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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 3 1352-1358
Copyright © 2004 by The Endocrine Society

Perilipin Expression in Human Adipose Tissue Is Elevated with Obesity

Philip A. Kern, Gina Di Gregorio, Tong Lu, Negah Rassouli and Gouri Ranganathan

Central Arkansas Veterans Healthcare System and Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Philip A. Kern, M.D., Central Arkansas Veterans Healthcare System, 598/151 LR, 4300 West 7th Street, Little Rock, Arkansas 72205. E-mail: kernphilipa{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The perilipins are highly phosphorylated adipocyte proteins that are localized at the surface of the lipid droplet. With activation by protein kinase A, perilipins translocate away from the lipid droplet and allow hormone-sensitive lipase to hydrolyze the adipocyte triglycerides to release nonesterified fatty acids (NEFA). Because of the potential importance of adipocyte lipolysis to obesity and insulin resistance, we measured perilipin protein and mRNA levels in nondiabetic subjects with varying degrees of insulin resistance. By Northern and Western blotting, we could detect perilipin A, but not perilipin B. Perilipin A protein and mRNA levels were quantitated and were highly correlated with each other. There was a significant positive relationship between perilipin expression and obesity (r = 0.55; P < 0.01, perilipin mRNA vs. percent body fat). However, there was no significant relationship between perilipin expression and blood NEFA, nor was there a significant relationship between perilipin expression and insulin resistance, using the insulin sensitivity index derived from the iv glucose tolerance test with minimal modeling. In addition, there was no significant relationship between perilipin and adipocyte or systemic inflammatory markers, such as TNF{alpha}, IL-6, and adiponectin. Thus, perilipin was elevated in obese subjects, perhaps as a compensatory mechanism to limit basal lipolysis. However, there was no relationship between perilipin and insulin resistance.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
AS THE PREVALENCE of obesity and type 2 diabetes continues to increase, much research has been focused on the development of obesity-related insulin resistance, which is an antecedent to type 2 diabetes. The expansion of adipose tissue mass with obesity probably plays a role in the development of peripheral insulin resistance (1), either through the dysregulation of adipokines such as TNF{alpha}, IL-6, and adiponectin or through the elevation of plasma nonesterified fatty acids (NEFA) by increased activity of hormone-sensitive lipase-mediated lipolysis (2, 3).

The perilipins are highly phosphorylated proteins in adipocytes that are not secreted, but are localized at the surface of the lipid droplet (4). After activation of protein kinase A by cAMP, perilipin is phosphorylated, resulting in translocation of the protein away from the surface of the lipid droplet, allowing hormone-sensitive lipase to hydrolyze the triglyceride core (5). In 3T3-L1 adipocytes, the stimulation of lipolysis by TNF{alpha} was associated with a dispersal of perilipin from the surface of the lipid droplet along with a decrease in the total cellular expression of perilipin (6). In addition, the overexpression of perilipin in adipocytes inhibited TNF-mediated lipolysis (6). In perilipin knockout mice, basal adipocyte lipolysis was increased, resulting in a lean mouse that was resistant to diet-induced obesity and reversed the obesity associated with the db/db mouse (7, 8). However, these mice also developed glucose intolerance and insulin resistance more readily, probably due to the elevated levels of NEFA (8).

Perilipin exists as two isomers, perilipin A and B, which share a common N-terminal amino acid sequence; perilipin A is the predominant form expressed in adipocytes (9). Little data exist on the regulation of perilipin, and few studies have examined perilipin expression in humans. Because of the potential importance of the perilipins as mediators of lipolysis, with potential importance to adipocyte lipid accumulation and insulin resistance, we examined the mRNA and protein expression of perilipin in humans covering a wide range of obesity and insulin resistance. An important goal of this study was to determine whether there were a relationship between perilipin and TNF{alpha} expression in adipocytes. TNF{alpha} expression is associated with insulin resistance (10), possibly through an enhancement of adipocyte lipolysis (11), and high expression of perilipin in humans could serve to limit lipolysis and therefore prevent insulin resistance (12, 13). However, in these studies we found increased mRNA and protein levels of perilipin in obese subjects; however, there was no consistent relationship with insulin resistance or TNF{alpha} expression.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

