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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0075
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2279-2285
Copyright © 2006 by The Endocrine Society

Glycosylphosphatidylinositol-Specific Phospholipase D in Nonalcoholic Fatty Liver Disease: A Preliminary Study

Naga Chalasani, Raj Vuppalanchi, Nandita S. Raikwar and Mark A. Deeg

Departments of Medicine (N.C., R.V., N.S.R., M.A.D.) and Biochemistry and Molecular Biology (M.A.D.), Indiana University School of Medicine and Department of Veterans Affairs (M.A.D.), Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Mark Deeg, M.D., Ph.D., Division of Endocrinology and Metabolism, Indiana University School of Medicine, 1481 West 10th Street, Indianapolis, Indiana 46202. E-mail: mdeeg{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Recent studies demonstrated that de novo lipogenesis is increased in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD also have plasma lipid abnormalities. These lipid abnormalities may in part be related to insulin resistance, which is common in patients with NAFLD. Insulin resistance is associated with alterations in proteins involved in lipid metabolism including glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), which is involved in triglyceride metabolism.

Objective: The objective of the study was to determine whether alterations in serum and hepatic levels of GPI-PLD occur in patients with NAFLD.

Design and Patients: We examined the following: 1) levels of serum GPI-PLD in nondiabetics with nonalcoholic steatohepatitis, compared with matched controls; 2) hepatic expression of GPI-PLD mRNA in patients with normal liver or NAFLD; and 3) effect of overexpressing GPI-PLD vs. ß-galactosidase (control) on global gene expression in a human hepatoma cell line.

Results: The serum levels of GPI-PLD were significantly higher in patients with nonalcoholic steatohepatitis than in matched controls (119 ± 24 vs.105 ± 15 µg/ml, P = 0.047). The hepatic expression of GPI-PLD mRNA was increased nearly 3-fold in NAFLD patients, compared with patients with normal liver (3.1 ± 2.6 vs. 1.1 ± 1.0 arbitrary units per microgram total RNA, P = 0.026). Finally, overexpressing GPI-PLD was associated with an increase in de novo lipogenesis genes.

Conclusions: Patients with NAFLD have elevated serum levels and hepatic expression of GPI-PLD, and its overexpression in vitro is associated with increased expression of de novo lipogenesis genes. These results suggest that GPI-PLD may play a role in the pathogenesis of NAFLD and/or its metabolic features and warrants further investigation.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
NONALCOHOLIC FATTY LIVER disease (NAFLD) is the leading cause of abnormal liver functions in Western society. Histologically, NAFLD can range from fatty liver to nonalcoholic steatohepatitis (NASH) to cirrhosis and is associated with altered lipid metabolism both in the liver and plasma compartment (1, 2). Hepatic de novo lipogenesis is significantly increased in patients with NAFLD (3). Patients with NAFLD also have elevations in serum triglycerides and lower levels of high-density lipoproteins. The etiology of these lipid abnormalities may in part be related to insulin resistance, which is common in patients with NAFLD (4). Insulin resistance is associated with alterations in proteins involved in lipid metabolism including glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (5).

GPI-PLD is an 815-amino acid protein expressed in numerous tissues and cells (6, 7). Liver has the highest level of GPI-PLD expression and is the primary organ contributing to GPI-PLD in the serum. GPI-PLD is abundant in serum (approximately 100 µg protein per milliliter) in which it associates with apolipoproteins AI and AIV (8, 9, 10). Increased serum GPI-PLD is associated with insulin resistance and elevated serum triglycerides (5), and overexpressing hepatic GPI-PLD in mice is associated with increases in fasting and postprandial triglycerides (11). This effect of GPI-PLD is mediated, at least in part, by reducing the catabolism of triglyceride-rich lipoproteins. Because patients with NAFLD frequently have insulin resistance and fasting and postprandial hypertriglyceridemia (4, 12), we hypothesized that subjects with NAFLD may have abnormal hepatic and serum levels of GPI-PLD as a contributor to the altered intra- and extrahepatic lipid metabolism.

