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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2240
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 4 1555-1559
Copyright © 2007 by The Endocrine Society

Human Adipose Tissue Cannabinoid Receptor 1 Gene Expression Is Not Related to Fat Cell Function or Adiponectin Level

Patrik Löfgren, Eva Sjölin, Kerstin Wåhlen and Johan Hoffstedt

Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden

Address all correspondence and requests for reprints to: Johan Hoffstedt, MD, Ph.D., Associate Professor, Karolinska Institutet, M61, Karolinska University Hospital, 141 86 Stockholm, Sweden. E-mail: johan.hoffstedt{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The cannabinoid receptor 1 gene (CNR1) is implicated in adipocyte function.

Objective: We investigated human adipose tissue CNR1 mRNA in relation to obesity, clinical and metabolic variables, adipocyte function, and adiponectin (ADIPOQ) levels.

Methods: We assessed sc fat biopsies from 96 obese and nonobese subjects and omental fat biopsies from 82 obese and nonobese subjects.

Results: The sc and omental adipose CNR1 gene expression were similar in obese and nonobese subjects. No association between either sc or omental adipose CNR1 mRNA levels and body mass index, waist circumference, plasma levels of glucose and insulin, lipids, or blood pressure was found. The sc and omental maximal adrenergic lipolytic activation as well as lipolytic adrenoceptor sensitivity were not related to CNR1 gene expression. Lipogenesis in sc adipocytes also showed no association with CNR1 mRNA levels. Finally, no relation was found between adipose CNR1 gene expression and ADIPOQ mRNA, adipose tissue adiponectin secretion, or circulating adiponectin.

Conclusion: We found no association of human adipose tissue CNR1 mRNA expression with measures of body fat, metabolic parameters, fat cell function, or ADIPOQ expression. These data do not suggest a major role of human adipose CNR1 in fat cell function or metabolic disease development.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CANNABINOID-1 (CB1) RECEPTOR, encoded by the CNR1 gene, is most abundantly expressed in the central nervous system but also in several peripheral organs including liver, muscle, and adipose tissue, whereas the CB2 receptor is restricted to immune cells (1). Both CB receptors belong to the family of G-coupled receptors that after ligand binding modulates the activity of various cell-signaling cascades including adenylate cyclase- and mitogen-activated protein kinase-related events (2). The observation of increased food intake in rodents given cannabinoid receptor ligands (3) together with the generation of a hypophagic and lean phenotype in mice lacking the CB1 receptor implicates a role of the endocannabinoid system in the treatment of diet-induced obesity (4). Moreover, the findings of increased adipocyte lipoprotein lipase activity (4) and reduced adiponectin, ADIPOQ, mRNA expression (5) and effects on lipolysis and energy expenditure (6) upon CB1 receptor activation also suggest a fat-cell-specific role of the endocannabinoid system. The human CNR1 gene is expressed in both omental and sc adipocytes at both the mRNA and protein levels and found to mediate an increase in intracellular cAMP levels after stimulation (7).

Whether adipose tissue CB1 receptors are important in the pathogenesis of obesity and associated metabolic disorders is unknown. In recent studies, obesity was found to be associated with increased levels of endocannabinoids but decreased amounts of CNR1 mRNA (8, 9). Here, we investigated human CNR1 mRNA levels from both abdominal sc and omental adipose tissue in relation to clinical variables, fat cell function, and adiponectin expression.


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

The Hospital’s committee on ethics approved the study. All subjects were apparently healthy and free of medication and ate a standard Swedish diet. Two separate cohorts were used. Cohort 1 was composed of 80 obese women with body mass index (BMI) of 31–53 kg/m2 (age, 21–63 yr) and 16 lean women with BMI of 20–24 kg/m2 (age, 26–52 yr). At approximately 0730 h after an overnight fast, a venous blood sample was obtained for analyses of plasma levels of glucose, insulin, triglycerides, cholesterol, high-density lipoprotein cholesterol, free fatty acids, and adiponectin. The analyses were performed by the hospital’s accredited chemistry laboratory except for serum levels of adiponectin, which were measured using an RIA method (Linco Research, Inc., St. Charles, MO) and were expressed as micrograms per milliliter. Insulin sensitivity was calculated from the homeostasis model assessment algorithm based on plasma glucose and plasma insulin (plasma insulin x plasma glucose/22.5). Systolic and diastolic blood pressures were measured in the supine position after 15 min of rest. Thereafter, an adipose sample (1–4 g) was obtained by needle biopsy from the abdominal sc area under local anesthesia (10). This tissue (1 g) was frozen in liquid nitrogen, kept frozen at –70 C, and used for subsequent mRNA analysis (see below). In obese subjects, there was adipose tissue available also for fat cell lipolysis and lipogenesis studies and in 30 obese subjects also for analysis of adipose tissue adiponectin secretion (see below).