This study involved 44 nondiabetic subjects, all of whom were in good health, weight stable, and euthyroid. All subjects gave informed consent, and this research was approved by the institutional review board. These subjects were taking no medications, with the exception of hormone replacement therapy in two women. Menstruating women were all studied during the follicular stage of their cycles. The ages ranged from 25–58 yr; 39 subjects were women, five were men, and six were African American. None of the subjects consumed significant alcohol, and three used tobacco, but no tobacco had been used for 12 h before the studies. All subjects had an oral glucose tolerance test using 75 g glucose. Of the 44 subjects, 18 had impaired glucose tolerance based on a 2-h glucose level of 140–200, and three of these subjects had impaired fasting glucose based on a fasting glucose level of 110–126. Subjects then underwent a frequently sampled iv glucose tolerance test and an adipose tissue biopsy, which were performed on separate days.

The characteristics of the subjects of this study are shown in Table 1Go. Blood lipids, glucose, and hemoglobin A1c were measured using standard clinical assays. The subjects ranged from lean to very obese [body mass index (BMI) range, 21–65]; some subjects demonstrated moderate dyslipidemia, but no subject demonstrated fasting triglycerides over 400 mg/dl. Body composition was determined using bioelectric impedance (14).


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  		TABLE 1. Baseline characteristics of the 44 subjects

 
Measurement of insulin sensitivity

Insulin sensitivity was measured in the fasting state using the classic tolbutamide-modified minimal model analysis of the frequently sampled iv glucose tolerance test (15, 16), which has been validated against the euglycemic clamp (17), as described previously (18, 19). Glucose was measured using the glucose oxidase method in a glucose analyzer, and insulin was measured using RIA (Indiana University School of Medicine, Indianapolis, IN). The insulin sensitivity index (SI) was calculated using the MINMOD program, along with the acute insulin response to glucose (16).

Adipose tissue biopsy

Abdominal sc adipose tissue was removed from each patient by an incisional biopsy from the lower abdominal wall. Some of the tissue was immediately frozen in liquid N2 for later RNA extraction; the rest of the tissue was placed into cold DMEM for other assays. To measure the secretion of cytokines from adipose tissue, approximately 500 mg adipose tissue were minced and placed into serum-free DMEM (pH 7.4; 10 mM HEPES) at 37 C for varying times, as described previously (10). To compare cytokine secretion among different subjects, we measured cytokine levels in the medium after 2 h at 37 C. All data were normalized to either adipose DNA content (20) or cell number to control for differences in fat cell size. Cell number was measured using the method described by DiGirolamo et al. (21).

Perilipin expression

Perilipin mRNA levels were measured by Northern blot. Adipose tissue was frozen at -80 C, and RNA was extracted according to modifications of the method described by Chomczynski and Sacchi (22). The quality of the RNA was verified by ethidium bromide staining of rRNA bands on a minigel, and Northern blotting was performed using 32P-labeled human perilipin cDNA (9). Loading of the blots was normalized to the rRNA levels, based on ethidium bromide staining; the image intensities were quantitated using the Eagle Sight 3.0 Image Capture and analysis software (Stratagene, La Jolla, CA); and the ratio of perilipin/rRNA was calculated.

To measure perilipin protein, Western blotting was performed. Approximately 500 mg frozen adipose tissue were homogenized in lysis buffer (6). To extract the perilipin from the adipocyte lipid, the samples were vigorously vortexed over 1 h at 37 C as described previously (4). The sample was centrifuged at 12,000 x g for 15 min at room temperature, and the solubilized material was separated from the lipid layer. This material was then electrophoresed using a 10% polyacrylamide gel. After transfer to nitrocellulose, the gel was blotted. Gels were blotted with a rabbit antiserum that was directed against a common region of perilipin A and B and therefore recognized both perilipin isoforms or with an antibody that was specific to perilipin A (6). The secondary antibody was an antirabbit peroxidase antibody. The antiperilipin antibodies were supplied by Dr. Andrew Greenberg (Tufts University, Boston, MA). Western blots were also performed using antiphosphoserine antibodies (Sigma-Aldrich Corp., St. Louis, MO). After blotting with antiperilipin antibodies, the nitrocellulose was stripped and reblotted with an antibody to ß-actin (Sigma-Aldrich Corp.). In additional methodological experiments, we found that ß-actin expression correlated strongly with the DNA content of the sample and therefore was a good indicator of cell number. The images were quantitated as described above, and perilipin protein levels were expressed in relation to actin.