To characterize GPI-PLD in patients with NAFLD, we conducted a study with three aims. First, we compared the serum levels of GPI-PLD in nondiabetic patients with NASH to age, gender, body mass index (BMI), and body fat-matched controls. Second, we measured the hepatic expression of GPI-PLD mRNA in patients with simple fatty liver and NASH, compared with normal hepatic histology. Third, to determine whether GPI-PLD may play a role in hepatic lipid metabolism, we examined the effect of overexpressing GPI-PLD vs. ß-galactosidase (control) on global gene expression in a human hepatoma cell line (HepG2). We found that serum levels and hepatic GPI-PLD mRNA levels are significantly higher in patients with NAFLD. In addition, overexpression of GPI-PLD in HepG2 cells is associated with increases in de novo lipogenesis genes, suggesting that GPI-PLD may have a role in the altered fatty acid and lipid metabolism in NAFLD.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Aim 1

Serum GPI-PLD levels were measured in 21 nondiabetic individuals with biopsy-proven NASH and 19 age-, gender-, BMI-, and body fat-matched nondiabetic individuals without liver disease. This cohort was studied previously, and it served as the basis for several papers related to NASH published by our group (13, 14, 15, 16, 17). These studies were conducted after these publications and in accordance with the guidelines in the Declaration of Helsinki. All patients provided informed consent to participate in these studies, all of which were approved by the Institutional Review Board and the Advisory Committee for the General Clinical Research Center of Indiana University School of Medicine. Fasting serum GPI-PLD levels were measured by ELISA from stored serum specimens (–80 C).

The details of clinical and histological criteria used to characterize the subjects with NASH and the controls in this cohort have been described previously (13, 14, 15, 16, 17). The anthropometric measurements available on this cohort of patients included height, weight, waist to hip ratio, body composition (measured by BODPOD method), and abdominal fat measurements (measured by single-slice computed tomography abdomen). Serum studies available for this analysis included liver biochemistries, fasting serum lipids, oxidized low-density lipoprotein (LDL), insulin, glucose, free fatty acids, leptin, and adiponectin. Insulin resistance was assessed by the homeostasis model assessment (HOMA) method, and individuals with HOMA greater than 3.5 were considered to have insulin resistance (18). Insulin resistance measured by HOMA correlates closely with euglycemic clamp techniques within our patient population, and in a previous study from this institution, HOMA greater than 3.5 was an accurate indicator of insulin resistance (18). The assay methods for these serum studies are described in previous publications (13, 14, 15, 16, 17).

Aim 2

The GPI-PLD mRNA was measured from the cell lysate of liver biopsy material of 13 subjects with NASH, 12 subjects with nonalcoholic steatosis, and 12 subjects with normal liver tissue (who had liver biopsies performed as part of donor evaluation for living related liver transplantation). These liver samples were obtained in the fasting state and collected as part of an institutional review board-approved protocol that allowed us to maintain a liver tissue bank. The majority of these liver samples were also used previously (13, 17). Serum samples were not available from this cohort. These subjects were not part of the cohort studied to address our first aim.

Total RNA was extracted and isolated from the core biopsy specimen using a RNeasy minikit (QIAGEN, Valencia, CA). DNase treatment was done by using DNA-free (Ambion, Austin, TX) to remove contaminating DNA in the extracted total RNA. Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) combined with the RNA 6000 Nano LabChip kit (Caliper Technologies Corp., Hopkinton, MA) was used to analyze the quality (28S to 18S ratio) and quantity of total RNA. RNA was stored at –80 C until used.

TaqMan real-time quantitative RT-PCR was used to quantitate GPI-PLD mRNA. The following sets of the primers and probes were designed using the Primer Express software (Applied Biosystems, Foster City, CA) to function according to the Taqman technology. GPI-PLD-specific primers and probes used for QPCR were: HumGPI-PLD forward, 5'-CCCAGCCTGAGCAACAAAG-3', HumGPI-PLD reverse, 5'-TTCCTCGCCTCTCACCGTC-3', and HumGPI-PLD probe, 5'-FAM-AAACTGAACGTGGAGGCGGCCAAC-TEMRA-3' synthesized from Integrated DNA Technologies (Coralville, IA). The amplicon was 720 bp and was verified by agarose gel electrophoresis. The RT-PCRs were performed in a Mx4000 multiplex quantitative PCR system (Stratagene, La Jolla, CA) using Brilliant Plus single-step quantitative RT-PCR core reagent kit (Stratagene) in a total reaction volume of 25 µl. The reaction contained 5 U StrataScript reverse transcriptase, 1.25 U SureStart Taq DNA polymerase, 0.2 mM GAUC mix, 5 mM MgCl2, and 0.3 µM of each primer and probe for GPI-PLD. dNTP cycling conditions were as follows: 1 cycle for 90 min at 45 C, 1 cycle for 10 min at 95 C followed by 40 cycles of 15 sec denaturation at 95 C, and a 1-min annealing/extension at 55 C. Standard curves were generated for GPI-PLD from serial dilutions of human fetal liver total RNA (BD Biosciences CLONTECH, San Diego, CA). Unknown sample values were determined from standard curves, and data were expressed as arbitrary units normalized to total RNA as determined by Ribogreen (Molecular Probes, Portland, OR).