Cohort 2 consisted of 66 obese (BMI, 30–60 kg/m2; age, 23–59 yr) men (n = 30) and women (n = 36) undergoing weight reduction surgery and 16 nonobese (BMI, 20–24 kg/m2; age, 21–59 yr) women undergoing gallstone surgery at the Department of Surgery, Karolinska University Hospital Huddinge. Before surgery, at approximately 0730 h after an overnight fast, a venous blood sample was obtained for plasma analyses, as described above. From these subjects, perioperative fat biopsies using laparoscopic technique from omental adipose tissue (1–2 g) were taken for subsequent lipolysis studies and mRNA analysis.

Fat cell lipolysis and lipogenesis

The lipolysis experiments were performed as described (11). In brief, fat cells were incubated in the absence (basal) or presence of increasing concentrations of either noradrenaline (a nonselective ß- and {alpha}2-adrenoceptor agonist), dobutamine (a selective ß1-adrenoceptor agonist), or terbutaline (a selective ß2-adrenoceptor agonist). After 2 h incubation, the medium was removed for measurement of glycerol, a quantitative marker for lipolysis. In studying lipogenesis, we used an indirect method, which is described in detail elsewhere (12). The method used reflects glucose transport because, at micromolar glucose concentrations, glucose transport is the rate-limiting step for lipogenesis in human fat cells as discussed (12).

Adipose tissue adiponectin secretion

In 30 obese subjects of cohort 1, there was tissue available also for measurement of sc adipose tissue adiponectin secretion. The adipose tissue was cut into small pieces (10–25 mg) and then incubated at 37 C (3.0 ml medium/300 mg tissue) in a medium consisting of sterile Krebs-Ringer phosphate buffer (pH 7.4), endotoxin-free BSA (4 g/100 ml), and glucose (1 mg/ml), with air as the gas phase. After a 2-h incubation, a 1-ml aliquot of medium was removed and stored at –70 C for subsequent analysis of adiponectin using the RIA described above.

mRNA analysis

cDNA synthesis and quantitative real-time PCR was performed as described (13). CNR1 mRNA was quantified using TaqMan (Hs00275009_s1; Applied Biosystems, Foster City, CA). Low-density lipoprotein receptor-related protein, LRP10, mRNA was quantified using either TaqMan (Hs00204094_m1) as a reference for CNR1 or SYBR Green-based real-time PCR (Bio-Rad Laboratories Inc., Hercules, CA) using primers 5'-GATGGAGGCTGAGATTGTG-3' (sense) and 5'-GAGTCATATCCTGGCGTAAG-3' (antisense) as reference for ADIPOQ. ADIPOQ mRNA (NM_004797) was quantified using SYBR Green-based real-time PCR with primers 5'-GGTCTCGAACTCCTGGCCTA-3' (sense) and 5'-TGAGATATCGACTGGGCATGGT-3' (antisense). Fatty acid amide hydrolase, FAAH, and monoglyceride lipase, MGLL, mRNA were quantified using TaqMan (Hs_01038659 and Hs_00996007, respectively). The Ct values were normalized to levels of LRP10 mRNA and data presented as arbitrary units. LRP10 was used as a reference gene based on a recent comparative study identifying LRP10 as the gene with the least variation in expression levels in human adipose tissue (14). Ct-values for CNR1 detection were 30.5 ± 0.6 (sc) and 30.9 ± 0.7 (omental).

Statistical analysis

Parameter distributions were normalized when necessary by logarithm10 transformation before statistical comparison. Values are mean ± SD. The Student’s unpaired t test, ANOVA, single regression analysis, and {chi}2 analysis were used for statistical evaluation. A P value less than 0.05 was considered statistically significant. The analyses were performed using StatView version 6.0 (Stata Corp., College Station, TX).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
No difference in CNR1 mRNA levels between obese and nonobese subjects in sc (2.4 ± 0.8 vs. 2.4 ± 1.0, P = 0.99) or omental (3.9 ± 1.6 vs. 4.2 ± 2.2, P = 0.60) adipose tissue was found. In contrast, sc (2.4 ± 0.7 vs. 3.2 ± 0.7, P = 0.0003) and omental (2.2 ± 0.7 vs. 2.8 ± 0.9, P = 0.0009) FAAH mRNA levels were reduced in obesity, whereas omental (3.8 ± 1.2 vs. 4.7 ± 1.9, P = 0.008) but not sc (2.9 ± 1.3 vs. 3.4 ± 1.6, P = 0.21) MGLL mRNA levels were down-regulated in obese subjects. No significant correlations between CNR1 and FAAH or MGLL mRNA levels were found in either sc or omental adipose tissue (data not shown).

After dividing the adipose CNR1 mRNA levels from obese subjects into tertiles based on high, intermediate, and low levels, respectively, no difference in clinical or metabolic variables was found between expression levels of sc or omental adipose tissue CNR1 mRNA (Table 1Go).


