) Quantitative real-time RT-PCR analysis of liver mRNA isolated from mice challenged with TM or vehicle control. Total liver mRNA was isolated at 8 h or 30 h after injection with vehicle or TM (2 μg/g body weight) for real-time RT-PCR analysis. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. Each bar denotes the mean ± SEM (n = 4 mice per group); **P < 0.01. Edem1, ER degradation enhancing, mannosidase alpha-like 1. (B) Oil-red O staining of lipid droplets in the livers of the mice challenged with TM or vehicle control (magnification: 200×). (C) Levels of TG in the liver tissues of the mice challenged with TM or vehicle control. (D) Levels of plasma lipids in the mice challenged with TM or vehicle control. TG, triglycerides; TC, total plasma cholesterol; HDL, high-density lipoproteins; VLDL/LDL, very low and low density lipoproteins. For C and D, each bar denotes mean ± SEM (n = 4 mice per group); *P < 0.05; **P < 0.01.

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Fig. 2. TM challenge leads to a quick NASH state in mice. (A) Histological examination of liver tissue sections of the mice challenged with TM (2 μg/g body weight) or vehicle control. Upper panel, hematoxylin–eosin (H&E) staining of liver tissue sections; the lower panel, Sirius staining of collagen deposition of liver tissue sections (magnification: 200×). (B) Histological scoring for NASH activities in the livers of the mice treated with TM or vehicle control. The grade scores were calculated based on the scores of steatosis, hepatocyte ballooning, lobular and portal inflammation, and Mallory bodies. The stage scores were based on the liver fibrosis. Number of mice examined is given in parentheses. Mean ± SEM values are shown. P-values were calculated by Mann–Whitney U-test.

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Fig. 3. TM challenge significantly increases levels of cleaved/activated forms of SREBP1a and SREBP1c in the liver. Western blot analysis of protein levels of SREBP1a (A) and SREBP1c (B) in the liver tissues from the mice challenged with TM (2 μg/g body weight) or vehicle control. Levels of GAPDH were included as internal controls. For A and B, the values below the gels represent the ratios of mature/cleaved SREBP signal intensities to that of SREBP precursors. The graph beside the images showed the ratios of mature/cleaved SREBP to precursor SREBP in the liver of mice challenged with TM or vehicle. The protein signal intensities shown by Western blot analysis were quantified by NIH imageJ software. Each bar represents the mean ± SEM (n = 3 mice per group); **P < 0.01. SREBP-p, SREBP precursor; SREBP-m, mature/cleaved SREBP.

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Fig. 4. TM challenge up-regulates expression of genes involved in lipogenesis but down-regulates expression of genes involved in lipolysis and FA oxidation. Quantitative real-time RT-PCR analysis of liver mRNAs isolated from the mice challenged with TM (2 μg/g body weight) or vehicle control, which encode regulators or enzymes in: (A) de novo lipogenesis: PGC1α, PGC1β, DGAT1 and DGAT2; (B) lipid droplet production: ADRP, FIT2, and FSP27; (C) lipolysis: ApoC2, Acox1, and LSR; and (D) FA oxidation: PPARα. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. Each bar denotes the mean ± SEM (n = 4 mice per group); **P < 0.01. (E and F) Isotope tracing analysis of hepatic de novo lipogenesis. Huh7 cells were incubated with [1-14C] acetic acid for 6 h (E) or 12 h (F) in the presence or absence of TM (20 μg/ml). The rates of de novo lipogenesis were quantified by determining the amounts of [1-14C]-labeled acetic acid incorporated into total cellular lipids after normalization to cell numbers.

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Fig. 5. TM activates the inflammatory pathway through NF-κB, but not JNK, in the liver. Western blot analysis of phosphorylated Iκ-B, total Iκ-B, phosphorylated JNK, and total JNK in the liver tissues from the mice challenged with TM (2 μg/g body weight) or vehicle control. Levels of GAPDH were included as internal controls. The values below the gels represent the ratios of phosphorylated protein signal intensities to that of total proteins.

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Fig. 6. TM induces expression of pro-inflammatory cytokines and acute-phase responsive proteins in the liver. Quantitative real-time RT-PCR analyses of liver mRNAs isolated from the mice challenged with TM (2 μg/g body weight) or vehicle control, which encode: (A) pro-inflammatory cytokine TNFα and IL6; and (B) acute-phase protein SAP and SAA3. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. (C–E) ELISA analyses of serum levels of TNFα, IL6, and SAP in the mice challenged with TM or vehicle control for 8 h ELISA. Each bar denotes the mean ± SEM (n = 4 mice per group); *P < 0.05, **P < 0.01.

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Corresponding author at: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201, USA. Tel.: +1 313 577 2669; fax: +1 313 577 5218.

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