Aim 3

HepG2 cells, a human hepatoma cell line, were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM (GIBCO-BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) and maintained at 37 C in a humidified atmosphere (5% CO2). For experiments, HepG2 cells were seeded in 100-mm culture plates (2.2 x 106 cells/plate) 2 d before viral transduction. Cells were transduced with recombinant adenovirus AdCMVGPI-PLD (AdGPI-PLD) or AdCMV-ß-galactosidase (AdLacZ) (control) (19) with a multiplicity of infection of 0.56. Five hours after the addition of the virus, the media were switched to low-glucose DMEM supplemented with fatty-acid free BSA (1 mg/ml). The cells were harvested 6 and 12 h after viral transduction, and the total RNA was extracted using TriPure (Roche Diagnostics, Indianapolis, IN) followed by further purification with Absolutely RNA miniprep kit (Stratagene) and stored at –80 C until used for microarray analysis or Northern blotting. Four independent culture plates were used for each condition for a total of 16 microarrays.

The microarray procedure was done by the Indiana University Center for Medical Genomics following standard Affymetrix protocols (Affymetrix GeneChip expression analysis technical manual; Affymetrix, Santa Clara, CA). cRNA was hybridized to human genome U133A GeneChips for 17 h, followed by standard washing, staining, and scanning. Data were extracted using Affymetrix MicroArray Suite 5.0 software (MAS5; Affymetrix MicroArray Suite 5.0 user’s guide, Santa Clara, CA). To confirm microarray observations, Northern blot analysis was performed on diacylglycerol acyltransferase (DGAT) 1 and DGAT2.

Northern blot analysis was performed to identify mRNA specific to DGAT1 and DGAT2. DGAT1- and DGAT2-specific probes were generated by PCR amplification from human liver QUICK-clone cDNA (BD Biosciences, San Diego, CA) using gene-specific primers. The primers used were: DGAT1 forward, 5'-CTTTCTGCTGCGACGGATCCTTGAGATGCT-3'; DGAT1 reverse, 5'-CTATTGGCTGTCCGATGATGAGCGACAGC-3'; DGAT2 forward, 5'-AGAGGCCACAGAAGTGAGCAAGAAG-3'; and DGAT2 reverse, 5'-CCCCAGGTGTCGGAGGAGAAGAGGCCTCGACCA-3'. The PCRs were performed in a total volume of 50 µl containing 250 nM each of dCTP, dGTP, dATP, and dTTP, 250 nM forward and reverse primers and Advantage cDNA polymerase mix (BD Biosciences CLONTECH). Cycling conditions were as follows: 1 cycle of 5 min at 94 C for initial denaturation followed by 30 cycles of 1 min denaturation at 94 C, 1 min annealing at 58 C, and 2 min of extension at 72 C. PCR products were run on agarose gel and purified by QLA quick gel extraction kit (QIAGEN). Purified PCR products were cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol and were verified by sequencing (Biochemistry and Biotechnology Core Facilities, Indiana University, Indianapolis, IN). Probes for Northern blot analysis were generated using DECAprime TM II kit (Ambion) as per the manufacturer’s instructions. Gene-specific bands were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and normalized to ß-actin.

Statistical analysis

Statistical analyses were performed using StatView software (SAS Institute Inc., Cary, NC). Depending on the data distribution, comparisons were made between the two groups using the Student’s t test or Mann-Whitney test. The association between continuous variables was tested using Spearman rank correlation. Data are presented as mean ± SD. P < 0.05 was considered statistically significant.

