1 The Department of Surgery, The George Washington University Medical ... 3 The George Washington University School of Medicine, Washington, D.C., USA.
Surg Endosc (2007) 21: 941–944 DOI: 10.1007/s00464-006-9098-3 Springer Science+Business Media, LLC 2007
Expression of ectonucleotide pyrophosphate phosphodiesterase and peroxisome proliferator activated receptor gamma in morbidly obese patients Fred Brody,1,2 Sarah Hill,3 Scott Celenski,1 Ryan Kar,3 Brian Kluk,2 Joe Pinzone,2 Sidney Fu2 1
The Department of Surgery, The George Washington University Medical Center, 2150 Pennsylvania Avenue, NW, Suite 6B, Washington, D.C., 20037, USA 2 The Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 Eye St., NW, Ross Hall 520, Washington, D.C., 20037, USA 3 The George Washington University School of Medicine, Washington, D.C., USA Received: 22 April 2006/Accepted: 12 October 2006/Online publication: 8 February 2007
Abstract Background: Recently, two genes, peroxisome proliferator activated receptor gamma (PPARc) and ectonucleotide pyrophosphate phosphodiesterase (ENPP1), have been localized and associated with diabetes and obesity. This report hypothesizes that there is a correlation between the genetic expression of ENPP1 and PPARc from gastrointestinal tissue and body mass index (BMI). Methods: Preoperative demographic data were collected from 16 severely morbidly obese patients. Extraneous gastrointestinal tissue was obtained during laparoscopic gastric bypass and gastric banding procedures. The tissue was snap frozen in liquid nitrogen. Initially, RNA extraction was performed on the tissue, followed by reverse transcription using appropriate primers and controls. Subsequently, the samples were subjected to quantitative polymerase chain reaction (PCR). Preoperative demographic data were analyzed for their influence on ENPP1 and PPARc expression using multivariate analysis and logistic regression models. Results: Expression of PPARc and ENPP1 was found in all samples. There was a higher level of PPARc expression in omental tissue than in enteric tissue. There was no significant difference in the expression of ENPP1 among the different tissue types. The relative level of PPARc expression in small bowel and gastric tissue was found to be inversely proportional to body mass index (BMI) using linear regression analysis (p = 0.01; r2 = 0.586). Similarly, PPARc expression from omental tissue showed an inverse relationship with BMI (p = 0.04; r2 = 0.576). The levels of ENPP1 expression did not show a correlation with BMI (p = 0.25). Conclusion: The results suggest that increasing obesity correlates with a decrease in PPARc expression. This Correspondence to: Fred Brody
decrease may induce dysfunctional adipocyte differentiation, maturation, and function, leading to diabetes and the metabolic syndrome. Similarly, the increased volume of adipose tissue may lead to a downregulation of PPARc. The lack of correlation between ENPP1 and BMI may suggest that glucose metabolism is more complex than lipid metabolism. Further evaluation is warranted to establish metabolic pathways for glucose and lipid biomarkers. Key words: Bariatric — ENPP1 — Obesity — PPAR
The obesity and diabetic epidemics have engendered a wave of research regarding fat and glucose metabolism. Over the past decade, key regulators of energy balance and insulin signaling have clarified many of the biologic pathways of fat and glucose metabolism [1]. Some of these regulators belong to the peroxisome proliferator activated receptor (PPAR) family of genes. These regulators play a role in lipid and glucose homeostasis. Notably, expression of PPAR in adipose tissue is 10 to 30 times higher than in muscle or liver [2]. As transcription factors, PPARs control the expression of a network of genes related to obesity [3, 4]. Furthermore, studies in mice document insulin resistance after deletion of PPARc in apidose and muscle tissue [5, 6]. In humans, activation of PPAR with the thiazolidinedione (TZD) family of oral antidiabetic medications increases insulin sensitivity in hepatic and adipose tissue. Clinically, this lowers postprandial glucose and fatty acid concentrations [7, 8]. Finally, PPARc is implicated in adipocyte development, differentiation, and lipid storage. This gene may be partially responsible for the seemingly unlimited capacity of adipocytes to store fat.
