AJP - Endo AJP: Heart and Circulatory Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 293: E958-E964, 2007. First published July 3, 2007; doi:10.1152/ajpendo.00235.2007
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/E958    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (21)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steinberg, G. R.
Right arrow Articles by Watt, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinberg, G. R.
Right arrow Articles by Watt, M. J.

Adipocyte triglyceride lipase expression in human obesity

Gregory R. Steinberg,1 Bruce E. Kemp,1,2 and Matthew J. Watt1

1St. Vincent's Institute and the Department of Medicine, The University of Melbourne, Fitzroy; and 2Commonwealth Scientific and Industrial Research Organisation Molecular Health Technologies, Parkville, Victoria, Australia

Submitted 16 April 2007 ; accepted in final form 22 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.

triglyceride metabolism; adipose triglyceride lipase; triglyceride hydrolase activity

THE ABILITY TO EFFICIENTLY regulate the storage and release of fatty acids contained in adipose triglyceride (TG) requires tight coupling between hydrolysis and esterification. Fat stored in subcutaneous and visceral depots is mobilized by activation of lipolytic enzymes, which degrade TGs and release free fatty acids (FFAs) into the circulation for use as an energy substrate. Obesity is characterized by excessive adipose tissue deposition and increased FFA release that exceeds metabolic demands (17). Elevated postprandial FFAs are a hallmark of obesity (33) and contribute to metabolic disturbances including insulin resistance in skeletal muscle and liver (4, 44), pancreatic beta-cell dysfunction (51), and increased VLDL triglyceride production (27).

Until recently, hormone-sensitive lipase (HSL) was considered the rate-limiting enzyme for lipolysis, but recent evidence suggests that adipose triglyceride lipase (ATGL) may also be important. HSL–/– mice have residual TG lipase activity (11, 31, 32) indicative of the presence of other TG lipases and maintain partial basal and catecholamine-stimulated lipolysis (41). Recent studies have identified the molecular lipolytic (and esterolytic) proteome in mouse adipose tissue (3) and 23 proteins that are incompletely characterized. Many of these proteins are predicted to contain an {alpha}/beta-hydrolase fold that accommodates catalytic residues typical of many lipases (36). Of these recently identified lipases (3), ATGL (also known as desnutrin and calcium-independent phospholipase, iPLA2{zeta}) has been demonstrated to be critical for both basal and catecholamine-stimulated TG hydrolysis, energy metabolism in cultured adipocytes, and weight maintenance in mice (10, 12, 15, 21, 40, 52). Homologues with similar functions have also been reported in Drosophila and Saccharomyces cerevisiae (9, 20, 38).

Aside from HSL, the role of other TG lipases in the pathogenesis of human obesity is unclear. HSL expression is reduced in obesity (23, 29, 42), which is inconsistent with the elevated basal lipolysis observed in these individuals, implying that other TG lipases may be upregulated. Two recent reports (22, 29) suggest that ATGL mRNA is not regulated in human obesity, whereas the same group reports that ATGL mRNA and protein expression are negatively associated with insulin resistance and not obesity per se (13). Suppression of ATGL with short interfering RNA in human adipocytes derived from human mesenchymal stem cells indicates that ATGL is important for basal lipolysis but does not contribute to catecholamine-induced lipolysis (34). Despite the uncertainty regarding ATGL in obesity, single nucleotide polymorphisms within the ATGL gene are associated with free fatty acid levels and increased risk of type 2 diabetes mellitus (35). Similarly, mutations in the lipase family member comparative gene identification-58 (CGI-58), an essential co-regulator of ATGL (24), result in TG accumulation in several tissues, resulting in serious pathologies including cardiomyopathy and liver steatosis (26). Another TG lipase, adiponutrin, has been reported to be either increased (14) or unchanged (28) with obesity. Taken together, these data suggest that the upregulation of basal adipose tissue lipolysis in obesity may be associated with the increased expression of alternative TG lipases.

The first aim of this study was to characterize the mRNA and protein expression of ATGL in relation to obesity and compare these levels with in vitro TG lipase activity. The second aim of the study was to assess the contribution of ATGL to in vitro TG lipase activity. To gain further insight into the molecular control of adipocyte lipolysis, the third aim was to assess the mRNA content of selected enzymes that were previously identified to possess murine TG lipase activity and contain an {alpha}/beta-hydrolase fold (3). Finally, we have extended previous studies (13) by examining both subcutaneous and visceral adipose tissue, because there is marked heterogeneity with regard to lipolysis regulation and fatty acid flux from these sites (17).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects and Study Design

Lean [n = 8, body mass index (BMI) range = 21.0–27.9, mass = 59–75 kg; 4 male, 4 female] and obese (n = 8, BMI range = 30.0–37.1, mass = 86–113 kg; 2 male, 6 female) subjects participated in this study. None was taking medication or involved in an exercise program, and their body weight was essentially stable for 12 mo before all experiments. All female subjects were postmenopausal. Participants were admitted to McMaster Health Sciences Center for a variety of abdominal surgical interventions after an overnight fast (~16 h) as previously described (5). Adipose tissue samples (from visceral and subcutaneous depots) were excised under general anesthesia and snap frozen in liquid N2 while a venous blood sample was obtained from the antecubital vein and stored on ice until processing (<5 min). All experimental procedures were performed according to the Declaration of Helsinki. Ethical clearance was granted by the McMaster University Ethics Committee, and informed consent was obtained after procedures were explained both verbally and in writing. All applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research.