Microarray analyses were carried out using Microarray Data Portal, a proprietary analytical and informatics tool developed by the Center for Medical Genomics. To eliminate noisy data from probe sets that reflect background signals, we analyzed only those probe sets called present by MAS5 in at least half of the arrays for at least one of the conditions (20). Then we carried out an ANOVA with one factor for the gene transduced (GPI-PLD vs. LacZ) using log-transformed signal values. Multifactor ANOVA was carried out for the gene transduced, one for time, and their interaction. Genes that were significant for the gene transduced or their interaction at P ≤ 0.05 were mapped onto Gene Ontology categories.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Aim 1

The demographic, anthropometric, and serological characteristics of individuals with NASH and their controls are shown in Table 1Go. These results have been published previously (13, 14, 15, 16, 17) but are presented here again for the interpretation of the GPI-PLD levels. There were no demographic differences between the groups, and all participants were Caucasian. BMI, percent body fat, and waist to hip ratio were similar between the groups, but patients with NASH had significantly greater visceral fat mass than the controls (Table 1Go). As expected, subjects with NASH had higher fasting levels of insulin and HOMA values than the controls.


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  		TABLE 1. Demographic, anthropometric, and biochemical measurements in NASH and control groups1

 
Serum GPI-PLD levels in patients with NASH were 119 ± 24 µg/ml and were significantly higher than the controls (105 ± 15 µg/ml, P = 0.047). There were no gender-specific differences in serum GPI-PLD levels in the total cohort (males 113 ± 16 vs. females 112 ± 26 mg/ml, P = 0.4). There was no statistically significant relationship between serum GPI-PLD levels and body weight, BMI, waist to hip ratio, percent body fat, percent visceral fat, or visceral fat mass. Serum GPI-PLD had a statistically significant negative correlation with age (r = –0.45, P = 0.006) as has been observed previously (21).

Aim 2

Hepatic GPI-PLD mRNA did not differ between patients with fatty liver or NASH; hence, the results from these two groups were combined (data not shown). Hepatic GPI-PLD mRNA was 3-fold higher in the combined group (NAFLD), compared with normal liver tissue (Fig. 1Go).


Figure 1
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  		FIG. 1. Liver GPI-PLD mRNA expression is significantly increased in patients with NAFLD. Liver tissue was obtained from healthy volunteers (n = 12) undergoing liver biopsies as part of live liver donor evaluation or patients with NAFLD (n = 25) undergoing liver biopsy as part of their clinical care. The mean age was 41 ± 5 yr in the normal group and 39 ± 8 yr in the NAFLD group. The BMI was significantly higher in the NAFLD group (32 ± 3.5 kg/m2), compared with the normal group (24 ± 3.2). Liver GPI-PLD mRNA was determined by real-time PCR as described in Patients and Methods. *, P = 0.026 by t test.

 
These results demonstrate that NAFLD is associated with elevations in liver expression of GPI-PLD mRNA. However, these results to do not distinguish whether increases in hepatic GPI-PLD expression are an epiphenomenon or may play a direct role in the pathogenesis of fatty liver or NASH.

Aim 3

To determine whether increased GPI-PLD expression may play an active role altering hepatic fatty acid and triglyceride metabolism, we overexpressed GPI-PLD in HepG2 cells, a human hepatoma cell line, and examined the effect on global gene expression. HepG2 cells were transduced with a control adenovirus expressing ß-galactosidase (AdLacZ) or an adenovirus expressing GPI-PLD (AdGPI-PLD), and global gene expression was determined using microarrays after 6 or 12 h. A total of 10,435 genes (47% of total gene probe set) were detected. Of these probe sets, 2340 differed at P < 0.05 (522 randomly expected) and 1270 genes at P < 0.01 (104 randomly expected).

Using a multifactor ANOVA, there were 2159 genes that were significantly different (P < 0.05) at 12 h, compared with 6 h, but were unrelated to the overexpression of GPI-PLD. Hence, the expression of these genes is related to time and/or cell cycle per se and was not analyzed further. Four hundred forty-one genes were significantly (P < 0.05) affected only by the overexpression of GPI-PLD independent of time. There were 773 genes that changed in a time- and GPI-PLD-dependent fashion. Analysis of the function of these genes using gene ontology demonstrated that the majority of these genes were intracellular and involved in metabolism (Tables 2Go and 3Go). The molecular function of these genes varied. The highest numbers of genes are involved in RNA metabolism and regulation of transcription. Numerous genes involved in signal transduction including the MAPK pathway and cAMP signaling pathway were also altered with GPI-PLD overexpression. This manuscript will focus on the effect of GPI-PLD on lipid metabolism.


