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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2247
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 7 2877-2884
Copyright © 2008 by The Endocrine Society

The Lack of the C-Terminal Domain of Adipose Triglyceride Lipase Causes Neutral Lipid Storage Disease through Impaired Interactions with Lipid Droplets

Kunihisa Kobayashi, Toyoshi Inoguchi, Yasutaka Maeda, Naoki Nakashima, Asako Kuwano, Erina Eto, Noriko Ueno, Shuji Sasaki, Fumi Sawada, Masakazu Fujii, Yuka Matoba, Shinji Sumiyoshi, Hisaya Kawate and Ryoichi Takayanagi

Departments of Medicine and Bioregulatory Science (K.K., T.I., Y.M., A.K., E.E., N.U., S.Sa., F.S., M.F., Y.M., H.K., R.T.) and Pathology (S.Su.), Graduate School of Medical Sciences, Kyushu University, and Department of Medical Informatics (N.N.), Kyushu University Hospital, Fukuoka 812-8582, Japan

Address all correspondence and requests for reprints to: Kunihisa Kobayashi, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Japan. E-mail: nihisak{at}med.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The molecular mechanisms by which triglycerides in lipid droplets (LDs) are synthesized, stored, and degraded need to be elucidated.

Objective: The objectives were to report siblings with neutral lipid storage disease with myopathy (NLSDM) with a novel mutation of adipose triglyceride lipase (ATGL) and determine whether the C-terminal part of ATGL containing the hydrophobic region plays a role in the interaction with LDs.

Design and Patients: Skin fibroblasts and peripheral blood leukocytes were obtained from NLSDM patients. In vitro experiments were performed with fibroblasts and COS7 cells.

Main Outcome Measures: Transfection studies were used to assess the effects of various recombinant ATGL proteins on lipase activities and lipid contents. Fluorescence microscopy were used for determination of intracellular distribution of ATGL proteins.

Results: The direct sequence of ATGL cDNA reveals that a patient is a homozygote for the 4-bp deletion, leading to a premature stop codon and causes the lack of the C terminus of the protein including the hydrophobic domain. Overexpressed control ATGL in NLSDM fibroblasts was found around the rims of LDs and caused significantly reduced cellular lipid accumulation. In contrast, NLSDM ATGL was homogeneously located in the cytoplasm despite the presence of LDs and had almost no effect on LD degradation despite its similar lipase activity. A series of C-terminal truncated ATGLs without the intact hydrophobic domain failed to localize around and degrade LDs.

Conclusions: These findings indicate that the domain including the hydrophobic region of ATGL was essential for association with LDs.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Lipid droplets (LDs) are the main energy reservoir for triglycerides in eukaryotic cells. The molecular mechanisms by which triglycerides in LDs are synthesized, stored, and degraded need to be elucidated to overcome health problems such as obesity, metabolic syndrome, and type 2 diabetes mellitus that are now issues in developed countries. Neutral lipid storage diseases (NLSDs) are characterized by the presence of intracellular triglyceride deposition in most tissues, including leukocytes (Jordans’ anomaly) (1), bone marrow, skeletal muscles, heart, and the liver. Chanarin-Dorfman syndrome (CDS: MIM 27630) (2, 3) is an autosomal recessive type of NLSD with ichthyosis. Lefevre et al. (4) reported that the mutation of comparative gene identification 58 (CGI-58) (5) was a cause of CDS (4). However, some NLSD patients have also been reported without ichthyosis or mutation in the CGI-58 gene (6).

Hormone sensitive lipase has been thought to be the main lipase in LDs. However, the fact that hormone sensitive lipase-deficient mice show a nonobese phenotype and accumulation of diglycerides means that there are several other triglyceride lipases (7, 8). In 2004 three groups independently reported a new lipase; named as adipose triglyceride lipase (ATGL) (9), desnutrin (10), and calcium-independent phospholipase A2{zeta} (11), which catalyzes the initial step in triglyceride hydrolysis.

Lass et al. (12) demonstrated that the triglyceride hydrolase activity of ATGL was activated by CGI-58 up to 20-fold. Smirnova et al. (13) showed that ATGL was located around LDs with a tail-interacting protein of 47 kDa, an LD-associated protein, and in nonadipocyte HeLa cells. Its overexpression caused a marked decrease in lipid droplet size, whereas short interfering RNA-induced knockdown resulted in an increase in size. They also showed an ATGL mutant lacking S47 in the phospholipase domain that reduces lipase activity existed around LDs but failed to decrease LD size. In addition, Haememerle et al. (14) reported that ATGL-depleted mice showed triglyceride deposition in most tissues. Their data suggested that ATGL is another causative gene for NLSDs. Recently Fischer et al. (15) reported a NLSD subgroup characterized by mild myopathy, an absence of ichthyosis, and mutations in both the alleles of ATGL [NLSD with myopathy (NLSDM)]. Three of these mutations were predicted to lead to a truncated ATGL protein with a patatin-like phospholipase domain and without the C-terminal domain including hydrophobic amino acid-rich region (residues 309–391). They showed that normal whole-cell lipase activity but low lipid droplet-associated lipase activity in NLSDM fibroblasts. These results suggest that the mutant ATGL they used for experiments keeps lipase activity but impairs the interaction with LDs. However, the detailed role of the C-terminal region of ATGL remains to be elucidated.