Analytical Methods

Adipose tissue mRNA analysis. Total RNA was extracted from adipose tissue in 750 µl of Qiazol extraction reagent followed by isolation using an RNeasy Lipid Tissue kit (Qiagen, Doncaster, Victoria, Australia) according to the manufacturer's instructions. RNA quantity was determined at 260 nm (NanoDrop p2000 Spectrometer, Biolab). RNA was reverse transcribed (Invitrogen, Mt. Waverley, Victoria, Australia), and gene products were determined by real-time quantitative RT-PCR (Stratagene Mx3000P, La Jolla, CA) using TaqMan Assays-on-Demand (Applied Biosystems, Scoresby, Victoria, Australia) as previously described (43). The final reaction mix consisted of 10 µl of TaqMan Universal PCR master mix, 0.5 ng of cDNA in 9 µl of RNase-free water, and 1 µl of supplied primers. PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of PCR reaction at 95°C for 15 s and 60°C for 45 s. 18S was used as a reference gene and did not vary between groups or adipose tissue depots [cycle threshold (CT) lean = 19.2 ± 0.1 vs. obese = 19.3 ± 0.2]. ATGL (PNPLA2) could not be identified using the TaqMan Assay-on-Demand probe. We therefore used 2x SYBR green PCR master mix, 2 ng of cDNA, and 10 µM primers (sense, 5'-GTGTCAGACGGCGAGAATG-3'; antisense, 5'-TGGAGGGAGGGAGGGATG-3') as described previously (29). The PCR conditions were as described above, and the primer pairs yielded a single amplicon based on dissociation curves. The mRNA levels were determined by a comparative CT method. For each sample, a {Delta}CT value was obtained by subtracting 18S CT values from those of the gene of interest. Similar values were obtained when subtracting RPL-32. The average {Delta}CT value of the lean group was then subtracted from the sample to derive a {Delta} – {Delta}CT value. The expression of each gene was then evaluated by 2–({Delta} – {Delta}CT).

Adipose tissue protein analysis. Adipose tissue was homogenized and processed as described previously (45). A rabbit ATGL polyclonal antibody was raised against the peptide CTNVAFPPDALRMRAPA based on the amino acid sequence of human ATGL (449–465) and was purified as described previously (7). The ATGL antibody showed one band at ~54 kDa (see GoGoFig. 3A) and in some instances a fainter band at ~48 kDa. The ATGL expressed from a mammalian expression vector was also detected with this antibody at a similar molecular mass (see Fig. 3A). After a 45-min washing, membranes were incubated with protein G-horseradish peroxidase-conjugated secondary antibody (1:2,000), and the immunoreactive proteins were detected with enhanced chemiluminescence and quantified by densitometry (Scion Image, Scion). In additional immunoprecipitation (IP) experiments, 1 mg of adipose tissue lysate was incubated for 2 h at 4°C in the presence of ATGL antibody conjugated to protein A-Sepharose beads. The post-IP lysate was recovered for TG lipase activity assays. For control experiments, some samples were immunoprecipitated with an antibody to insulin receptor substrate-2 (IRS2; Upstate, Lake Placid, NY).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 1. A–H: mRNA expression of triglyceride (TG) lipases in subcutaneous adipose tissue of lean and obese subjects. I: correlation between PNPLA2 and LIPE mRNA expression levels in subcutaneous adipose tissue. Values are means ± SE (n = 8 subjects).

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. A–H: mRNA expression of TG lipases in visceral adipose tissue of lean and obese subjects. I: correlation between PNPLA2 and LIPE mRNA expression levels in visceral adipose tissue. Values are means ± SE (n = 8 subjects).

 

Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Adipose triglyceride lipase (ATGL) protein expression and neutral lipase activity in subcutaneous and visceral adipose tissue of lean and obese subjects. A, left: representative immunoblot demonstrating a single band at ~54 kDa in human adipose tissue lysates using a polyclonal ATGL antibody. SC, subcutaneous; Vis, visceral. Middle: immunoblot showing specificity of the polyclonal ATGL antibody for His-ATGL expressed in HEK293 cells compared with untransfected cells. Right: immunoblot showing ATGL protein from human adipose tissue lysates before (Pre-IP) and after (Post-IP) immunoprecipitation of ATGL. B: ATGL protein content in subcutaneous and visceral adipose tissue. C: TG lipase activity in subcutaneous and visceral adipose tissue. D: TG lipase activity in adipose tissue lysates of lean and obese individuals before and after immunoprecipitation of ATGL. The success of the IP is shown in A. Values are means ± SE (n = 8 subjects).