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  		TABLE 2. Gene ontology analysis of GPI-PLD-dependent gene expression

 

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  		TABLE 3. Gene ontology analysis of time- and GPI-PLD-dependent gene expression

 
There were 32 genes involved in fatty acid and lipid metabolism that were affected by overexpressing GPI-PLD (Table 4Go). These genes are involved in de novo lipogenesis, triglyceride synthesis, and lipoprotein metabolism. The largest effect was on DGAT. DGAT1 increased nearly 3-fold with overexpressing GPI-PLD. To confirm the microarray results, Northern blot analysis of both isoforms of DGAT, DGAT1 and DGAT2, was performed. This confirmed that GPI-PLD overexpression increased DGAT1 but not DGAT2 mRNA levels (Fig. 2Go).


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  		TABLE 4. Effect of GPI-PLD overexpression on lipid gene expression

 

Figure 2
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  		FIG. 2. GPI-PLD increases DGAT1 but not DGAT2 expression in HepG2 cells. HepG2 cells were transduced with AdLacZ or AdGPI-PLD and total RNA extracted after 6 or 12 h as described in Materials and Methods. DGAT1 and DGAT2 mRNA levels were determined by Northern blotting and normalized to ß-actin mRNA. Results are from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The accumulation of triglycerides in liver results from an imbalance between the uptake of fatty acids, de novo lipogenesis, and export as very low-density lipoproteins or fatty acid oxidation. Numerous biochemical and hormonal defects result in fatty liver (4). In this report, we demonstrate that GPI-PLD expression is elevated in patients with NAFLD and provide evidence that it may play a role in enhancing de novo lipogenesis in liver.

Donnelly et al. (3) demonstrated that the primary lipid defect in patients with NAFLD is a 5-fold increase in de novo lipogenesis. Consistent with this in vivo observation, a recent analysis of fatty acid synthesis and oxidation genes in patients with NAFLD demonstrated an increase in genes involved in de novo lipogenesis [acetyl coenzyme A carboxylase (ACC) 1 and 2] and triglyceride synthesis (DGAT1) as well as alterations in genes involved in ß-oxidation of fatty acids [carnitine palmitoyltransferase 1a decreased, whereas long-chain acyl-coenzyme A dehydrogenase and long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase (HADH)-{alpha} increased] (22). An increase in fatty oxidation genes may be a compensatory effect in response to the increase in de novo lipogenesis. The predicted net effect is an accumulation of intracellular fatty acids and triglycerides. Many of these same genes altered in NASH are similarly affected by overexpressing GPI-PLD in vitro. Genes affected by GPI-PLD overexpression that are involved in de novo lipogenesis and triglyceride synthesis include ATP citrate lyase (ACLY), ACC2, elongation of very long-chain fatty acids, stearoyl coenzyme-A desaturase (SCD), and DGAT1, whereas those involved in ß-oxidation of fatty acids include short-chain HADH, HADH{alpha}, and acetyl-coenzyme A acyltransferase 2. These results are expected to result in a net increase in fatty acid synthesis, which may contribute to the increase in serum triglycerides observed when hepatic GPI-PLD expression is increased (11). This raises the possibility that the increase in hepatic GPI-PLD mRNA that occurs in patients with NAFLD may play an active role in the development of fatty liver. Many of these genes (ACC, SCD, ACLY, 3-hydroxy-3-methylglutaryl-coenzyme A reductase) are regulated by sterol-response element binding proteins (SREBP). The SREBP family of transcription factors regulates cholesterol and triglyceride synthesis. It is conceivable that overexpressing GPI-PLD alters the lipid composition of the endoplasmic reticulum resulting in activation of SREBPs. However, this was not examined in our study because sufficient liver tissue was not available.

In addition to altering genes involved in lipid metabolism, overexpressing GPI-PLD in HepG2 cells had a large effect on global gene expression. More than 100 genes involved in RNA metabolism and regulation of transcription were affected including 20 transcription factors and 18 corepressors/coactivators. Altering that many transcription factors likely results in many downstream effects. In addition, more than 30 growth factors or their receptors were altered including TGFß1 (up 1.74-fold), IL-8 (up 1.6-fold), vascular endothelial growth factor (up 1.2-fold), IGF-I receptor (up 2.4-fold), and fibroblast growth factor receptor 1 (up 1.5-fold). Many of these growth factors have been implicated in the progression of fatty liver to the fibrosis and cirrhosis seen in NASH (23, 24). Clearly, overexpressing GPI-PLD affects many genes involved in a multitude of pathways and biological processes. Using global gene expression makes it difficult to determine which, if any, of these pathways or processes affected by GPI-PLD may contribute to the development of fatty liver and/or NASH. Our observation that liver GPI-PLD mRNA is elevated to the same extent in patients with fatty liver and NASH suggests that GPI-PLD is more related to the former rather than the latter. More direct experiments are needed to determine how and at what point GPI-PLD plays in the development and progression of NAFLD.