Measurement of cytokine expression

Adiponectin was measured by RIA (Linco Research, Inc., St. Charles, MO). TNF{alpha} and IL-6 protein was measured using ELISAs (R&D Systems Minneapolis, MN), and leptin was measured by RIA (Linco Research, Inc.). TNF{alpha} mRNA levels were measured as described previously, using competitive RT-PCR with a cRNA probe (23). Data were expressed as the copy number per microgram of total RNA.

Statistics

All data are expressed as the mean ± SEM. Analysis of trends was performed using linear regression.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The characteristics of the study subjects are shown in Table 1Go. The 44 subjects covered a spectrum fromn lean to obese, and none was older than 58 yr. In keeping with a population of normal and obese subjects, blood glucose and triglycerides ranged from low to the moderately elevated range.

To demonstrate the presence of perilipin mRNA in human adipose tissue, Northern blotting was performed. As shown in Fig. 1Go, a single band was recognized at 3.0 kb, which corresponded to the mRNA of perilipin A. Using this method, we could detect no evidence of the larger 3.9-kb band for perilipin B, which has been described in rat adipocytes (9). To demonstrate the presence of the perilipin protein in adipose tissue, Western blotting was performed. Initial Western blots used an antibody raised to the common region of both perilipin A and B. In Western blots with this antibody, a predominant band at 64 kDa was identified, with a minor band at 50 kDa (Fig. 2Go, left panel). Because rodent adipose tissue expresses both perilipin A and B, we wanted to better determine the identity of the faster migrating band in these gels. Because both perilipin A and B are highly phosphorylated, Western blotting was performed using antiphosphoserine antibodies. As shown in Fig. 2Go (right panel), only the 62-kDa band was identified by the antiphosphoserine antibodies. Finally, additional blots were performed with an antibody that was specifically directed against the unique region of perilipin A, which would not be expected to identify perilipin B. As shown in Fig. 3Go, most gels demonstrated only the 64-kDa band, although occasional samples also contained the 50-kDa band. Thus, the 50-kDa band represented an unphosphorylated protein that was identified by antibodies to perilipin A/B and also in some samples with antibodies that were specific to perilipin A. Together with the Northern blots, these data suggest that perilipin B is not expressed in human adipose tissue at levels detectable using these methods.



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  		FIG. 1. Northern blotting for perilipin. The 3.0-kb perilipin mRNA is demonstrated in this representative Northern blot of two lean and two obese subjects. Equal loading was confirmed by ethidium bromide staining for rRNA.

 


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  		FIG. 2. Western blotting of perilipin. Human adipose tissue samples were analyzed by SDS-PAGE and blotted with either antiperilipin antibodies, using an antibody to the common region of both perilipin A and B, or an anti-phosphoserine antibody, as indicated.

 


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  		FIG. 3. Western blotting using antiperilipin A antibodies. A, Using antibodies specific to perilipin A, samples from lean and obese subjects were analyzed by SDS-PAGE and Western blotting. The samples shown are from two lean and two obese subjects. B, The blots were stripped and reblotted with antibodies to ß-actin.

 
The mRNA and protein expressions of perilipin were quantitated as described in Subjects and Methods. As shown in Fig. 4Go, there was a strong correlation between the level of expression of perilipin mRNA and that of perilipin protein, providing no evidence for regulation of perilipin translation. Both perilipin protein levels and mRNA levels demonstrated a significant positive relationship with measures of obesity. As shown in Fig. 5AGo, there was a significant positive relationship between perilipin mRNA levels and obesity, using percent body fat (r = 0.55; P < 0.01). A similar relationship was observed with perilipin protein and percent body fat (Fig. 5BGo). There was also a significant correlation between BMI and perilipin protein (r = 0.47; P < 0.01; data not shown). These relationships remained statistically significant when only females were analyzed, except for perilipin mRNA, for which the correlation decreased to r = 0.47 (P < 0.1). To determine whether there were any differences between males and females in perilipin expression, we matched five male subjects to five female subjects according to age (±5 yr) and BMI (±3 U). As shown in Table 2Go, there were no significant differences between the males and females in age or BMI or in the expected increase in percent body fat in the females. In these subjects, there was no significant gender-related difference in perilipin protein.



