Endocannabinoid Dysregulation in the Pancreas and Adipose Tissue of Mice

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Endocannabinoid Dysregulation in the Pancreas and Adipose Tissue of Mice Fed With a High-fat Diet


Katarzyna M. Starowicz1,2,5, Luigia Cristino1,3, Isabel Matias1,2, Raffaele Capasso1,4, Alessandro Racioppi1,2, Angelo A. Izzo1,4 and Vincenzo Di Marzo1,2

1Endocannabinoid Research Group, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy
2Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy
3Institute of Cybernetics, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy
4Department of Experimental Pharmacology, University of Naples "Federico II", Naples, Italy
5Present address: Department of Pain Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland.

Correspondence: Vincenzo Di Marzo (vdimarzo@icmib.na.cnr.it)

The first three authors contributed equally to this work.

Received 11 July 2007; Accepted 16 November 2007; Published online 17 January 2008.
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Abstract
Objective:


In mice, endocannabinoids (ECs) modulate insulin release from pancreatic beta-cells and adipokine expression in adipocytes through cannabinoid receptors. Their pancreatic and adipose tissue levels are elevated during hyperglycemia and obesity, but the mechanisms underlying these alterations are not understood.
Methods and Procedures:


We assessed in mice fed for up to 14 weeks with a standard or high-fat diet (HFD): (i) the expression of cannabinoid receptors and EC biosynthesizing enzymes (N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) and DAGLalpha) and degrading enzymes (fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) in pancreatic and adipose tissue sections by immunohistochemical staining; (ii) the amounts, measured by liquid chromatography—mass spectrometry, of the ECs, 2-AG, and anandamide (AEA).
Results:


Although CB1 receptors and biosynthetic enzymes were found mostly in alpha-cells, degrading enzymes were identified in beta-cells. Following HFD, staining for biosynthetic enzymes in beta-cells and lower staining for FAAH were observed together with an increase of EC pancreatic levels. While we observed no diet-induced change in the intensity of the staining of EC metabolic enzymes in the mesenteric visceral fat, a decrease in EC concentrations was accompanied by lower and higher staining of biosynthesizing enzymes and FAAH, respectively, in the subcutaneous fat. No change in cannabinoid receptor staining was observed following HFD in any of the analyzed tissues.
Discussion:


We provide unprecedented information on the distribution of EC metabolic enzymes in the pancreas and adipose organ, where their aberrant expression during hyperglycemia and obesity contribute to dysregulated EC levels.
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Introduction

Among the signaling systems suggested to play a role in the control of energy homeostasis, the endocannabinoid (EC) system is one of the most recently discovered. It comprises (i) the two cloned cannabinoid receptors, CB1 and CB2; (ii) their endogenous ligands, named ECs, the best studied of which are anandamide (N-arachidonoyl-ethanolamine, AEA) and 2-arachidonoyl-glycerol (2-AG) (1,2,3,4,5); (iii) the enzymes responsible for the formation of AEA and 2-AG from their most important biosynthetic precursors, N-arachidonoyl-phosphatidyl-ethanolamine and 2-arachidonoyl-sn-1-acyl-glycerols (DAGs), respectively (6,7), and for their degradation. AEA is obtained from N-arachidonoyl-phosphatidyl-ethanolamine mostly through the action of the N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD), although alternative pathways exist for this conversion. Instead, 2-AG formation from DAGs occurs uniquely through the catalytic action of the sn-1-selective diacylglycerol lipases alpha and beta (DAGLalpha and beta), the former of which is the most abundant DAGL isoform in the adult brain. Regarding EC metabolism, an amidase known as fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of AEA and 2-AG (8). At least one isoform of monoacylglycerol lipases (MAGLs), cloned from several mammalian species, also plays a major role in the enzymatic hydrolysis of 2-AG (9). EC biosynthesizing and degrading enzymes have been identified not only in neurons but also in peripheral cells including white adipocytes and rat insulinoma cells (10,11,12).