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Similarly, ectonucleotide pyrophosphate phospodiesterase (ENPP1) has been identified as an insulinresistant gene associated with obesity. Variants of this gene mediate insulin resistance as well as the development of obesity and type 2 diabetes. These two components, PPARc and ENPP1, interact in the overall homeostasis of obesity and its associated physiologic changes. At this writing, little data exist regarding actual tissue levels in humans [9]. By understanding the role of PPARc and ENPP1 in various tissues from obese patients, it may be possible to control fat and glucose metabolism pharmacologically [10]. This study sought to characterize the expression of PPARc and ENPP1 in obese patients by analyzing extraneous gastrointestinal tissue from morbidly obese patients undergoing Roux-en-Y gastric bypasses and LapBand procedures. Methods Before this project began, institutional review board approval was obtained from the George Washington University Medical Center Review Board (IRB#050408ER).
Tissue collection Extraneous gastrointestinal tissue from morbidly obese patients, with and without diabetes, undergoing bariatric surgery was collected and examined. The tissue was snap frozen in liquid nitrogen and stored in a )80C freezer for further analysis. No buffers were added at this time. The tissue samples included gastric body, omentum, and full-thickness samples of small intestine.
RNA extraction Tissue samples measuring 50 to 100 mg were placed in a flat-bottom tube with 1 ml of TriZol reagent (Invitrogen; Carlsbad, CA). The tissue was homogenized with the reagent. The homogenate was transferred to a 1.7-ml tube and centrifuged at 12,000 rpm at 4C for 10 min. Next, 500 ll of the supernatant containing RNA was transferred to a fresh 1.7-ml tube and incubated at room temperature for 5 min. By vigorous hand shaking, 200 ll of chloroform was mixed with the sample and incubated at room temperature for 3 min. The tube then was centrifuged at 12,000 rpm at 4C for 15 min. The aqueous phase, which now contained the RNA, was transferred to a fresh 1.7-ml tube. To precipitate the RNA, 500 ll of isopropyl alcohol was added, and the sample was incubated at room temperature for 10 min. The sample was centrifuged at 7,500 rpm at 4C for 10 min. The supernatant was discarded, and the RNA pellet was dried at room temperature for 5 min. The pellet was redissolved in 10 ll of RNase free water and incubated at 58C for 10 min. Total RNA quality and concentration then were measured using spectrophotometry. Next, the RNA samples were purified of residual DNA using DNase I (Invitrogen) and converted to cDNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).
Reverse transcription and QPCR Real-time PCR (RT-PCR) was performed on all cDNA samples for PPARc and ENPP1. The 18S gene was used as an internal control to normalize the expression of PPARc and ENPP1. A series dilution of RT-PCR product of the target gene was used to generate a standard curve to quantitate the expression level of each gene. The ABI 7300 System (Applied Biosystems, Foster City, CA). was used for quantitative analysis. Reverse transcription reaction was performed as follows. Initially, 1 lg of DNase-treated total RNA, 0.5 lg of anchored oligo(dT) pri-
mer, and 500 mol/l of dNTPs (NEB) were heated for 5 min at 65C. Next, 1x first-strand buffer (Invitrogen), 0.01 mol/l of dithiothreitol, and 200 units of Superscript II were added, and reverse transcription was performed in a 20-ll reaction for 50 min at 42C and terminated by heating for 15 min at 70C. To assess for potential contamination of solutions, a control containing all reagents, but devoid of RNA, was included. In addition, a control containing all reagents except Superscript II was included for each sample to monitor for possible residual genomic DNA in the RNA preparations. The quantitative RT-PCR was performed using the fluorescent dye SYBR Green Master Mix, Valencia, CA following standard protocols on an ABI 7300 sequence detection system. The primers used for the PCR were designed using Integrated DNA Technologies, Inc. (Coralville, IA, USA). The following primers were used: ENPP1 forward (5¢-CCCTCTTTGTTGGCTATGGA-3¢), ENPP1 reverse (5¢-TAGGAGCCGGTGTCAAATTC-3¢), PPARc forward (5¢-TTCAGAAATGCCTTGCAGTG-3¢), and PPARc reverse (5¢-CCAACAGCTTCTCCTTCTCG-3¢). The data were analyzed using the Sequence Detector Software SDS 2.0 (Applied Biosystems). Coamplification of the 18S housekeeping gene was used to normalize the amount of RNA present. The 18S primers were 18S forward (5¢-CCGCAGCTAGGAATAATGGA3¢) and reverse (5¢-CCCTCTTAATCATGGCCTCA-3¢). All the PCRs were performed twice in triplicate.
