Biochim Biophys Acta. 2014 Oct;1842(10):1513-24. doi: 10.1016/j.bbalip.2014.07.017.

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver.

 

Asimakopoulou A1, Borkham-Kamphorst E1, Henning M1, Yagmur E2, Gassler N3, Liedtke C4, Berger T5, Mak TW6, Weiskirchen R7.
  • 1Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Aachen, Germany.
  • 2MVZ Medical Laboratory Center, Dr. Stein and Partner, Mönchengladbach, Germany.
  • 3Institute of Pathology, RWTH University Hospital Aachen, Aachen, Germany.
  • 4Department of Internal Medicine III, RWTH University Hospital Aachen, Aachen, Germany.
  • 5The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON, Canada.
  • 6The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON, Canada; Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.
  • 7Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Aachen, Germany. Electronic address: rweiskirchen@ukaachen.de.

 

Abstract

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2’s functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2(-/-)) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2(-/-) mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2(-/-) primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.

KEYWORDS: Fatty liver; Hepatocyte; Lipid droplet; MCD diet; NGAL; PPAR-γ

PMID: 25086218

 

Supplement:

Lipocalin-2 (LCN2), also known as neutrophil gelatinase associated lipocalin (NGAL) or 24p3, belongs to the widespread group of transport proteins for small hydrophobic molecules. Mice knock out studies have shown that this lipocalin is involved in innative immunity and has the capacity to limit bacterial growth by iron sequestration and binding to bacterial siderophores (1). Moreover, it was proposed that LCN2 deficiency during dietary-induced obesity in mice protects from developing aging- and obesity-induced insulin resistance largely by modulating 12-lipoxygenase and TNF-α levels in adipose tissue (2). However, at the same time the accumulation of lipid peroxidation products was significantly attenuated in the adipose tissues of respective mice (2). Therefore, it is suggested that LCN2 plays a causal role in the development of insulin resistance through modulating the inflammatory responses in adipose tissue.

During the last years my laboratory has investigated LCN2 expression and function in hepatic inflammation. We found that (i) LCN2 expression correlates with hepatic damage and inflammation, (ii) hepatocytes are the major source of hepatic LCN2, (iii) LCN2 expression in hepatic inflammation is triggered by IL-1β through the NF-κB pathway, and finally (iv) increased LCN2 expression is an intrinsic hepatoprotective “HELP-me” signal that upon hepatic injury, develops an activity necessary to recruit inflammatory cells into the damaged tissue (3-5).

 

Figure 1

Figure 1: LCN2 in the methionine and choline deficient diet. (A) LCN2 mainly consists of an eight-stranded antiparallel β-barrel forming a cavity in which hydrophobic substances can be incorporated. (B) The amino acid methionine and the quaternary ammonium salt choline are essential nutrients that mice must consume through their diet to remain healthy. (C) Wild type and LCN2 deficient mice were subjected to a MCD diet for 4 weeks. The liver histology was comparatively analysed in H & E and Oil Red O stains to highlight the presence of fat droplets.

 

In the present project we extended our work and analyzed the biological significance of LCN2 in another dietary hepatic disease model. We fed mice with a methionine and choline deficient (MCD) diet for 4 weeks. Importantly, this diet contains 10% corn-oil enriched in mono- and polyunsaturated fatty acids representing important target structures for lipid oxidation. This nutritional regimen provokes strong hepatic steatosis that is characterized by abnormal retention of lipids in the tissue due to impairment of mitochondrial β-oxidation and hampered VLDL production. However, in contrast to other disease models, mice that are subjected to this dietary treatment lose weight, are not obese, and show no signs of insulin resistance.

When we comparatively analysed MCD-fed wild type and LCN2-deficient mice, we found that the intrahepatic lipid accumulation and lipid droplet formation was significantly reduced in mice that lacked LCN2 (Figure 1). Moreover, the basal expression of perilipin 5 (PLIN5) that is involved in cellular lipid metabolism and lipid droplet formation was significantly reduced in these mice suggesting that LCN2 has a key role in stimulating PLIN5 expression. To confirm the close connection of LCN2 and PLIN5, we isolated primary hepatocytes from both genotypes and overexpressed LCN2 in respective cells, either by adenoviral expression technology or by conventional cell transfection methods. In line with our assumption, we found that the transient overexpression of LCN2 normalized cellular PLIN5 expression and resulted in the formation of more distinct fat droplets (Figure 2).

 

 

BW Figure 2

Figure 2: LCN2 induces PLIN5 expression in primary hepatocytes. (A) Primary hepatocytes isolated from LCN2 null mice were infected with the adenoviral vector Ad5-CMV-mLCN2 or left uninfected (mock). Subsequently the cells were stained with antibodies against LCN2 (green) and PLIN5 (red). Nuclei were counterstained with DAPI (blue). (B) Nile Red staining of lipid droplets in LCN2 deficient hepatocytes that were transfected with empty vector (control) or vector expressing LCN2 (pCMV-mLCN2). The microscope settings were chosen such that Nile Red was displayed in green. Nuclei were counterstained with DAPI (blue).