Here we report two NLSDM siblings that have a novel homozygous ATGL mutation together with clinical investigations and show that the C-terminal part of ATGL containing the hydrophobic region has an important role in localizing around LDs.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Sequencing of ATGL cDNA of the patient

Both patients signed informed consent forms for this study, which was approved by the Ethics Review Committee of Kyushu University. Total RNA samples were prepared from the patient’s fibroblasts and white blood cells from a control individual using Isogen (Nippon Gene, Tokyo, Japan). Oligo dT primer-primed cDNA was made from total RNA with a Superscript first-strand synthesis system for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA). These cDNAs were used as templates for PCR. The primers used were based on the human patatin-like phospholipase domain-containing protein 2 nucleotide sequence (GenBank accession no. NM 020376) and were as follows: 5'-AGCGAGCGAGCGGCGAGCAG-3' and 5'-GGCGTCTCAGGCAGGGTTCC-3'. PCRs were carried out in a 50-µl volume containing 5% dimethylsulfoxide using KOD-Plus-version 2 (Toyobo, Osaka, Japan): the initial denaturation step was performed at 94 C for 2 min, followed by 35 cycles of 10 sec at 98 C, 30 sec at 64 C, 1.5 min at 68 C, and a 5-min terminal elongation step. The nucleotide sequences of the PCR products were determined using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Tokyo, Japan). For subcloning to expression plasmids, the sequences containing the complete open reading frame of human ATGL were amplified by PCR. The primers were designed to create endonuclease cleavage sites (underlined): human ATGL forward 5'-CGGGATCCTTTCCCCGCGAGAAGACGTG-3', and human ATGL reverse 5'-CCCTCGAGCTCACAGCCCCAGGGCCCC-3'. The PCR fragments were digested with BamHI or XhoI and subcloned into the eukaryotic expression vector pcDNA4/HisMax C (Invitrogen). A pcDNA4/HisMax vector with β-galactosidase (LacZ) was provided by the manufacturer. After confirming the sequences, plasmids were prepared for transfection using a QIAfilter plasmid maxikit (QIAGEN, Tokyo, Japan). A series of truncated control ATGLs were generated using a Kilo-sequence deletion kit (Takara BIO, Tokyo, Japan) and subcloned into the green fluorescence protein (GFP)-expression vector, pEGFP-C1 (CLONTECH, Palo Alto, CA).

Cell culture and transfection with expression vectors

Fibroblasts obtained from the patient’s tissue and Simian virus-40-transformed African green monkey kidney cell lines (COS7) were cultured with DMEM (400 mg/dl glucose; Sigma, St. Louis, MO) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM [SCAP];l-glutamine. In some experiments, cells were incubated overnight with 300 µM oleate complexed to BSA to increase the synthesis and storage of triglycerides in LDs. The patient’s fibroblasts and COS7 cells were transfected with various expression vectors using Lipofectamine LTX and Plus Reagent (Invitrogen). After transfection for 24–48 h, cells were used for experiments.

Fluorescence microscopy

For determination of GFP-ATGL fluorescence, cells were cultured on 35-mm coverslip-bottomed dishes (Magtek, Ashland, MA) and transfected with the appropriate plasmids. Fluorescence imaging of GFP-ATGL was assessed by confocal laser-scanning microscopy (TCS-SP system; Leica Microsystems, Heidelberg, Germany). Cells were imaged for GFP by excitation with the 488-nm line from an argon laser and emission viewed through a 496-to 550-nm band-pass filter. To correlate the localization of GFP-ATGL with the intracellular structure, cells were viewed with fluorescence and Oil red O staining images.