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  		TABLE 1. Clinical data of obese subjects in relation to tertiles of CNR1 mRNA expression

 
With respect to adipocyte function, there was no difference in sc basal or maximal lipolysis induced by noradrenaline, dobutamine, or terbutaline between tertiles of CNR1 mRNA (Table 2Go). Also, EC50 (representing adrenoceptor sensitivity) for noradrenaline-, dobutamine-, and terbutaline-induced lipolysis were not different between levels of CNR1 mRNA. The results from omental fat cell lipolysis displayed a similar pattern. Results from lipogenesis experiments revealed that basal and maximal insulin-induced lipogenesis as well as EC50 for insulin receptor agonist activation were similar between tertiles of CNR1 mRNA (Table 2Go).


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  		TABLE 2. Obese fat cell function in relation to tertiles of CB1 mRNA expression

 
No association between mRNA expression of CNR1 and ADIPOQ was found in either sc or omental adipose tissue (Fig. 1Go). We also investigated the relation between serum and adipose secretion levels of adiponectin and sc adipose tissue CNR1 gene expression (cohort 1). As seen in Fig. 2Go, there were no associations between adiponectin serum or secretion levels and CNR1 mRNA.


Figure 1
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  		FIG. 1. Relation between CNR1 mRNA (CNR1/LRP10) and ADIPOQ mRNA (ADIPOQ /LRP10) in sc and omental adipose tissue from obese and nonobese subjects. Values are compared using simple regression analysis.

 

Figure 2
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  		FIG. 2. The sc adipose tissue CNR1 mRNA (CNR1/LRP10) in relation to serum adiponectin (µg/ml) and sc adipose tissue adiponectin secretion (ng/107 cells) in obese subjects of cohort 1. Values are compared using simple regression analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
A principal function of adipose tissue is to store or release energy depending on nutritional status. These processes, termed lipogenesis and lipolysis, respectively, are subject to intense hormonal regulation (15, 16). Although it has been shown that the endocannabinoids may also play an important role in fat cell function (17), very few data have been presented regarding the pathophysiological significance of the endocannabinoid system in obesity and insulin resistance development.

To further explore this issue, we investigated the sc and omental adipose tissue CNR1 gene expression in relation to obesity and various clinical and metabolic variables as well as both lipogenesis and lipolysis. In contrast to previous studies, in which both a reduced (8, 9) and increased (5) expression of CNR1 mRNA in obesity has been demonstrated, we found no difference in adipose tissue CNR1 mRNA levels between obese and lean subjects. Moreover, there was no association between either sc or omental adipose CNR1 gene expression and clinical and metabolic variables. The reason for these conflicting results is unknown. Nevertheless, the results from adipocyte functional studies displayed a similar pattern; the sc and omental fat cell function was not different in subjects with either a high or a low CNR1 gene expression level. However, we measured lipogenesis in sc adipocytes only. Thus, we cannot exclude that CNR1 mRNA expression may be related to omental adipocyte lipogenesis given the recent finding of positive visceral fat CNR1 and SREB1c mRNA correlation (9). Unfortunately, there was not enough tissue available for analysis of protein levels of CNR1 to confirm the mRNA data. Nevertheless, although the present results are based on associative data only, they do not speak in favor of a clear role of the CNR1 gene in fat cell function or metabolic disease development.

Interestingly, we found decreased adipose mRNA levels of the endocannabinoid-degrading enzymes in obesity, at least for FAAH. This finding is in line with the report by Engeli et al. (8) and may at least in part be a mechanistic explanation for the observed increase in circulating endocannabinoids seen in obesity (8, 9).

It has been suggested that the endocannabinoids may exert their action in adipocytes also by reducing the expression of adiponectin. In CNR1 knockout mice, an increased adipose tissue mRNA level of ADIPOQ was demonstrated (5), and in murine cell lines, ADIPOQ gene expression was reduced after CB1 receptor activation (18, 19) but increased after CB1 receptor antagonism (19). In contrast, in a recent weight reduction study, no difference in serum adiponectin levels was found between rats treated with CB1 receptor antagonist or given a low-calorie diet, despite a similar degree of weight loss (20). In accordance with the latter study, the present negative data do not speak in favor of a clear association between human adipose CNR1 expression and ADIPOQ levels in man.

In conclusion, we found no relation between sc or omental adipose tissue gene expression of CNR1 and various clinical variables. Moreover, fat cell function measured as lipogenesis and lipolysis and various measures of ADIPOQ expression were not associated with adipose CNR1 mRNA. Consequently, these data do not provide evidence for a consistent role of CNR1 in human adipocyte function.


   Footnotes
 
This study was supported by grants from the Swedish Research Council and the Swedish Medical Society.

Disclosure Statement: P.L., E.S., and K.W. have nothing to declare. J.H. has received lecture fees from Astra Zeneca, GlaxoSmithKline, Novo Nordisk, and Sanofi-Aventis.

First Published Online February 6, 2007

Abbreviations: BMI, Body mass index; CB1, cannabinoid-1.

Received October 13, 2006.

Accepted January 26, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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