Our results are also the first to demonstrate acute regulation of DGAT1 expression. It is unclear how GPI-PLD activity may regulate DGAT1 and other genes. GPI-PLD-mediated cleavage of GPIs generates phosphatidic acid, which leads to an increase in diacylglycerol and the activation of protein kinase C{alpha} (25). In addition, inositol glycans would also be released. Inositol glycans have been postulated to serve as insulin mediators and have been suggested to stimulate de novo lipogenesis and triglyceride synthesis (26, 27). Whether these signals are responsible for the effects of GPI-PLD on gene expression is unknown at this time.

Why GPI-PLD expression is increased in NAFLD is unknown. Oxidative stress alters GPI-PLD expression in macrophage cell lines (28, 29). This might explain an alteration in NASH as increased oxidative stress is suggested to mediate the transition from fatty liver to NASH but does not explain the increase in patients with fatty liver. Glucose and insulin regulate GPI-PLD expression in pancreatic islet ß-cells (30), raising the possibility that increased expression of hepatic GPI-PLD may be related to serum insulin levels in NAFLD patients. Increased expression of GPI-PLD not only will have effects within the hepatocyte but also will be expected to increase GPI-PLD secretion into the plasma. In mice, serum GPI-PLD is under genetic control (7, 31), and we have found that fasting serum GPI-PLD levels do correlate to levels of hepatic levels of GPI-PLD mRNA (32). However, serum levels of GPI-PLD also appear to be acutely regulated because serum GPI-PLD levels rise and fall during the postprandial state without changes in liver GPI-PLD mRNA levels (11). Hence, the increased serum levels of GPI-PLD in NASH patients may result from a combination of increased hepatic expression as well as the altered lipoprotein metabolism in the plasma compartment that occurs with insulin resistance.

Limitations of this study include use of stored samples [although serum GPI-PLD levels are stable frozen for at least 3 yr (Deeg, M. A., unpublished observation)], no data with respect to actual protein levels in the liver, and not being able to have serum and hepatic samples from the same cohort. Hence, we were not able to test whether there was a significant correlation between hepatic GPI-PLD mRNA and serum or hepatic GPI-PLD protein. In addition, overexpressing GPI-PLD in HepG2 cells may not entirely reflect the changes in gene expression that would occur in normal hepatocytes. In addition, the changes in gene expression after 6–12 h of overexpressing GPI-PLD may be different from that which occurs after prolonged increased expression of GPI-PLD. Nonetheless, these studies have yielded important preliminary findings to support GPI-PLD as a target for further evaluation.

In summary, patients with NAFLD have elevated serum levels and hepatic expression of GPI-PLD. Because increased expression of hepatic GPI-PLD is associated with increases in serum triglycerides and increased expression of de novo lipogenesis genes, GPI-PLD may play an active role in the pathogenesis of NAFLD or its metabolic features and warrants further investigation.


   Acknowledgments
 
We thank Rosario Bowen for excellent technical assistance and Drs. David Crabb and Steve Miller for helpful comments.


   Footnotes
 
This work was supported by funds from a Veterans Affairs Merit Review (to M.A.D.), K24 DK 072101 (to N.C.), and RR00750 (Indiana University General Clinical Research Center). Microarray studies were carried out in the Center for Medical Genomics at Indiana University School of Medicine, which is supported in part by grants from the Indiana 21st Century Research and Technology Fund and the Indiana Genomics Initiative of Indiana University (supported in part by the Lilly Endowment, Inc.).

First Published Online April 4, 2006

Abbreviations: ACC, Acetyl coenzyme A carboxylase; ACLY, ATP citrate lyase; AdGPI-PLD, adenovirus expressing GPI-PLD; BMI, body mass index; DGAT, diacylglycerol acyltransferase; GPI-PLD, glycosylphosphatidylinositol-specific phospholipase D; HADH, L-3-hydroxyacyl-coenzyme A dehydrogenase; HOMA, homeostasis model assessment; LDL, low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SCD, stearoyl coenzyme-A desaturase; SREBP, sterol-response element binding protein.

Received January 13, 2006.

Accepted March 28, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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