For Oil red O staining (16), cells were washed twice with PBS and then fixed by soaking in 10% formalin for 10 min at room temperature. After being washed with PBS and incubated with 60% isopropanol for 1 min, cells were stained for 20 min at room temperature in freshly diluted Oil red O (Sigma) solution (six parts of Oil red O stock and four parts of H2O; Oil red O stock solution is 0.3% Oil red O in isopropanol). The staining solution was removed and the cells washed with 60% isopropanol and then twice with PBS. Next, nuclei were stained with hematoxylin (Muto Pure Chemicals, Tokyo, Japan). Stained cells were examined under a light microscope (Nikon, Tokyo, Japan). Fluorescence imaging of GFP-ATGL and Oil red O was assessed by confocal laser-scanning microscopy. Imaging of Oil red O was provided by excitation with the 568-nm line, and the emission was viewed through a 580- to 660-nm band-pass filter.

The measurement of intracellular lipid contents

For the determination of triglyceride contents in the patient’s fibroblasts, cells were washed with PBS and total lipids were extracted with 3:2 hexan-isopropanol, brought to dryness, and solubilized in 0.1% Triton X-100. Triglyceride was measured using triglyceride E test (Wako, Osaka, Japan). Protein concentrations were determined with BCA protein assay reagent kit (Pierce, Rockford, IL) using BSA as standard.

Sample preparation and assay for lipase activity

For the preparation of total cell extracts, transfected COS7 cells were collected, washed three times with PBS, and disrupted by sonication in a buffer [0.25 M sucrose, 1 mM EDTA (pH 7.0)] containing protease inhibitor cocktails (Sigma). After centrifugation at 15,000 rpm at 4 C for 5 min, supernatants were used for the experiments. Lipase activities of various recombinant ATGL proteins with or without the CGI-58 expressing extract (100 µg protein) were determined by the methods described by Lass et al. (12) or Lehner and Verger (17). LD-associated lipase activities in transfected COS7 cells were determined by the method described by Fischer et al. (15).

Western blotting analysis

Samples were separated in 5–15% SDS-PAGE gels (Bio-Rad, Tokyo, Japan). Proteins were transferred to 0.2 µm polyvinyl difluoride membranes (Bio-Rad) in Tris/glycine buffer and the membranes probed with anti-ATGL antibody (Cell Signaling Technology, Beverly, MA) at a dilution of 1:1000 or Anti-Xpress antibody (Invitrogen) at a dilution of 1:5000. A secondary antibody (mouse antirabbit IgG-horseradish peroxidase; Amersham Biosciences, Piscataway, NJ) was used at a dilution of 1:5000. Signals were detected by a chemiluminescent reaction (ECL Plus; Amersham Biosciences).

Isolation and immunostaining of peripheral blood leukocytes

Peripheral blood leukocytes were obtained by Ficoll-Hypaque gradient centrifugation (18). Recovered leukocytes were washed in Hank’s balanced salt solution and adjusted to a final concentration of 1 x 106/ml for each cytospin preparation; 150,000 cells were distributed on each slide. Slides were then cytocentrifuged at 800 rpm for 10 min with a Shandon centrifuge (Shandon Inc., Pittsburgh, PA), air dried for 30 min, and fixed in cold acetone. In sample sections, endogenous peroxidase was inactivated with 3% hydrogen peroxide in methanol and nonspecific sites inactivated with 0.1% NaN3 and 1% BSA (Sigma) in PBS. Samples were incubated at room temperature for 1 h with anti-ATGL antibody (Cell Signaling Technology). Immunoreactivity was visualized with 3, 3'-diaminobenzidine tetrahydrochloride using Histofine Simplestain MAX-PO(R) (Nichirei Ltd., Tokyo, Japan) and the samples counterstained with hematoxylin.

Euglycemic-hyperinsulinemic clamp

The insulin-stimulated rate of glucose turnover was assessed as a glucose infusion rate with the infusion of 1.25 mU/kg·min of human insulin (Novo Nordisk, Tokyo, Japan) using an STG 22 artificial pancreas model (Nikkiso Co., Tokyo, Japan) (19). Blood glucose levels were determined every 5 min during the study, and euglycemia (5.5 mM) was maintained by infusion of 20% glucose.

Statistical analysis

Values are presented as mean + SEM. Results were analyzed with Statview Ver. 5 (SAS Institute Inc., Cary, NC) using one-way ANOVA followed by comparisons using Tukey-Kramer’s method. P < 0.05 denoted the presence of a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Case reports