 
Adipose tissue TG lipase activity assay. Adipose tissue TG lipase activity was determined using an emulsified substrate consisting of 5 mmol/l triolein, 6.7 x 106 counts/min [9,10-3H]triolein, 0.6 mg of phospholipid (phosphatidylcholine-phosphatidylinositol, 3:1, wt/wt), 0.1 M potassium phosphate, and 20% BSA. Procedures for this assay have been described previously (42).

Plasma metabolite determination. One portion of heparinized whole blood was deproteinized 1:5 with 0.6% (wt/vol) perchloric acid and centrifuged. The extract was analyzed for blood glucose (2). A second portion of whole blood was centrifuged, and the plasma was removed for the determination of FFA by an enzymatic colorimetric method (Wako NEFA C test kit, Wako Chemicals) and insulin by radioimmunoassay (Coat-a-Count insulin test kit, Diagnostics Products).

Calculations and statistics. Insulin sensitivity of subjects was evaluated using the homeostasis model assessment of insulin resistance [HOMA-IR; (fasting glucose x fasting insulin)/22.5] (where glucose is expressed as mmol/l and insulin as µU/l). Comparisons were made between lean and obese subjects by unpaired t-tests. Values are reported as means ± SE. Statistical significance was set a priori at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gene Expression of Lipolytic Enzymes in Subcutaneous and Visceral Adipose Tissue of Lean and Obese Subjects

The clinical characteristics of lean and obese subjects after an overnight fast are listed in Table 1. Obese subjects had a greater body mass, BMI, and HOMA index relative to lean controls. Although plasma glucose and insulin were not different between groups, obese subjects displayed whole body insulin resistance as demonstrated by an increased HOMA index.


View this table:
[in this window]
[in a new window]

  		Table 1. Clinical characteristics of lean and obese subjects

 
Subcutaneous adipose tissue. Subcutaneous fat located in the upper body is thought to contribute ~65% of systemic FFA flux and is likely the dominant source of the excess systemic fatty acids released in obesity (17). The mRNA expression of lipolytic proteins in subcutaneous adipose tissue of lean and obese subjects is shown in Fig. 1. We first examined members of the "patatin-like phospholipase domain-containing proteins" (PNPLA) family. These proteins contain a patatin-like domain that shows acyl-hydrolase activity and are highly conserved across all life forms. PNPLA proteins are expressed in mouse adipose tissue (18) and demonstrate marked TG lipase activity in vitro (21, 52). PNPLA2 (ATGL) mRNA expression levels tended to be higher in obese (Fig. 1A, P = 0.08) than in lean subjects. PNPLA3 and PNPLA4 mRNA contents were increased 2.7- and 1.8-fold, respectively, in obese vs. lean subjects (Fig. 1, B and C). PNPLA8 mRNA expression was unaltered between groups (Fig. 1D). We did not probe for PNPLA1 because its physiological function is unknown, and we were unable to detect PNPLA5 mRNA with the assay conditions listed, consistent with a previous report using Northern blot analysis (21). LIPE (HSL) mRNA expression was not altered by obesity (Fig. 1E). The human carboxylesterase-1, or triacylglycerol hydrolase, has been shown to play a role in hepatic lipid metabolism (1). The expression of its gene, CES1, was increased 2.2-fold in obesity (Fig. 1F). There were no changes in ABHD5 mRNA (Fig. 1G), whereas LYPLAL1 mRNA was increased in obesity (Fig. 1H).

The abundance of ATGL and HSL transcripts was highly correlated (R2 = 0.74, P = 0.004; Fig. 1I). There were no significant relationships between the other transcripts assessed. Further correlative analysis with BMI as the independent variable indicated that PNPLA2 (ATGL) and LIPE (HSL) are not obesity dependent (BMI vs. ATGL: R2 = 0.08, P = 0.30). Previous studies have demonstrated that peroxisome proliferator-activated receptor (PPAR){gamma} increases ATGL transcription (15, 16, 19). PPAR {gamma} protein was decreased (P < 0.01) by 48 ± 6% in subcutaneous adipose tissue of obese subjects (data not shown). No significant correlation was reported between ATGL mRNA and PPAR {gamma} protein (R2 = 0.11, P = 0.56).

Visceral adipose tissue. Visceral fat is positively associated with whole body insulin resistance (25) and contributes ~15% to systemic FFA flux (17). The gene expression profile of the PNPLA family was similar to our observations in subcutaneous adipose tissue. PNPLA2 was increased 2.2-fold (Fig. 2A) and PNPLA3 2.8-fold (Fig. 2B), and PNPLA4 tended to increase (P = 0.11, Fig. 2C). PNLAP8 was not different between groups (Fig. 2D). LIPEwas elevated 2.3-fold in the visceral adipose tissue of obese individuals (Fig. 2E), whereas CES1 (Fig. 2F), ABHD5 (Fig. 2G), and LYPLAL1 (Fig. 2H) were not different between groups. Consistent with our observations in subcutaneous adipose tissue, PNPLA2 and LIPE were positively correlated (Fig. 2I).