 

When we further stimulated hepatocytes with Rosiglitazone, we found that this PPAR agonist failed to suppress significantly LCN2 and PLIN5 expression, while the expression of adipose triglyceride lipase (ATGL) representing a PPAR-sensitive gene was strongly inhibited. This suggests that the LCN2-dependent regulation of PLIN5 in parenchymal liver cells is PPAR independent. We further found that compared to wild type mice the hepatic expression of mitochondrial marker genes (Tfam, Nrf1, CTP1-β, Cox IV, ATP5-β, and CPT1) in LCN2 null mice was significantly lowered when fed with the MCD diet, while there was a tendency that respective animals had already lower numbers of mitochondria under normal feeding conditions (Figure 3).

All these findings offer some molecular hints how LCN2 help to protect the liver against injury. In the proposed scenario, LCN2 expression becomes increased in response to stress, thereby stimulating expression of PLIN5. PLIN5 then protects the mitochondria against excessive exposure to fatty acids by oxidative lipid droplet hydrolysis and control of fatty acid flux. This lipid storage protein attempts to maintain cellular integrity by storing fatty acids transiently in the lipid droplet compartment, preventing them from further triggering pro-inflammatory pathways and formation of reactive oxygen species.

 

BW Figure 3

Figure 3: LCN2 in the control of mitochondrial marker gene expression. The hepatic expression of indicated mitochondrial marker genes was analysed by quantitative real-time PCR in livers taken from wild type and LCN2 null mice that received control or a MCD diet.

 

As mentioned above, we have previously shown that LCN2 expression is massively induced during hepatic inflammation and controlled by the NF-κB pathway in hepatocytes. We speculate that an increase in intracellular fatty acid concentration in hepatocytes may first trigger the expression of inflammatory acting cytokines and chemokines that subsequently activate the NF-κB pathway triggering LCN2 expression. Subsequently, LCN2 stimulates PLIN5 expression in concert with other factors. LCN2 (24p3) was already two decades ago shown to act as an acute phase response protein that is stimulated by TNF-α (6) underpinning the important role of LCN2 in hepatic homeostasis.

Other studies have shown that rosiglitaone treatment and PPAR-γ activation is associated with the control of lipid homeostasis and energy expenditure (7). Conversely, a deficit in PPAR-γ activity induces steatosis and reduces serum triglycerides in type 2 diabetes patients while rosiglitazone improves the atherogenic dyslipidemic profile (8,9). Although we found no association of LCN2 and PPAR-driven pathways in our study in hepatocytes, we speculate that a decrease in PPAR-γ expression associated with the loss of LCN2 function might synergistically trigger the observed hepatic steatosis and elevated serum triglycerides in hepatocytes in LCN2 null mice when fed with MCD.

To sum up, our findings demonstrate that LCN2 and PLIN5 form a regulatory network controlling lipid droplet homeostasis, lipolysis and mitochondrial balance. The key regulator role of LCN2 in hepatic lipid homeostasis is mediated, at least in part, by inducing PLIN5 expression. The findings of our study give reason to believe that the inflammation-associated LCN2/PLIN5 axis and its involvement in lipid droplet formation can be considered as a novel therapeutic target in diseases associated with fatty livers.

Acknowledgement: The author is supported by grants from the German Research Foundation (SFB TRR57) and the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (IZKF).

 

 References:

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  6. Liu Q, Nilsen-Hamilton M. Identification of a new acute phase protein. J Biol Chem. 1995;270:22565-70.
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  8. Seber S, Ucak S, Basat O, Altuntas Y. The effect of dual PPAR α/γ stimulation with combination of rosiglitazone and fenofibrate on metabolic parameters in type 2 diabetic patients. Diabetes Res Clin Pract. 2006;71:52-8.
  9. Rogue A, Anthérieu S, Vluggens A, Umbdenstock T, Claude N, de la Moureyre-Spire C, Weaver RJ, Guillouzo A. PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells. Toxicol Appl Pharmacol. 2014;276:73-81.

 

Figure 4Contact:

Ralf Weiskirchen, Ph.D

Professor and Chair

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC)

RWTH University Hospital Aachen

Pauwelsstr. 30

D-52074 Aachen, Germany

E-mail: rweiskirchen@ukaachen.de

http://www.ukaachen.de/kliniken-institute/institut-fuer-molekulare-pathobiochemie-experimentelle-gentherapie-und-klinische-chemie-ifmpegkc.html

 

 

 

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