A 63-yr-old female patient was the seventh child of consanguineous parents. From aged 36 yr, she became aware of muscle weakness and easy fatigability. At age 42 yr, she and her brother were diagnosed with NLSD. Her brother was frequently hospitalized with heart failure, dying of ventricular tachycardia at age 59 yr. At this time she was hospitalized for shortness of breath. She was 149 cm tall and weighed 50.3kg (body mass index 22.7 kg/m2). Physical examination revealed no ichthyosis, mild hepatomegaly, and mild generalized hypotonia. Laboratory investigations showed vacuolated leukocytes, elevated serum levels of aspartate aminotransferase (322 IU/liter), alanine aminotransferase (177 IU/liter), lactate dehydrogenase (1309 IU/liter), {gamma}-glutamyl transpeptidase (240 IU/liter), and creatinine kinase (742 U/liter). Her plasma myoglobin (170 ng/ml) and aldolase (20.2 IU/liter, 37 C) were elevated. Serum levels of lactate, pyruvate, serum carnitine fraction, and long-chain fatty acid profile were within normal ranges. Serum thyroid stimulating hormone was elevated (8.50 µU/ml), and a heterogeneous echo pattern was detected in the thyroid gland. Fasting plasma glucose was 117 mg/dl and glycated hemoglobin A1c 7.6%. Impaired insulin secretion and normal insulin resistance were suggested by oral glucose loading test (insulinogenic index: (insulin 30 min – insulin 0 min)/(glucose 30 min – glucose 0 min) = 0.23 < 0.4) and hyperinsulinemic-euglycemic clamp study [glucose infusion rate 5 mg/kg·min at insulin infusion rate 1.25 mU/kg·min; normal 4.83 ± 1.70 mg/kg·min (19)]. Urine C-peptide excretion was 69.6 µg/d. Mild fatty liver was suggested from ultrasonography and computed tomography images. Endoscopic ultrasonography showed an atrophic pancreas with heterogeneous parenchyma and no dilatation of the main duct. Brain natriuretic peptide level was markedly elevated (2891 pg/ml). Chest x-ray revealed mild cardiomegaly. Echocardiography showed left ventricular hypertrophy and low diastolic function. Her clinical course is shown in Supplement Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org. Insulin secretory capacity was assessed as {Delta}CPR (the increase of serum CPR for 5 min after 1 mg of glucagon iv injection). {Delta}CPR markedly decreased with age whereas homeostasis model assessment for insulin resistance, an insulin resistance index, stayed almost constant throughout her course.

Optical microscope images of biopsy and autopsy samples and cultured fibroblasts

Biopsy specimens from the liver (Fig. 1Go, A and B) and muscle (Fig. 1Go, C and D) of the patient showed marked triglyceride droplets. Autopsy samples of the patient’s brother also showed LDs in cardiac muscle (supplemental Fig. 1B), an atrioventricular node (supplemental Fig. 1C), pancreatic acinar cells (supplemental Fig. 1D) and islet cells (supplemental Fig. 1E), renal tubular epithelium, glomerulus (supplemental Fig. 1F), and thyroid gland (supplemental Fig. 1G). Fibroblasts from the patient grew and presented LDs in DMEM with 10% fetal bovine serum. Addition of oleic acid (300 µM) caused more and larger LDs in the cells, compared with control fibroblasts (Fig. 1EGo).


Figure 1
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  		FIG. 1. Light microscopy of formalin-fixed sections of biopsy samples from an individual with NLSDM. Biopsy samples from the patient (A–D) show typical cytosolic lipid deposition. A case report of the patient is described in Results. A, Liver; hematoxylin and eosin (H&E), bar, 100 µm. B, Liver; H&E, bar, 50 µm. C, Muscle; H&E, bar, 50 µm. D, Muscle; Sudan, bar, 50 µm. E, Fibroblasts obtained from control and an individual with NLSDM were cultured in DMEM (400 mg/dl of glucose) with 10% fetal bovine serum in the presence and absence of 300 µM oleate complexed to BSA. Oil red O staining was performed as described in Patients and Methods. Bar, 100 µm.

 
Detection of ATGL mutation in both alleles of the ATGL gene

The direct sequence of ATGL cDNA revealed that the patient was a homozygote for a 4-bp deletion (799–802delGCCC) (Fig. 2AGo), leading to a premature stop codon at amino acid position 318, which caused a lack of a C terminus of the protein including the hydrophobic domain (Fig. 2BGo). This deletion (underlined) occurred at a unique GC sequence (GCCCCCCGCCCGCCCC) of the gene, near which individuals 1 and 2 reported by Fischer et al. (15) also had heterozygous and homozygous single base pair deletions, respectively. We detected no mutation in the sequence of the CGI-58 gene, another causative gene of NLSDs (4) (data not shown).


Figure 2
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  		FIG. 2. ATGL mutation in the patient with NLSDM. The direct sequence of ATGL cDNA reveals that the patient is a homozygote for a 4-bp deletion (799–802delGCCC) (A), leading to frameshift (green) and a premature stop codon at amino acid position 318, which results in the lack of the C terminus of the protein including the hydrophobic domain (B).