ATGL Protein Content and Neutral Lipase Activity in Adipose Tissue of Lean and Obese Subjects

We first confirmed the specificity of our antibody. The ATGL polyclonal antibody revealed a single band at the expected molecular mass of ~54 kDa (Fig. 3A). In some samples, a faint band was also detected at ~48 kDa. The prevalence of this 48-kDa band was not prominent in any particular group. Further experiments revealed that ATGL expressed from a mammalian expression vector was detected with our antibody at the expected molecular mass (Fig. 3A). We next performed IP experiments and reprobed, using a commercially available ATGL antibody (Cell Signaling, Danvers, MA). In the post-IP fraction, no ATGL was observed, suggesting that all ATGL protein was immunoprecipitated (Fig. 3A). There was no reduction in HSL protein after ATGL IP (data not shown). To further evaluate the role of ATGL in adipose tissue lipolysis, we measured its protein expression in subcutaneous and visceral adipose tissue samples using immunoblotting. ATGL protein expression was lower (24%, P = 0.04) in the subcutaneous adipose tissue of obese compared with lean subjects (Fig. 3B). ATGL protein expression in visceral adipose tissue was not different between groups (P = 0.09).

TG lipase activity was assessed in adipose tissue lysates in vitro. TG lipase activity was decreased in the subcutaneous (P = 0.02), but not visceral, adipose tissue of obese compared with lean subjects (Fig. 3C). Overall, TG lipase activity did not show any significant correlations with ATGL protein in either adipose tissue depot (R2 = 0.06). Even when subjects were divided into groups based on high (18.8 ± 0.7 nmol·h–1·mg protein–1, n = 8) or low (7.5 ± 1.0 nmol·h–1·mg protein–1, n = 8) TG lipase activity, ATGL protein expression was remarkably similar (high-TG lipase group, 1.79 ± 0.15 arbitrary units; low-TG lipase group, 1.79 ± 0.17 arbitrary units). We directly tested the importance of ATGL for total TG lipase activity in human adipose tissue in vitro. To do this, we examined total TG lipase activity in adipose lysates before and after IP of ATGL. We were unable to measure both visceral and subcutaneous depots because of a lack of tissue; hence, the data are pooled (n = 4 visceral and n = 4 subcutaneous for each group). IP removed the majority of ATGL protein from the adipose lysate as determined by Western blot analysis (Fig. 3A). TG lipase activity was reduced post-IP by 83 ± 7% and 70 ± 6% in lean and obese subjects, respectively (Fig. 3D). The percent reduction in TG lipase activity post-IP was not different between groups (P = 0.15). TG lipase activity was unaffected in the post-IP lysates when an unrelated antibody (IRS2) was used in control experiments (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we investigated the mRNA expression profile of several novel TG lipases and ATGL protein expression and the relationship with in vitro TG lipase activity in subcutaneous and visceral adipose tissue from lean and obese subjects. We have assessed adipose tissue from male and female subjects. Although it is documented that lipolysis control varies according to sex, owing mainly to differences in sex hormones and adrenergic receptor sensitivity (46), this remains controversial (6). In fact, estrogen treatment in postmenopausal women does not affect the expression of proteins and genes involved in adipocyte signal transduction (49). Regardless, adipose tissue in this study was obtained from postmenopausal women, which most likely minimizes potential sex differences. The results of this study indicate the following. 1) The mRNA expression of ATGL (P = 0.08 in subcutaneous) and HSL is increased in adipose tissue of obese subjects, but this increase is not related to BMI. 2) ATGL protein expression is reduced in subcutaneous adipose tissue despite increases in ATGL mRNA. 3) The mRNA of several novel lipolytic proteins is elevated in obese subcutaneous and visceral adipose tissue. 4) TG lipase activity is reduced in subcutaneous adipose tissue of obese subjects despite increases in lipase gene expression. And 5) ATGL is essential for efficient TG lipase activity, but ATGL protein expression is not related to TG lipase activity in subcutaneous or visceral adipose tissue from lean or obese subjects.