A function of the EC system in the regulation of energy balance, at the level of both food intake and peripheral control of metabolism, was suggested by the observation that genetic and/or pharmacological impairment of cannabinoid CB1 receptors causes reduction of body weight in lean animals in part independently from the inhibition of food intake (13). CB1 blockade also normalizes several dysregulated metabolic parameters (i.e., low high-density-lipoprotein cholesterol, high triglycerides, low adiponectin, high glucose and insulin, etc.) in obese animals and patients in a way partly independent from weight loss (13,14,15). In agreement with its role in the regulation of metabolism independently from its effects on food intake, the CB1 receptor is expressed in mouse and human adipocytes, and in mouse pancreatic islets, hepatocytes, and skeletal muscle (11,12,13, 16,17,18,19,20, and ref. 21 for review). In particular, in adipocytes, EC and CB1 receptor levels were found to increase during differentiation (11,17,22), and CB1 stimulation was shown to lead to more rapid differentiation of preadipocytes (11), stimulation of lipoprotein lipase activity (16), upregulation of glucose uptake (22,23), inhibition of AMP-activated protein kinase (24), and stimulation of fatty acid synthase (18). These pro-lipogenetic actions of CB1 in adipocytes might explain in part why CB1 knockout mice fed with the same amount of food as wild-type mice still exhibit less fat mass (16). On the other hand, the role of the EC system in the endocrine pancreas is less well understood. In the beta-cells from isolated mouse pancreatic islets, both CB1 (25) and CB2 (20) stimulation have been reported to inhibit insulin release, whereas in insulinoma cells grown in a high glucose concentration, CB1, but not CB2, stimulation causes the enhancement of glucose-induced insulin release (11). Despite the increasing evidence for a metabolic function of ECs in both the adipose organ and pancreas, little is known about their regulation under dietary conditions leading to hyperglycemia and obesity; and, moreover, no data exist on the exact localization of EC metabolic enzymes in pancreatic islets.

There is increasing evidence for the overactivity of the EC system (in terms of upregulation of either CB1 receptor or EC levels, or both) during conditions of unbalanced energy homeostasis (e.g., obesity and hyperglycemia), and for its causative role in these disorders (13,26). This overactivity occurs at the level of both the hypothalamus (27) and peripheral tissues, including the liver, pancreas, and epididymal adipose tissue in animals fed with a high-fat diet (HFD), and in the visceral fat and blood of obese patients (10,11,18,28,29,30,31). We know that it is associated with, and underlies in part, some of the metabolic dysfunctions that accompany obesity and hyperglycemia (11,18,29), thus explaining why CB1 receptor blockade is effective at reducing these dysfunctions in obese individuals (13). On the other hand, still very little is known on the possible biochemical mechanisms underlying the overactive EC system in obesity. Impairment of FAAH expression or activity seems to correlate with obesity and overweight in humans ((32); but see also ref. 33 for discrepant results), and with the elevated EC levels found in the liver of mice fed with a HFD (18) or in the blood of obese women (10,28). It has also been found that changes in small intestine levels of AEA following food consumption or deprivation are due to biosynthetic precursor availability rather than to changes in the activity of biosynthesizing and degrading enzymes (34). Also with regard to the effect of obesity on CB1 receptor expression in the adipose tissue, there are discrepant results in the literature, with decreases (10) and no changes (11) in obese patients and increases in rats (30) having been reported to date.