 
Localization of endogenous ATGL in peripheral blood leukocytes and GFP-fused ATGL expression in NLSDM fibroblasts

Peripheral blood leukocytes from the patient showed cytoplasmic vacuoles (Jordans’ anomaly) (Fig. 3AGo) and Oil red O-positive LDs (Fig. 3BGo). Immunostaining suggested that endogenous ATGL of the patient was located in the cytoplasm homogeneously and not around the rims of LDs (Fig. 3DGo), whereas endogenous ATGL protein was not clearly detected in control leukocytes (Fig. 3EGo). We next investigated the distribution of GFP-fused control and NLSDM ATGL proteins in the patient’s fibroblasts. It has been reported that endogenous ATGL is localized around the rims of LDs (13). By contrast, localization of overexpressed ATGL is unclear, such as homogenous distribution (10) or a granular appearance (20) in the cytoplasm. In the present study, GFP-control ATGL protein was located in cytoplasm and appeared as a ring shape around LDs. By contrast, GFP-fused NLSDM ATGL was homogeneously located in the nucleus and cytoplasm, and LDs remained after transfection, suggesting that NLSDM ATGL is not capable of approaching and degrading LDs (Fig. 3FGo).


Figure 3
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  		FIG. 3. Localization of endogenous ATGL in peripheral blood leukocytes obtained from an NLSDM patient. Leukocytes from the patient’s peripheral blood show cytoplasmic lipid droplets (Jordans’ anomaly). A, Hematoxylin and eosin; bar, 5 µm. B, Oil red O; bar, 5 µm. Immunoreactivity was visualized with 3, 3'-diaminobenzidine tetrahydrochloride staining (brown) and counterstained with hematoxylin (violet-blue). C, A negative control; bar, 5 µm. D, NLSDM leukocytes; bar, 5 µm. E, Control leukocytes; bar, 5 µm. F, Overexpression of control and NLSDM ATGL is differently distributed in NLSDM fibroblasts. Fibroblasts established from the NLSDM patient were transfected with plasmids expressing GFP-control ATGL or GFP-NLSDM ATGL and assessed by confocal laser-scanning microscopy.

 
Effects of control and NLSDM ATGL expression on the lipid contents in fibroblasts and different distribution of both ATGL

To investigate the effects of the mutation on lipid degradation activity, we carried out transfection studies using COS7 cells (Fig. 4Go, A, C, and D) or fibroblasts from the patient’s skin (Fig. 4BGo). Transfection of control ATGL in NLSDM fibroblasts significantly reduced cellular lipid accumulation. Lipid contents in NLSDM ATGL-transfected cells showed no significant difference from those in β-galactosidase (LacZ)-transfected cells (Fig. 4BGo). We next investigated intracellular distribution of both control and NLSDM ATGL. In total cell extracts, NLSDM ATGL-transfected COS7 cells showed similar lipase activity to control. In addition, both ATGL activities were stimulated by CGI-58 (Fig. 4CGo). In contrast, LD-associated NLSDM ATGL activity was significantly lower than control (Fig. 4DGo).


Figure 4
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  		FIG. 4. Effect of overexpression of control or NLSDM ATGL on lipid contents and lipase activities in NLSDM fibroblasts. A, Western blotting analysis of ATGL expression in transfected NLSDM fibroblasts. Fibroblasts established from the NLSDM patient were transfected with cytomegalovirus promoter-driven expression plasmids containing control, NLSDM ATGL, and β-galactosidase (LacZ). B, Intracellular lipid contents in transfected NLSDM fibroblasts. Lipase activities in total cell extracts (C) and lipid-associated fractions (D) were determined with COS7 cells as described in Patients and Methods. Values are means ± SE (n = 4–6).

 
Effects of expression of deletion mutants of ATGL on LDs in NLSDM fibroblasts

To investigate details of the role of the C-terminal region in lipid degradation, we made a series of C-terminal deletion mutants (Fig. 5AGo) and carried out transfection studies. In total cell extracts prepared from transfected COS7 cells, deletion mutants (mutant 1–3) showed similar lipase activities, which were stimulated by CGI-58. On the contrary, mutant 4, which lacks total hydrophobic region and partial patatin-like phospholipase domain, showed almost no lipase activity or no enhancing effect of CGI-58 (Fig. 5BGo). LDs in transfected cells was diminished completely by control ATGL and mostly diminished by mutants 1 and 2, which have the intact hydrophobic domain. In contrast, the expression of mutants 3 and 4 showed no effect on LDs and significantly higher intracellular lipid contents (Fig. 6Go).