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  		FIG. 4. Relationship between perilipin protein, using perilipin Western blots, and perilipin mRNA levels, using Northern blotting (r = 0.74; P < 0.001).

 


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  		FIG. 5. Relationship between obesity and perilipin expression. Relationship between perilipin mRNA levels (A) and perilipin protein levels (B) in adipose tissue in relation to obesity using percent body fat.

 

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  		TABLE 2. Perilipin protein in men and women matched for age and BMI

 
Many previous studies have demonstrated significant relationships between obesity, using either BMI or percent body fat, and insulin resistance or markers of insulin resistance, and the subjects in this study demonstrated a significant inverse relationship between BMI and SI (r = -0.58; P < 0.001). However, in this study we found no significant relationship between either perilipin mRNA levels or perilipin protein and markers of insulin sensitivity. Table 3Go lists the partial correlation coefficients between perilipin expression and markers of insulin resistance. The relationships between perilipin protein or mRNA and SI, TNF, IL-6, adiponectin, and NEFA were not statistically significant. There was a significant association between perilipin protein expression and plasma leptin (r = 0.41; P < 0.05); however, this relationship was not supported by an association between leptin and perilipin mRNA levels.


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  		TABLE 3. Correlation coefficients with perilipin expression

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Perilipin is bound to the lipid droplet in adipocytes, and phosphorylation of perilipin by protein kinase A results in improved access of hormone-sensitive lipase to the lipid droplet and, hence, increased lipolysis (4, 24). Studies involving the overexpression of perilipin in adipocytes resulted in decreased lipolysis (25), and perilipin knockout mice demonstrated increased levels of basal lipolysis, resulting in a lean mouse (8). In addition, an important interaction has been described between perilipin and TNF{alpha}. Overexpression of perilipin in 3T3-L1 adipocytes resulted in decreased TNF{alpha}-mediated lipolysis (6). TNF{alpha} expression from human adipose tissue is elevated in obese humans (23, 26), and elevated TNF{alpha} secretion from adipose tissue is associated with insulin resistance (10). However, the mechanism by which TNF{alpha} may be associated with insulin resistance is not known. Because TNF{alpha} stimulates lipolysis (27), however, it is possible that elevated TNF expression leads to elevated plasma NEFA, leading to insulin resistance (28, 29). Perilipin could therefore play an important role in preventing part of the TNF{alpha}-mediated lipolysis and insulin resistance. To date, perilipin has been measured in two studies involving humans (30, 31). In one study, sc fat from very obese subjects undergoing bariatric surgery was compared with fat from lean subjects, from whom fat was obtained by biopsy (30). Perilipin mRNA levels were lower in obese subjects, and perilipin protein was decreased in obese subjects when expressed as perilipin per unit surface area, but perilipin protein was unchanged with obesity when expressed as perilipin per 105 cells. In another study perilipin protein levels in isolated adipocytes of lean and obese women were compared (31). The obese subjects demonstrated less perilipin protein, and this lower level of adipocyte perilipin was associated with a higher level of lipolysis in the cells.

In this study perilipin expression by human sc adipose tissue was examined in nondiabetic subjects who covered a broad array of obesity and insulin sensitivity. We found that perilipin mRNA levels correlated well with perilipin protein levels measured by Western blotting. Although perilipin is a highly phosphorylated protein that undergoes considerable posttranslational modification (4), these data suggest that translational regulation of perilipin does not occur, as has been described with leptin and lipoprotein lipase (32, 33).

Previous studies have demonstrated that adipocytes express predominantly perilipin A, although rat adipose tissue expresses a small amount of perilipin B (9). In this study we could find no evidence for the expression of perilipin B in human adipose tissue. A perilipin B transcript was not found by Northern blotting. In Western blots, a band at 50 kDa, which is the approximate size of perilipin B, was identified in some samples. However, this band was not phosphorylated, as would be expected with perilipin B, and was identified in some samples even when blotted with a perilipin A-specific antibody. Thus, these data suggest that this faster migrating band is a perilipin A breakdown product, an unphosphorylated form of perilipin A, or a cross-reacting irrelevant protein.