ATGL belongs to the patatin-like phospholipase family and shares a close evolutionary relationship with other lipid acyl-hydrolases (21). ATGL possesses several key structural features including an NH2-terminal patatin homology domain, which is a feature of proteins that possess acyl-hydrolase activity on lipid substrates (50), and an {alpha}/beta-hydrolase fold and an active site GXSXG consensus sequence, both common features of many lipases (36). ATGL specifically hydrolyzes TG (12, 21, 52) and is predominantly expressed in adipose tissue. Inhibition of ATGL reduces TG breakdown in cell culture (15, 38) and mice in vivo (10). In the present investigation, we examined the influence of obesity on ATGL mRNA expression, as ATGL mRNA is induced during adipogenesis in murine adipocytes (15, 16, 40, 52) and is elevated in genetically obese mice (21, 40). We found that ATGL mRNA expression was increased in subcutaneous (P = 0.08) and visceral adipose tissue of obese compared with lean humans; however, ATGL mRNA expression was not related to BMI. The obese subjects in this study were mildly insulin resistant, and ATGL mRNA was positively correlated with HOMA (R2 = 0.306, P < 0.05). So, rather than obesity per se, these findings in obese, mildly insulin-resistant humans are consistent with studies demonstrating transcriptional inhibition of ATGL by insulin (15, 16, 19) and increased ATGL mRNA expression in murine models of insulin deficiency (streptozotocin-induced diabetes) and insulin resistance (fat-specific insulin receptor knockout) (15). Also, preliminary data from our laboratory show a marked upregulation of ATGL mRNA (2.2-fold, n = 5) in the subcutaneous adipose tissue of nonobese type 2 diabetes patients. Collectively, these data support the concept that ATGL gene expression becomes resistant to the inhibitory action of insulin and is not regulated by obesity. In contrast to our results, it was reported that ATGL mRNA was reduced in insulin-resistant individuals, independent of fat mass in obese individuals (13). It is difficult to reconcile our results in obese individuals with those of Jocken et al. (13). It must be stressed that the increased ATGL mRNA in the obese subjects from the present study is entirely consistent with the premise that insulin's demonstrated inhibitory effects were obviated in these insulin-resistant individuals, thus resulting in increased ATGL mRNA expression.

As an extension of previous human studies (22, 29), we examined ATGL protein expression in adipose tissue and compared this with TG lipase activity in fasted individuals. ATGL protein content was decreased in the subcutaneous adipose tissue of obese individuals, whereas no differences were observed in visceral fat. The reduced ATGL protein in subcutaneous fat is consistent with a previous report (13) and may reflect increased degradation, since ATGL mRNA expression was increased in obesity. The factors underpinning this apparent mismatch are presently unclear but may involve proteasome degradation via a ubiquitination pathway, as has been described previously for other proteins important for lipolytic regulation including perilipin and adipose differentiation-related protein (47, 48).

HSL content was previously shown to be associated with total neutral lipase activity (23, 42), and, given the central role of ATGL in murine TG lipolysis (10, 15, 52), we hypothesized that ATGL content would also be positively related to TG lipase activity in humans. Surprisingly, we found no correlation between ATGL content and in vitro TG lipase activity despite finding that total TG lipase activity was reduced by 70–85% after ATGL immunoprecipitation, indicating that ATGL is essential for efficient TG lipase activity. Our observations agree with a recent study in isolated human adipocytes that demonstrated a 50% reduction in basal lipolysis when ATGL expression was reduced by short interfering RNA administration in vitro (34). Subcutaneous ATGL protein and total TG lipase activity were concomitantly decreased in obesity; however, there were no significant correlations between ATGL protein content and TG lipase activity. The discordant regulation between ATGL protein expression and TG lipase activity may be due to a combination of factors. First, the interactions of ATGL with co-factors such as perilipin (8, 30) and CGI-58 (24, 39) are clearly important for lipolytic control in vitro and are likely to be relevant in vivo. Second, the regulation of ATGL activity, like HSL, may be more dependent on posttranslational modifications such as phosphorylation and translocation (8) than expression levels. Further studies examining the regulation of ATGL by these co-factors may be informative on the level/s of aberrant lipolytic control in human obesity.

We report increased gene expression of a number of novel TG lipases (PNPLA2, PNPLA3, PNPLA4, CES1, and LYPLAL1) in subcutaneous adipose tissue of obese subjects, several of which were not previously described in humans. Surprisingly, total TG lipase activity was reduced, suggesting that increased lipase gene expression in obesity may be a homeostatic mechanism intended to enhance TG metabolism, perhaps to facilitate the disposal of the enlarged adipose mass. Several studies, including our own, demonstrate a close correlation between PNPLA2 and LIPE mRNA expression that indicates co-regulation of these two major lipolytic proteins in humans (22, 29); however, it is presently unclear whether a single transcription factor can mediate generic changes in both lipolytic genes. With respect to the other lipolytic genes assessed, the observed responses are unlikely to be due to insulin, glucose, or PPAR{gamma}, as these have opposite effects on PNPLA2 and PNPLA3 transcription (15). While this paper was in preparation, Schweiger et al. (37) reported that ATGL and HSL account for ~95% of total murine TG lipase activity, indicating that additional known or unknown lipases may play a quantitatively minor role in fat cell lipolysis. The present data do not conflict with this report; rather, they provide valuable information with respect to the expression of alternative lipolytic genes in humans in vivo. Indeed, we report in human adipose lysates that ATGL is important for TG lipase activity, and that non-ATGL lipases are unable to compensate for the loss of ATGL in human adipose tissue. In light of the observed dissociation between ATGL mRNA and protein content in this study, and the previous observation of proteosomal degradation of other proteins involved in lipolysis (47, 48), future studies examining the protein expression of these novel lipases combined with studies examining TG lipase activity in the presence of HSL and ATGL inhibitors will reveal whether these newly identified lipases are important in the regulation of human adipose tissue lipolysis.