Figure 5
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  		FIG. 5. Effects of C-terminal deletion mutants on lipase activities in transfected COS7 cells. A, Predicted structures of control, NLSDM, and C-terminal truncated ATGL mutants. The red, yellow, and green boxes indicate a patatin-like phospholipase domain, a hydrophobic region, and frameshift, respectively. Numbers indicate amino acid residues of ATGL. B, Lipase activities in total cell extracts from COS7 cells transfected with various ATGL mutants with or without CGI-58. *, P < 0.05 vs. control. n.s., Not significant.

 

Figure 6
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  		FIG. 6. Hydrophobic domain-deleted mutants cause residual LDs. Control, NLSDM, and truncated ATGLs were subcloned into GFP expression plasmids. NLSDM fibroblasts were transiently transfected with recombinant plasmids and stained with Oil red O and assessed by confocal laser-scanning microscopy. Intracellular lipid contents were determined as described in Patients and Methods. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
CDS (MIM 27630) (2, 3) is an autosomal recessive form of NLSD characterized by the presence of ichthyosis and intracellular triglyceride droplets in most tissues. In 2001 Lefevre et al. (4) identified CGI-58, of the esterase/thioesterase/lipase superfamily, as a causative gene using linkage-disequilibrium analysis. CGI-58 binds to intracellular LDs together with perilipin A (21, 22) and is demonstrated to stimulate ATGL activity (12). ATGL, also known as desnutrin or calcium-independent phospholipase A2{zeta}, has been reported by three independent groups (9, 10, 11) and shown to have a pivotal role in the initial step of triglyceride hydrolysis. The N-terminal part of ATGL contains a patatin-like phospholipase domain and GXSXG consensus sequence for serine lipases. On the other hand, Brummer lipase, a Drosophila homolog of human ATGL, has a pivotal region for localizing around LDs called the Brummer box (23). Smirnova et al. (13) showed that an ATGL mutant-S47A in phospholipase domain, which lacks lipase activity, failed to decrease the LD size but still was present around the LDs. These results suggest that ATGL also contains the putative lipid-binding site. Recently Fischer et al. (15) demonstrated mutations of ATGL in a NLSD subgroup with myopathy and without ichthyosis (NLSDM). These mutations lead to a truncated ATGL protein with a patatin-like phospholipase domain but without the C-terminal region including the hydrophobic amino acid-rich domain (residues 309–391). They showed that whole-cell neutral lipase activity in NLSDM fibroblasts was the same as in control fibroblasts but that lipid droplet-associated lipase activity was eventually diminished. However, the mutated ATGL of individual 2 they used in the lipase studies lacks the C-terminal half of the protein; therefore, it is possible that the defective part other than hydrophobic region plays a role in ATGL association with LDs. In addition, it is also possible that a C-terminal nonsense sequence caused by a frameshift of this mutant ATGL inhibits the targeting of ATGL to LDs. The ATGL of our patient showed a novel mutation and lacked the C-terminal hydrophobic domain but also had a nonsense sequence at C terminus (Fig. 2BGo). In the present study, we sought to confirm that the C-terminal region of ATGL that includes the hydrophobic domain was essential for localization around LDs.

First, we checked the localization of ATGL in the patient’s leukocytes with Jordans’ anomaly. Smirnova et al. (13) reported that endogenous ATGL showed a pattern of small rings that colocalized with tail-interacting protein of 47 kDa. Contrarily, the patient’s ATGL was located homogenously in cytoplasm of peripheral blood leukocytes (Fig. 3DGo). We used a GFP fusion expression plasmid and the patient’s fibroblasts to determine the distribution of control and the patient’s mutant ATGL. GFP-control ATGL fusion protein was located ring-like around small LDs, whereas GFP-NLSDM ATGL showed homogenous distribution in the cytoplasm and residual LDs. (Fig. 3FGo). Next, we investigated the effects of the mutation on intracellular localization and lipid degradation activity. NLSDM fibroblasts transfected with NLSDM ATGL showed significantly higher lipid contents than control ATGL-transfected cells. In COS7 cells transfected with NLSDM ATGL, LD-associated lipase activities were significantly lower than control-transfected cells, whereas lipase activities in total cell extracts show no difference including enhancing effect of CGI-58. These results suggest that the patient’s ATGL was not capable of association with LDs, thereby resulting in inefficient lipid degradation. Then we generated a series of ATGL deletion mutants (Fig. 5AGo) to undertake a detailed analysis of the C-terminal part of ATGL protein without an additional nonsense sequence. Mutants 1 and 2, which have the intact hydrophobic domain, showed similar lipase activity in total cell extracts to control ATGL and trace amount of LDs. In contrast, mutant 3, which lacks partial hydrophobic domain, showed significantly higher lipase activity than control but was not capable of degrading LDs to the same extent as NLSDM ATGL, and mutant 4, which lacks complete hydrophobic domain and partial lipase domain (Figs. 5BGo and 6Go). These results suggested that the C-terminal part of ATGL, in particular the hydrophobic region, was essential for association with LDs. Granneman et al. (24) proposed a working model of lipolytic trafficking. ATGL is both cytoplasmic and bound to LDs in the basal state. Protein kinase A activation by stimulation causes phosphorylation of perilipin A, which frees CGI-58 to recruit and activate ATGL on the surface of LDs, thereby leading to triglyceride breakdown. According to our present study, the C-terminal hydrophobic domain of ATGL does not seem to be important for interaction with CGI-58. We then carried out an immunoprecipitation study but were not able to detect any association of control or mutant ATGL with CGI-58, maybe because of the limited detection sensitivity (data not shown). The effects of the N-terminal part of ATGL including patatin-like phospholipase domain and residues 178–267 (between the patatin-like domain and a C-terminal nonsense sequence caused by a frame shift) on its interaction with CGI-58 remain to be elucidated in detail.