The subjects in this study covered a broad range of obesity and were well characterized. In these subjects, perilipin mRNA and protein expression correlated with the degree of obesity, with obese subjects demonstrating higher levels of perilipin protein and mRNA. These data differ from those reported by others, where obese subjects demonstrated decreased levels of perilipin (30, 31). The reasons for these differences are not readily apparent. This study examined perilipin in whole adipose tissue in subjects covering a continuum of BMI rather than in separate subjects from lean and very obese groups. Only sc adipose tissue was examined, and therefore, this study can draw no conclusions about other adipose tissue depots, nor can this study address perilipin expression in relation to lipolysis in isolated adipocytes, as was measured by Mottagui-Tabar et al. (31).

Because of the well described association between leptin and obesity (34), it is not surprising that there were also a significant relationship between perilipin protein levels and blood leptin. Obesity is associated with higher levels of blood NEFA and increased adipocyte basal lipolysis (35). Although other studies demonstrated an inverse correlation between perilipin expression and lipolysis (31), this study did not observe such a relationship. Although the reason for this apparent discrepancy is not clear (36), these data would suggest that the elevated expression of perilipin in obesity is not sufficient to restrain lipolysis to levels seen in leaner subjects.

Even though the obese subjects in this study were more insulin resistant than the lean subjects, there was no association between perilipin expression and insulin sensitivity, using the SI derived from the IVGTT and the minimal model. In addition, there was no significant relationship between perilipin expression and TNF expression, and blood levels of NEFA, IL-6, or adiponectin and in vitro adipocyte secretion of IL-6, leptin, and adiponectin also were not significantly associated with perilipin expression.

In previous studies plasma levels of IL-6 and adiponectin and tissue secretion of TNF{alpha} were highly correlated with insulin resistance independent of any association with obesity (10, 18, 37). However, the mechanism of obesity-associated insulin resistance and the potential role of adipocyte secretory cytokines are not well understood. One potential mechanism for the relationship between obesity and insulin resistance is increased adipose tissue lipolysis, which would then induce peripheral insulin resistance (38). An increase in perilipin expression could play an important role in controlling adipocyte lipolysis in obesity, particularly in response to increased adipose tissue TNF{alpha} expression. However, this study failed to find evidence that perilipin was associated with any index of cytokine expression or insulin sensitivity.

In summary, perilipin A was highly expressed by human adipose tissue, and the level of expression was determined in nondiabetic subjects. Obese subjects demonstrated higher levels of perilipin expression; however, there was no relationship between perilipin and insulin sensitivity or cytokine expression. Although perilipin knockout and overexpression experiments demonstrated the importance of perilipin in lipolysis, it is unclear what role perilipin plays in the regulation of human obesity and insulin resistance.


   Acknowledgments
 
We thank Dr. Andrew Greenberg for supplying the antiperilipin antibodies and cDNA. We especially thank the many subjects who volunteered for this project. We also thank Leslie Miles and the staff and nurses on the General Clinical Research Center for their assistance with recruiting and patient care, and Sarah Dunn for secretarial assistance.


   Footnotes
 
This work was supported by a Merit Review Grant from the Veterans Administration, Grant M01-RR-14288 from the General Clinical Research Center, and NIH Grant DK-39176.

Abbreviations: BMI, Body mass index; NEFA, nonesterified fatty acid; SI, insulin sensitivity index.

Received August 18, 2003.

Accepted December 4, 2003.


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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Hyperinsulinemia Induces Insulin Resistance on Glucose and Lipid Metabolism in a Human Adipocytic Cell Line: Paracrine Interaction with Myocytes
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Regulation of Adipocyte Lipolysis by Degradation of the Perilipin Protein: NELFINAVIR ENHANCES LYSOSOME-MEDIATED PERILIPIN PROTEOLYSIS
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Obese Subjects Carrying the 11482G>A Polymorphism at the Perilipin Locus Are Resistant to Weight Loss after Dietary Energy Restriction
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