In conclusion, we demonstrate that ATGL is an important enzyme for efficient TG lipase activity in human adipose tissue. ATGL mRNA content is increased in the adipose tissue of obese individuals, and this appears to be related to insulin resistance rather than adiposity. Surprisingly, ATGL protein content is decreased in subcutaneous but not visceral adipose tissue in obesity and was not associated with changes in TG lipase activity assessed in vitro. Collectively, these observations suggest that small reductions in ATGL protein expression are not important for human adipose tissue lipolysis in obesity; rather, posttranslational modifications may be more critical for the regulation of lipolysis.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
M. J. Watt is supported by an NHMRC RD Wright Biomedical Fellowship; G. R. Steinberg is a Canadian Institutes of Health Research, Canadian Diabetes Association and Heart and Stroke Foundation "Target Obesity" Research Fellow; and B. E. Kemp is an ARC Federation Fellow.

We acknowledge the support of the Australian Research Council (ARC), National Health and Medical Research Council (NHMRC), and Ramaciotti Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Watt, St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, Victoria, Australia 3065 (e-mail: mwatt{at}svi.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alam M, Ho S, Vance DE, Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase. Protein Expr Purif 24: 33–42, 2002.[CrossRef][Web of Science][Medline]
  2. Bergmeyer HU. Methods in Enzymatic Analysis. New York: Academic, 1974.
  3. Birner-Gruenberger R, Susani-Etzerodt H, Waldhuber M, Riesenhuber G, Schmidinger H, Rechberger G, Kollroser M, Strauss JG, Lass A, Zimmermann R, Haemmerle G, Zechner R, Hermetter A. The lipolytic proteome of mouse adipose tissue. Mol Cell Proteomics 4: 1710–1717, 2005.[Abstract/Free Full Text]
  4. Boden G, Chen X. Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest 96: 1261–1268, 1995.[Web of Science][Medline]
  5. Bonen A, Tandon NN, Glatz JF, Luiken JJ, Heigenhauser GJ. The fatty acid transporter FAT/CD36 is upregulated in subcutaneous and visceral adipose tissues in human obesity and type 2 diabetes. Int J Obes 30: 877–883, 2006.[CrossRef][Web of Science][Medline]
  6. Bulow J, Gjeraa K, Enevoldsen LH, Simonsen L. Lipid mobilization from human abdominal, subcutaneous adipose tissue is independent of sex during steady-state exercise. Clin Physiol Funct Imaging 26: 205–211, 2006.[CrossRef][Web of Science][Medline]
  7. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285–289, 1999.[CrossRef][Web of Science][Medline]
  8. Granneman JG, Moore HP, Granneman RL, Greenberg AS, Obin MS, Zhu Z. Analysis of lipolytic protein trafficking and interactions in adipocytes. J Biol Chem 282: 5726–5735, 2007.[Abstract/Free Full Text]
  9. Gronke S, Mildner A, Fellert S, Tennagels N, Petry S, Muller G, Jackle H, Kuhnlein RP. Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metab 1: 323–330, 2005.[CrossRef][Web of Science][Medline]
  10. 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. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science 312: 734–737, 2006.[Abstract/Free Full Text]
  11. Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E, Sattler W, Magin TM, Wagner EF, Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem 277: 4806–4815, 2002.[Abstract/Free Full Text]
  12. Jenkins CM, Mancuso DJ, Yan W, Sims HF, Gibson B, Gross RW. 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, 2004.[Abstract/Free Full Text]
  13. Jocken JW, Langin D, Smit E, Saris WH, Valle C, Hul GB, Holm C, Arner P, Blaak EE. Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin resistant state. J Clin Endocrinol Metab 92: 2292–2299, 2007.[Abstract/Free Full Text]
  14. Johansson LE, Hoffstedt J, Parikh H, Carlsson E, Wabitsch M, Bondeson AG, Hedenbro J, Tornqvist H, Groop L, Ridderstrale M. Variation in the adiponutrin gene influences its expression and associates with obesity. Diabetes 55: 826–833, 2006.[Abstract/Free Full Text]
  15. Kershaw EE, Hamm JK, Verhagen LA, Peroni O, Katic M, Flier JS. Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 55: 148–157, 2006.[Abstract/Free Full Text]
  16. Kim JY, Tillison K, Lee JH, Rearick DA, Smas CM. The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-{alpha} in 3T3-L1 adipocytes and is a target for transactivation by PPAR{gamma}. Am J Physiol Endocrinol Metab 291: E115–E127, 2006.[Abstract/Free Full Text]
  17. Koutsari C, Jensen MD. Thematic review series: patient-oriented research. Free fatty acid metabolism in human obesity. J Lipid Res 47: 1643–1650, 2006.[Abstract/Free Full Text]