The two patients reported here showed similar clinical features to those of individual 2 reported by Fischer et al. (15), such as cardiomyopathy and chronic pancreatitis besides myopathy and liver dysfunction. In addition, both had hypothyroidism. All these characteristics were probably associated with lipid deposition because LDs were seen in cardiac muscle (supplemental Fig. 1B), an atrioventricular node (supplemental Fig. 1C), and the thyroid gland (supplemental Fig. 1F). Notably, the two cases here and individual 2 had type 2 diabetes mellitus. Several investigators reported that intramyocellular lipid content is correlated with insulin resistance in humans (25, 26). However, our female patient showed no insulin resistance from the euglycemic-hyperinsulinemic clamp study and homeostasis model assessment for insulin resistance. Instead, she showed decreasing insulin secretory capacity with age (supplemental Fig. 1A). It is possible that lipid deposition in pancreatic cells (supplemental Fig. 1, D and E) caused dysfunction, thereby leading to the onset of diabetes mellitus. On the other hand, this notion is inconsistent with the finding that ATGL knockout mice showed increased glucose tolerance and increased insulin sensitivity, regardless of lipid deposition in muscle (14). The clinical significance of ATGL on glucose metabolism should be investigated and hopefully revealed in future studies.

In summary, we have identified a novel homozygous mutation of the ATGL gene in a NLSDM patient. The mutation caused a lack of the C-terminal of the protein including the hydrophobic region. A lack of the C-terminal region of ATGL leads to dissociation from LDs and lipid deposition, whereas gene supplementation diminished LDs in NLSDM fibroblasts.


   Acknowledgments
 
We thank Mikiko Sato for her research assistance. We also thank Ms. Eri Nagashima and Ms. Tamaki Amano for secretarial help.


   Footnotes
 
Disclosure Information: All authors have nothing to declare.

First Published Online April 29, 2008

Abbreviations: ATGL, adipose triglyceride lipase; CDS, Chanarin-Dorfman syndrome; CGI-58, comparative gene identification 58; GFP, green fluorescence protein; LD, lipid droplet; NLSD, neutral lipid storage disease; NLSDM, NLSD with myopathy.

Received October 9, 2007.