Based on this background, we have investigated here, in mice fed for different periods of time either a standard diet (STD) or a HFD, the expression and localization of cannabinoid CB1 and CB2 receptors and of EC metabolic enzymes in pancreatic and visceral or subcutaneous adipose tissue sections by using immunohistochemistry and immunofluorescence, in parallel with the measurement of EC amounts by liquid chromatography—mass spectrometry.
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Methods and Procedures
Animals and diets

Male, 7-week-old C57Bl/6J mice were purchased from Harlan (Corezzana, Italy). After 1-week acclimatization, animals were fed a diet containing 25.5% fat (49% of calories), 22% protein, and 38.4% carbohydrate (TD97366, Harlan Italy) for 14 weeks. Control mice received STD containing 5.7% fat (10.9% of calories), 18.9% protein, and 57.3% carbohydrate (2018, Harlan Italy). Body weight was measured weekly. Mice were fed ad libitum, except for the 12-h period immediately preceding the killing, which occurred after 3, 8, and 14 weeks. An overnight, 12-h fasting period was chosen, because it is known that blood EC levels decrease postprandially in human volunteers (11). Fasting plasma glucose levels were determined in 12-h-fasted animals, using the glucose test kit with an automatic analyzer (AQccu-Chek Active, Roche) in blood samples obtained from tail vein (35). Measurements were performed at time 0 and after 3, 8, and 14 weeks of dietary treatment. Experiments were also performed in Wistar rats (Harlan, CorezzanaItaly) to compare the basal expression of receptors and enzymes in alpha- and beta-cells to those in the mouse. Rats received STD and were fed ad libitum, except for the 12-h period immediately preceding the killing and perfusion.

After killing, the pancreas and adipose (subcutaneous, visceral, and epididymal) tissues were removed and immediately immersed into liquid nitrogen, to be stored at -70 °C until extraction and purification of ECs. Visceral fat was taken from the mesenteric area. Care was taken to dissect as much fat as possible for each depot, also in order to calculate the changes in fat distribution following the diets. For immunohistochemistry studies, the animals were instead first perfused, and then the aforementioned tissues prepared as detailed below.
Immunohistochemistry and immunofluorescence

Experiments were performed following international guidelines on the ethical use of animals from European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering. Four C57Bl/6J male adult mice per each time point tested (3, 8, and 14 weeks of HFD) and their respective controls (n = 4) were used. Animals were deeply anesthetized (pentobarbital, 60 mg/kg, intraperitoneal) and perfused transcardially with saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Pancreas, visceral, and subcutaneous adipose fat tissues were removed, postfixed for 2 h, and then washed. Tissues to be cut at cryostat were cryoprotected overnight in PB containing 30% (w/vol) sucrose at 4 °C until they sank. Pancreas serial cryostat were cut into three alternate series at 8 mum, visceral and subcutaneous adipose fat tissue were cut at 10 mum, and mounted onto gelatine-coated slides. Each tissue dissected and its respective control were also paraffin-embedded; microtome sections were cut at a thickness of 6 mum, collected on slides in three serial sections. Experiments were also performed in Wistar rats. Briefly, animals were perfused transcardially as described above; pancreas (n = 3) were removed, postfixed, cryoprotected, and processed for cryostat sectioning (three alternate series of 8 mum mounted onto gelatin-coated slides).

Single and double immunofluorescence labeling in pancreas. For single immunofluorescence, pancreas sections were incubated for 1 h in 10% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) in PB containing 0.3% Triton X-100 (block solution). Subsequently, the sections were incubated for 2 days at 4 °C in a humid chamber with the respective polyclonal antibodies (all diluted in block solution): rabbit anti-CB1 receptor: 1:50 (Abcam, Cambridge, UK); rabbit anti-CB2 receptor 1:200 (Abcam, Cambridge, UK); rabbit anti-FAAH 1:50 (Cayman Chemicals, Ann Arbour, MI); rabbit anti-DAGLalpha 1:50 (generously provided by Prof. Patrick Doherty, King's College London, UK); guinea pig anti-insulin 1:50 (Abcam, Cambridge, UK); and rabbit anti-glucagon 1:50 (Abcam, Cambridge, UK). For double immunofluorescence, sections were incubated under the same conditions in a mixture of the same primary antibodies mentioned above, except for double staining with glucagon, due to incompatibility with the species from which the antibody was obtained. In this case, polyclonal goat anti-glucagon (Abcam, Cambridge, UK) diluted at 1:50 was used. Primary antibody combinations were as follows: anti-insulin and anti-glucagon; anti-CB1 and anti-insulin; anti-CB1 and anti-glucagon; anti-DAGLalpha and anti-insulin; anti-DAGLalpha and anti-glucagon; anti-FAAH and anti-insulin; anti-FAAH and anti-glucagon. After three washes in PB, single and double immunofluorescence was revealed by incubation for 2 h in the appropriate fluorochrome-conjugated secondary antibody: Alexa Fluor488 anti-rabbit (for CB1, CB2, DAGLalpha, FAAH); Alexa Fluor546 anti-rabbit or Alexa Fluor546 anti goat (for single and double glucagon staining, respectively), and Alexa Fluor546 or Alexa Fluor488 anti guinea pig (for insulin) diluted 1:100 in block solution. For the purpose of double staining, a mixture of the respective secondary antibodies was used. Thereafter, sections were washed with PB and coverslipped with Aquatex mounting medium (Merck, Darmstadt, Germany).