Statistical analysis Preoperative demographic and laboratory data were used for their influence on ENPP1 and PPARc expression using univariate analysis and logistic regression models. Only those factors with a p value less than 0.1 were included in the multivariate analysis.
Results Demographic data including comorbidities such as diabetes and body mass index (BMI) were documented for 16 patients together with their preoperative laboratory values. A variety of tissue samples including gastric, omental, hepatic, and small intestine were obtained from patients during the gastric bypass or LapBand procedure. There were 15 females with a mean age of 45 years. The mean BMI was 45.04, and there were seven diabetic patients. For each tissue sample, total RNA was exacted using TriZol reagent (Invitrogen) and reverse transcribed into cDNA. Real-time PCR (ABI 7300 System, Foster City, CA) was performed using primers specific to ENPP1 and PPARc, and the expression level was normalized using the 18S gene. The expression of PPARc and ENPP1 was present in all samples. The relative level of PPARc expression from gastric and small intestine was found to be inversely proportional to BMI using linear regression analysis (p = 0.01; r2 = 0.586) (Fig. 1). Similarly, PPARc expression from omental tissue showed an inverse relationship with BMI (p = 0.04; r2 = 0.576). The levels of ENPP1 expression did not show a correlation with BMI (p = 0.25). The level of expression was higher in the omental tissue than in small intestine and gastric tissue samples. Conclusion The obesity epidemic has reached worldwide proportions because of sedentary lifestyles, diets, cultures, and
PPAR
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Dysfunctional Adipocytes
PPAR expression vs. BMI omental and small bowel/gastric tissue Omental Tissue Small Boweland Gastric
↓ PPAR Y
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48
50
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- ↑ BMI (visceral adipose tissue) - Insulin Resistance - Type 2 Diabetes
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↑ Insulin Sensitivity
BMI
Fig. 1. The relative expression of peroxisome proliferator activated receptor gamma (PPARc) from omental, small bowel, and gastric tissue versus body mass index (BMI). The expression of PPARc from these tissue samples was inversely proportional to BMI. The expression of PPARc from omental tissue was higher than in enteric tissue.
TZDs
↑ PPAR Y ↑ Subcutaneous adipose tissue
Fig. 2. The relationship between obesity, adipocytes, type 2 diabetes, and thiazolidinediones.