  18. Kraemer FB, Shen WJ. Hormone-sensitive lipase knockouts. Nutr Metab (Lond) 3: 12, 2006.[CrossRef][Medline]
  19. Kralisch S, Klein J, Lossner U, Bluher M, Paschke R, Stumvoll M, Fasshauer M. Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes. Mol Cell Endocrinol 240: 43–49, 2005.[CrossRef][Web of Science][Medline]
  20. Kurat CF, Natter K, Petschnigg J, Wolinski H, Scheuringer K, Scholz H, Zimmermann R, Leber R, Zechner R, Kohlwein SD. Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast. J Biol Chem 281: 491–500, 2006.[Abstract/Free Full Text]
  21. Lake AC, Sun Y, Li JL, Kim JE, Johnson JW, Li D, Revett T, Shih HH, Liu W, Paulsen JE, Gimeno RE. Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members. J Lipid Res 46: 2477–2487, 2005.[Abstract/Free Full Text]
  22. Langin D, Dicker A, Tavernier G, Hoffstedt J, Mairal A, Ryden M, Arner E, Sicard A, Jenkins CM, Viguerie N, van Harmelen V, Gross RW, Holm C, Arner P. Adipocyte lipases and defect of lipolysis in human obesity. Diabetes 54: 3190–3197, 2005.[Abstract/Free Full Text]
  23. Large V, Arner P, Reynisdottir S, Grober J, Van Harmelen V, Holm C, Langin D. Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells. J Lipid Res 39: 1688–1695, 1998.[Abstract/Free Full Text]
  24. Lass A, Zimmermann R, Haemmerle G, Riederer M, Schoiswohl G, Schweiger M, Kienesberger P, Strauss JG, Gorkiewicz G, Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome. Cell Metab 3: 309–319, 2006.[CrossRef][Web of Science][Medline]
  25. Lebovitz HE, Banerji MA. Point: visceral adiposity is causally related to insulin resistance. Diabetes Care 28: 2322–2325, 2005.[Free Full Text]
  26. 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. 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, 2001.[CrossRef][Web of Science][Medline]
  27. Lewis GF, Uffelman KD, Szeto LW, Weller B, Steiner G. Interaction between free fatty acids and insulin in the acute control of very low density lipoprotein production in humans. J Clin Invest 95: 158–166, 1995.[Web of Science][Medline]
  28. Liu YM, Moldes M, Bastard JP, Bruckert E, Viguerie N, Hainque B, Basdevant A, Langin D, Pairault J, Clement K. Adiponutrin: a new gene regulated by energy balance in human adipose tissue. J Clin Endocrinol Metab 89: 2684–2689, 2004.[Abstract/Free Full Text]
  29. Mairal A, Langin D, Arner P, Hoffstedt J. Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity. Diabetologia 49: 1629–1636, 2006.[CrossRef][Web of Science][Medline]
  30. Miyoshi H, Perfield JW 3rd, Souza SC, Shen WJ, Zhang HH, Stancheva ZS, Kraemer FB, Obin MS, Greenberg AS. Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes. J Biol Chem 282: 996–1002, 2007.[Abstract/Free Full Text]
  31. Okazaki H, Osuga J, Tamura Y, Yahagi N, Tomita S, Shionoiri F, Iizuka Y, Ohashi K, Harada K, Kimura S, Gotoda T, Shimano H, Yamada N, Ishibashi S. Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases. Diabetes 51: 3368–3375, 2002.[Abstract/Free Full Text]
  32. Osuga J, Ishibashi S, Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoiri F, Yahagi N, Kraemer FB, Tsutsumi O, Yamada N. 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, 2000.[Abstract/Free Full Text]
  33. Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD. Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37: 1020–1024, 1988.[Abstract]
  34. Ryden M, Jocken J, van Harmelen V, Dicker A, Hoffstedt J, Wiren M, Blomqvist L, Mairal A, Langin D, Blaak EE, Arner P. Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis. Am J Physiol Endocrinol Metab 292: E1847–E1855, 2007.[Abstract/Free Full Text]
  35. Schoenborn V, Heid IM, Vollmert C, Lingenhel A, Adams TD, Hopkins PN, Illig T, Zimmermann R, Zechner R, Hunt SC, Kronenberg F. The ATGL gene is associated with free fatty acids, triglycerides, and type 2 diabetes. Diabetes 55: 1270–1275, 2006.[Abstract/Free Full Text]
  36. Schrag JD, Cygler M. Lipases and alpha/beta hydrolase fold. Methods Enzymol 284: 85–107, 1997.[Web of Science][Medline]
  37. Schweiger M, Schreiber R, Haemmerle G, Lass A, Fledelius C, Jacobsen P, Tornqvist H, Zechner R, Zimmermann R. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism. J Biol Chem 281: 40236–40241, 2006.[Abstract/Free Full Text]
  38. Smirnova E, Goldberg EB, Makarova KS, Lin L, Brown WJ, Jackson CL. ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells. EMBO Rep 7: 106–113, 2006.[CrossRef][Web of Science][Medline]
  39. Subramanian V, Rothenberg A, Gomez C, Cohen AW, Garcia A, Bhattacharyya S, Shapiro L, Dolios G, Wang R, Lisanti MP, Brasaemle DL. Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes. J Biol Chem 279: 42062–42071, 2004.[Abstract/Free Full Text]