Immunofluorescence labeling in mouse visceral and subcutaneous adipose tissue. For immunofluorescence, visceral and subcutaneous adipose fat serial sections were incubated for 1 h in 10% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) in PB containing 0.3% Triton X-100 (block solution). Then the sections were incubated for 1 day at room temperature in a humid chamber with the respective antibodies (all diluted in block solution): rabbit anti-CB1 receptor: 1:100; rabbit anti-CB2 receptor 1:200; rabbit anti-DAGLalpha 1:100; rabbit anti-FAAH 1:100; rabbit anti-MAGL 1:100. In addition, a rat monoclonal (FA-11) antibody against CD68 (Abcam, Cambridge, UK), diluted at 1:100, was used for visceral adipose fat tissue staining in order to visualize CD68, which is specifically expressed by tissue macrophages; this antibody was incubated for 1 h at room temperature. After three washes in PB, immunofluorescence was revealed by incubation for 2 h in the appropriate secondary antibody: for CB1, CB2, DAGLalpha: Alexa Fluor488 anti-rabbit diluted at 1:200; for FAAH and MAGL: Alexa Fluor546 anti-rabbit diluted 1:200 in block solution; for CD68: rabbit polyclonal against rat fluorescein isothiocyanate (Abcam, Cambridge, UK). Slides were mounted and processed for microscope observation.

Single immunoperoxidase labeling in pancreas. For single NAPE-PLD and MAGL antigen immunohistochemistry, the sections were dewaxed and rehydrated and then proceeded like with all other antibodies used. For anti-CB1, anti-CB2, anti-DAGLalpha, anti-FAAH, anti-MAGL, and anti-NAPE-PLD immunoperoxidase, the sections were reacted for 10 min in 0.3% H2O2 to inactivate endogenous peroxidase activity and incubated for 1 h at room temperature in 10% normal goat serum (Vector Laboratories, Burlingame, CA) in 0.1M Tris—HCl-buffered saline, pH 7.3 (Tris-buffered saline), containing 0.3% Triton X-100 and 0.05% sodium azide (Sigma-Aldrich, Germany). The sections were then incubated for 2 days at 4 °C with individual rabbit polyclonal antibody as already used for the immunofluorescence technique, and were diluted in normal goat serum as indicated: rabbit anti-CB1 : 1:50; rabbit anti-CB2 1:200; rabbit anti-DAGLalpha 1:50, rabbit anti-FAAH 1:50; rabbit anti-MAGL 1:50, and rabbit anti-NAPE-PLD 1:50. After three rinses, the sections were incubated for 2 h in biotinylated goat anti-rabbit IgGs (Vector Laboratories, Burlingame, CA) diluted 1:100 in normal goat serum, followed by incubation for 1 h at room temperature in the avidin—biotin—peroxidase solution (ABC kit; Vectastain, Vector, Burlingame, CA) in Tris-buffered saline, and then in 0.05permil 3-3'diaminobenzidine (DAB; Sigma Fast, Sigma-Aldrich, Germany) in 0.01 M Tris-buffered saline. Then, the pancreas sections were washed in water, and all sections were dehydrated in alcohol, cleared in xylene, and mounted in dibutylpthalate polystyrene xylene (Merck, Germany).