Accepted April 18, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Jordans GH 1953 The familial occurrence of fat containing vacuoles in the leukocytes diagnosed in two brothers suffering from dystrophia musculorum progressiva (ERB.). Acta Med Scand 145:419–423[Medline]
  2. Dorfman ML, Hershko C, Eisenberg S, Sagher F 1974 Ichthyosiform dermatosis with systemic lipidosis. Arch Dermatol 110:261–266[Abstract/Free Full Text]
  3. Chanarin I, Patel A, Slavin G, Wills EJ, Andrews TM, Stewart G 1975 Neutral-lipid storage disease: a new disorder of lipid metabolism. Br Med J 1:553–555[Free Full Text]
  4. Lefevre C, Jobard F, Caux F, Bouadjar B, Karaduman A, Heilig R, Lakhdar H, Wollenberg A, Verret JL, Weissenbach J, Ozguc M, Lathrop M, Prud'homme JF, Fischer J 2001 Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome. Am J Hum Genet 69:1002–1012[CrossRef][Medline]
  5. Lai CH, Chou CY, Ch'ang LY, Liu CS, Lin W 2000 Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. Genome Res 10:703–713[Abstract/Free Full Text]
  6. Wessalowski R, Schroten H, Neuen-Jacob E, Reichmann H, Melnik BC, Lenard HG, Voit T 1994 Multisystem triglyceride storage disorder without ichthyosis in two siblings. Acta Paediatr 83:93–98[Medline]
  7. Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E, Sattler W, Magin TM, Wagner EF, Zechner R 2002 Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem 277:4806–4815[Abstract/Free Full Text]
  8. Osuga J, Ishibashi S, Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoiri F, Yahagi N, Kraemer FB, Tsutsumi O, Yamada N 2000 Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci USA 97:787–792[Abstract/Free Full Text]
  9. Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R 2004 Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306:1383–1386[Abstract/Free Full Text]
  10. Villena JA, Roy S, Sarkadi-Nagy E, Kim KH, Sul HS 2004 Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis. J Biol Chem 279:47066–47075[Abstract/Free Full Text]
  11. Jenkins CM, Mancuso DJ, Yan W, Sims HF, Gibson B, Gross RW 2004 Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J Biol Chem 279:48968–48975[Abstract/Free Full Text]
  12. Lass A, Zimmermann R, Haemmerle G, Riederer M, Schoiswohl G, Schweiger M, Kienesberger P, Strauss JG, Gorkiewicz G, Zechner R 2006 Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome. Cell 3:309–319
  13. Smirnova E, Goldberg EB, Makarova KS, Lin L, Brown WJ, Jackson CL 2006 ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells. EMBO Rep 7:106–113[CrossRef][Medline]
  14. Haemmerle G, Lass A, Zimmermann R, Gorkiewicz G, Meyer C, Rozman J, Heldmaier G, Maier R, Theussl C, Eder S, Kratky D, Wagner EF, Klingenspor M, Hoefler G, Zechner R 2006 Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science 312:734–737[Abstract/Free Full Text]
  15. Fischer J, Lefevre C, Morava E, Mussini JM, Laforet P, Negre-Salvayre A, Lathrop M, Salvayre R 2007 The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy. Nat Genet 39:28–30[CrossRef][Medline]
  16. Imamura M, Inoguchi T, Ikuyama S, Taniguchi S, Kobayashi K, Nakashima N, Nawata H 2002 ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts. Am J Physiol Endocrinol Metab 283:E775–E783
  17. Lehner R, Verger R 1997 Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase. Biochemistry 36:1861–1868[CrossRef][Medline]
  18. Riccobon A, Gunelli R, Ridolfi R, De Paola F, Flamini E, Fiori M, Saltutti C, Petrini M, Fiammenghi L, Stefanelli M, Granato AM, Cuzzocrea DE, Amadori D 2004 Immunosuppression in renal cancer: differential expression of signal transduction molecules in tumor-infiltrating, near-tumor tissue, and peripheral blood lymphocytes. Cancer Invest 22:871–877[CrossRef][Medline]
  19. Yokoyama H, Emoto M, Mori K, Araki T, Teramura M, Koyama H, Shoji T, Inaba M, Nishizawa Y 2006 Plasma adiponectin level is associated with insulin-stimulated nonoxidative glucose disposal. J Clin Endocrinol Metab 91:290–294[Abstract/Free Full Text]
  20. Baulande S, Lasnier F, Lucas M, Pairault J 2001 Adiponutrin, a transmembrane protein corresponding to a novel dietary-and obesity-linked mRNA specifically expressed in the adipose lineage. J Biol Chem 276:33336–33344[Abstract/Free Full Text]
  21. Yamaguchi T, Omatsu N, Matsushita S, Osumi T 2004 CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome. J Biol Chem 279:30490–30497[Abstract/Free Full Text]
  22. Subramanian V, Rothenberg A, Gomez C, Cohen AW, Garcia A, Bhattacharyya S, Shapiro L, Dolios G, Wang R, Lisanti MP, Brasaemle DL 2004 Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3–L1 adipocytes. J Biol Chem 279:42062–42071[Abstract/Free Full Text]
  23. Gronke S, Mildner A, Fellert S, Tennagels N, Petry S, Muller G, Jackle H, Kuhnlein RP 2005 Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metab 1:323–330[CrossRef][Medline]
  24. Granneman JG, Moore HP, Granneman RL, Greenberg AS, Obin MS, Zhu Z 2007 Analysis of lipolytic protein trafficking and interactions in adipocytes. J Biol Chem 282:5726–5735[Abstract/Free Full Text]
  25. Perseghin G, Scifo P, De Cobelli F, Pagliato E, Battezzati A, Arcelloni C, Vanzulli A, Testolin G, Pozza G, Del Maschio A, Luzi L 1999 Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H–13C nuclear magnetic resonance spectroscopy assessment in offspring of type 2 diabetic parents. Diabetes 48:1600–1606[Abstract]
  26. Jacob S, Machann J, Rett K, Brechtel K, Volk A, Renn W, Maerker E, Matthaei S, Schick F, Claussen CD, Haring HU 1999 Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects. Diabetes 48:1113–1119[Abstract]



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