genetics [11]. In the United States, diets and exercise are used to control obesity. However, both of these methods fail to sustain long-term weight loss. Only surgery can induce a sustained long-term weight loss with resolution of comorbidites [17–19]. Because of the obesity epidemic, bariatric surgery has proliferated over the past decade. Yet these bariatric surgery procedures entail a definitive rate of morbidity and mortality [19]. Concurrently, obesity research in the past decade has delineated detailed mechanisms regarding gene and hormonal expression associated with adipogenesis [20, 21]. Several of these mechanisms entail regulators associated with energy balance, inflammation, and insulin expression [12]. Genetically altered obese (ob/ob) mice have provided a large amount of data regarding these various mechanisms. Quite a few of the genes expressed during normal adipocyte differentiation are decreased significantly in obese mice. These genes include the transcription factors PPARc, sterol regulatory element binding protein (SREBP), and CCAAT/enhancer-binding proteins (C/EBP). Moreover, certain proteins such as collagen and pro-a are increased significantly with obesity [16]. Overall, obese mice exhibit a reverse pattern of gene expression and protein synthesis. Ultimately, the adipocytes of obese mice are abnormal in terms of fatty acid synthesis and insulin response. The abnormal fatty acid synthesis shifts the lipogenic burden to the liver, whereas the altered insulin response is associated with type 2 diabetes. Thus, obesity is related to a complex set of gene expression that results in altered adipocyte differentiation, maturation, expression, and insulin resistance. However, it is unclear whether obese patients with higher BMI inherently express low levels of PPARc or whether low PPARc levels cause excess adipose tissue and an increase in BMI. Regardless of the relationship, decreased PPARc levels and increased BMI are associated with an increase in dysfunctional adipocytes. Ultimately, this scenario creates a cycle that results in glucose intolerance, which in some patients may lead to type 2 diabetes (Fig. 2). Although, our research did not
analyze the functionality of these adipocytes, we did document the inverse relationship between PPARc and BMI. Interestingly, we documented a higher expression of PPARc from omental tissue than from enteric tissue. As noted earlier, this is consistent with previous data. As the number of functional adipocytes decreases with increasing BMI, there is a decrease in the lipogenic expression of these cells as well. The decrease in lipogenic activity causes an obligatory increase in lipogenic gene expression in the liver [15]. This obligatory increase induces hepatic steatosis and is associated with hyperglycemia and type 2 diabetes. Quite possibly, patients who can upregulate their hepatic lipogenic activity without hepatic steatosis may be able to avoid type 2 diabetes. Alternatively, the addition of normal, mature adipocytes or a decrease in the number of abnormal adipocytes may prevent type 2 diabetes. These theories have yet to be tested in humans, and no data exist to unify these theories in animals. Medications such as TZDs that activate PPARc increase subcutaneous adipose tissue and improve postprandial glucose and fatty acid levels. Theoretically, by activating PPARc and increasing the number of functionally normal adipocytes, TZDs address the underlying cause of insulin resistance (Fig. 2). Our data point to a correlation between PPARc levels and BMI, further supporting the use of PPARc-activating medications in the treatment of obese individuals, regardless of diabetic status. At this writing, there is some limited human data to support the expansive research documented in mice. A significant association has been documented between ENPP1 and childhood and adult obesity. Furthermore, findings show that ENPP1 directly inhibits the insulininduced conformational changes in the insulin receptor [13, 14]. These changes affect the activation of the insulin receptor and the downstream signaling associated with glucose homeostasis. Thus, ENPP1 provides genetic evidence that links the molecular mechanisms of childhood obesity and type 2 diabetes [15]. However, a single gene theory to explain the link between obesity and type 2 diabetes is difficult to comprehend. More
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than likely, a series of concurrent genetic cascades contribute to this complex interaction. The lack of correlation with ENPP1 and BMI in our study suggests that glucose metabolism may be more complex than lipid metabolism. Obviously, further evaluation of these relationships is warranted to establish metabolic pathways for glucose and lipid biomarkers. Our tissue samples were obtained from several different organ systems and entailed all tissue layers. There was no separation of serosa, muscle, and mucosa with the enteric samples. The separation and validation of these tissue components could elucidate postoperative analysis for long-term follow-up evaluation. Although, endoscopic surveillance of mucosal tissue is a viable option, we are pursuing analyses of pre-and postoperative blood samples. Obviously, this latter technique represents the least invasive approach for following this cohort. By understanding the role of PPARc and ENPP1 in various tissues from obese patients, it may be possible to control glucose and lipid metabolism pharmacologically. The development of drugs to modulate the activity of PPARs and ENPP1 as well as other receptors associated with the metabolic cascade will undoubtedly assist in targeting therapies to a patientÕs specific risk factors and comorbid conditions. Currently, TZDs such as troglitazone and pioglitazone are used clinically to target the PPARc and a isoforms, respectively. These drugs reduce hyperglycemia and improve insulin sensitivity by increasing the number of functional adipocytes [22]. Our study delineates more information on the relationship between adipocyte and lipid metabolism. Currently, the genetic expression of these factors and the metabolic pathways associated with these entities have not been elucidated from enteric and omental tissue. Our initial data are promising and engender the potential to develop personalized therapies for obese patients.
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