Single immunoperoxidase labeling in visceral and subcutaneous adipose tissue. Visceral and subcutaneous adipose fat serial sections were processed with the antibodies already indicated for pancreas immunohistochemistry for anti-CB1 (1:100), CB2 (1:200), DAGLalpha (1:100), FAAH (1:100), MAGL (1:100), and NAPE-PLD (1:100) staining with the ABC method described above. Briefly, the sections were reacted in 0.3% H2O2, blocked, incubated with respective primary and secondary antibodies and finally dehydrated in alcohol, cleared in xylene, and mounted in dibutylpthalate polystyrene xylene.

Controls and image processing. Controls included (i) pre-absorption of diluted antibodies with their respective immunizing peptides (if not commercially available, control peptides were synthesized upon custom request by Inbios, Italy); and (ii) omission of either the primary antisera or the secondary antibodies. These control experiments did not show staining. The sections processed for immunofluorescence were studied with an epifluorescence microscope (Leica DM IRB); settings for the excitation of fluorescein isothiocyanate (488 nm) and Texas Red (543 nm) were identical throughout the analysis. All other materials were investigated under bright-field illumination. Images were acquired using the digital camera Leica DFC 320 connected to the microscope and the image analysis software Leica IM500, which allows both single and merged pictures acquisitions. Digital images were processed in Adobe Photoshop, with brightness and contrast being the only adjustments made.

Densitometric analysis. Quantitative analyses of the intensity of CB1, FAAH, DAGLalpha, and NAPE-PLD immunostaining in pancreas islets and subcutaneous fat in STD and HFD mice were performed, for each marker at the different times, by using a digital camera working on gray levels (JCV FC 340FX, Leica) for image acquisition and the image analysis software Image Pro Plus 6.0 for Windows, MediaCybernetics, working on logarithmic values scale of absorbance for densitometric evaluation. All measures were performed on the sections processed for single immunoperoxidase reaction and blind with respect to the type of immunoreaction marker and the time of diet under analysis. A sample of 60 immunopositive cells with nuclei (unstained or lightly stained) in the focal plane were randomly identified per each animal from N = 3 animals per groups at each marker, time, and type of diet, as alpha- or beta-cells with respect to the adjacent section labeled with glucagone or insulin antibodies. The images were acquired under constant light illumination and at the same magnification. In each section, the zero value of optical density was assigned to the background, i.e., a portion of pancreas or of subcutaneous fat tissue devoid of stained cell bodies. On the same sample designed for the densitometric analysis, and with the same criteria of cellular identification (i.e., cells with nuclei, unstained or lightly stained, in the focal plane and alpha or beta type identification with respect to the corresponding adjacent section double insulin/glucagon labeled), we performed the counting of beta-cells whose number changed with respect to enzymatic immunoexpression after HFD. For each immunophenotype, the percentage value was obtained from the ratio between the mean of immunolabeled cells and the mean of beta-cells (insulin-labeled, n = 100 plusminus 20) per animal in STD and HFD islets at each time-points.
EC measurement

Pancreas and visceral/subcutaneous fat from mice fed with a STD and a HFD were removed and immediately frozen in liquid nitrogen until quantitative determination of ECs. The extraction, purification, and quantification of AEA and 2-AG from tissues require several biochemical steps as described previously (36). First, tissues were dounce-homogenized and extracted with chloroform/methanol/Tris—HCl 50 mmol/l, pH 7.5, (2:1:1, vol/vol) containing internal standards ([2H]8 AEA and of [2H]5 2-AG, 100 pmol each). The lipid containing organic phase was dried down, weighed, and prepurified by open-bed chromatography on silica gel. Fractions were obtained by eluting the column with 9:1 and 1:1 (by vol) chloroform/methanol. After lipid extraction and separation, the prepurified lipids were then analyzed by liquid chromatography—atmospheric pressure chemical ionization—mass spectrometry by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2010) quadrupole mass spectrometry through a Shimadzu APCI interface as previously described (27). The amounts of ECs in the tissues, quantified by isotope dilution with the above-mentioned deuterated standards, are expressed as picomole per mg of total lipid extract or as picomole per gram of wet tissue.
Statistical analysis