  40. Villena JA, Roy S, Sarkadi-Nagy E, Kim KH, Sul HS. 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, 2004.[Abstract/Free Full Text]
  41. Wang SP, Laurin N, Himms-Hagen J, Rudnicki MA, Levy E, Robert MF, Pan L, Oligny L, Mitchell GA. The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice. Obes Res 9: 119–128, 2001.[Web of Science][Medline]
  42. Watt MJ, Carey AL, Wolsk-Petersen E, Kraemer FB, Pedersen BK, Febbraio MA. Hormone-sensitive lipase is reduced in the adipose tissue of patients with type 2 diabetes mellitus: influence of IL-6 infusion. Diabetologia 48: 105–112, 2005.[CrossRef][Web of Science][Medline]
  43. Watt MJ, Dzamko N, Thomas WG, Rose-John S, Ernst M, Carling D, Kemp BE, Febbraio MA, Steinberg GR. CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK. Nat Med 12: 541–548, 2006.[CrossRef][Web of Science][Medline]
  44. Watt MJ, Hevener A, Lancaster GI, Febbraio MA. Ciliary neurotrophic factor prevents acute lipid-induced insulin resistance by attenuating ceramide accumulation and phosphorylation of c-Jun N-terminal kinase in peripheral tissues. Endocrinology 147: 2077–2085, 2006.[Abstract/Free Full Text]
  45. Watt MJ, Holmes AG, Pinnamaneni SK, Garnham AP, Steinberg GR, Kemp BE, Febbraio MA. Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue. Am J Physiol Endocrinol Metab 290: E500–E508, 2006.[Abstract/Free Full Text]
  46. Williams CM. Lipid metabolism in women. Proc Nutr Soc 63: 153–160, 2004.[CrossRef][Web of Science][Medline]
  47. Xu G, Sztalryd C, Londos C. Degradation of perilipin is mediated through ubiquitination-proteasome pathway. Biochim Biophys Acta 1761: 83–90, 2006.[Medline]
  48. Xu G, Sztalryd C, Lu X, Tansey JT, Gan J, Dorward H, Kimmel AR, Londos C. Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem 280: 42841–42847, 2005.[Abstract/Free Full Text]
  49. Zang H, Ryden M, Wahlen K, Dahlman-Wright K, Arner P, Hirschberg AL. Effects of testosterone and estrogen treatment on lipolysis signaling pathways in subcutaneous adipose tissue of postmenopausal women. Fertil Steril 88: 100–106, 2007.[CrossRef][Web of Science][Medline]
  50. Zechner R, Strauss JG, Haemmerle G, Lass A, Zimmermann R. Lipolysis: pathway under construction. Curr Opin Lipidol 16: 333–340, 2005.[Web of Science][Medline]
  51. Zhou YP, Grill VE. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93: 870–876, 1994.[Web of Science][Medline]
  52. Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306: 1383–1386, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
K. Kantartzis, A. Peter, F. Machicao, J. Machann, S. Wagner, I. Konigsrainer, A. Konigsrainer, F. Schick, A. Fritsche, H.-U. Haring, et al.
Dissociation Between Fatty Liver and Insulin Resistance in Humans Carrying a Variant of the Patatin-Like Phospholipase 3 Gene
Diabetes, November 1, 2009; 58(11): 2616 - 2623.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
R. C. R. Meex, P. Schrauwen, and M. K. C. Hesselink
Modulation of myocellular fat stores: lipid droplet dynamics in health and disease
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2009; 297(4): R913 - R924.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. Schweiger, A. Lass, R. Zimmermann, T. O. Eichmann, and R. Zechner
Neutral lipid storage disease: genetic disorders caused by mutations in adipose triglyceride lipase/PNPLA2 or CGI-58/ABHD5
Am J Physiol Endocrinol Metab, August 1, 2009; 297(2): E289 - E296.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
G. Mantovani, S. Bondioni, L. Alberti, L. Gilardini, C. Invitti, S. Corbetta, M. A. Zappa, S. Ferrero, A. G. Lania, S. Bosari, et al.
Protein Kinase A Regulatory Subunits in Human Adipose Tissue: Decreased R2B Expression and Activity in Adipocytes From Obese Subjects
Diabetes, March 1, 2009; 58(3): 620 - 626.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
R. Zechner, P. C. Kienesberger, G. Haemmerle, R. Zimmermann, and A. Lass
Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores
J. Lipid Res., January 1, 2009; 50(1): 3 - 21.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
P. Wang, E. Smit, M. C G J Brouwers, G. H Goossens, C. J H van der Kallen, M. M J van Greevenbroek, and E. C M Mariman
Plasma pigment epithelium-derived factor is positively associated with obesity in Caucasian subjects, in particular with the visceral fat depot
Eur. J. Endocrinol., December 1, 2008; 159(6): 713 - 718.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/E958    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (21)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steinberg, G. R.
Right arrow Articles by Watt, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinberg, G. R.
Right arrow Articles by Watt, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.