All quantitative results were expressed as mean plusminus s.e.m. or plusminuss.d. The statistical significance of differences in mean values was assessed by one-way ANOVA followed by the Bonferroni's post-hoc analysis.
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Results
Effect of HFD on mouse body weight and fasting plasma glucose levels

The progressive body weight gain in mice fed with HFD and STD is shown in Figure 1. At the beginning of the treatments (time 0), the weight of the mice was 22.44 plusminus 0.15 g. At the end of the treatment (14 weeks), HFD-fed mice weighed 41.5% more than age-matched animal fed with STD. Compared to aged-matched STD-fed animals, HFD showed higher glucose levels after 3, 8, and 14 weeks, although statistical significance (P < 0.05), even when calculated after numerical transformations, was achieved only starting from 8 weeks.
Figure 1.
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(a) Body weight and (b) glucose levels at killing of mice fed with a standard and high-fat diet (HFD). Effect of a standard diet (STD) and HFD on body weight (a) and blood glucose levels (b). Body weight was measured weekly; glucose levels were measured at times 0, 3, 8, and 14 weeks on diet. Blood samples were obtained following 12-h fasting. Values are means plusminus s.e.m. (a, N = 38; b, N = 6—8 animals). *P < 0.05 vs. corresponding animals fed with STD.
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Expression of CB1and CB2 receptors in pancreas

With both techniques used, we found that CB1 receptors are mostly expressed in cells that, by the use of double immunofluorescence, were identified as glucagon-expressing alpha-cells (Figure 2a—f). Similar results were obtained using rat pancreas, although in this case CB1 receptor-immunoreactivity was also seen inside the islets, in some insulin-secreting beta-cells (Figure 2j,k). CB2 receptors were, instead, found inside mouse pancreatic islets, co-expressed with both glucagon and insulin, and hence present also in beta-cells (Figure 2g,h and data not shown). In all islets examined (see Methods and Procedures), exactly the same phenotype of cannabinoid receptor staining was found.
Figure 2.
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Distribution of CB1and CB2 receptors within the mouse (a—h) and rat (i—k) pancreas islets of Langerhans. Photomicrographs demonstrating localization of CB1 with the outer layer alpha-cells by (a) immunofluorescence and (b) immunoperoxidase staining as established with (c) insulin and (d) glucagon co-immunostaining. CB1displays an immunoreactivity pattern complementary to that of insulin (beta-cells) and matching with that of glucagon (alpha-cells) (see merged panels (e) and (f), respectively). CB2shows to some extent both glucagon- (arrows) and insulin-secreting cells (asterisks) co-localization, as indicated in g (immunofluorescence) and h (immunoperoxidase labeling). Additionally, in pancreatic islets prepared from 2-month-old male Wistar rats, CB1 localizes with both non-beta and, to a lesser extent, insulin-expressing beta-cells (arrows) as demonstrated by immunofluorescencnt photomicrographs (i—k): (i) describes the staining for CB1, (j) the staining for insulin, and (k) is the merged image. Note some co-localization of CB1 with insulin (arrows and yellow color in (k)). All scale bars (b, f, and h) correspond to 40 mum (f relates to a and c—f; g to g and h; b only to itself and k relates to j,k). Images are representative of the islets obtained from three different mice or rats.
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Expression of EC metabolic enzymes in the pancreas

As shown in Figures 3 and 4, using only DAB staining, we could detect specific immunoreactivity for NAPE-PLD, which seemed to be significantly more abundant in external layer cells. With both staining techniques used, we could identify in mouse pancreatic islets clear signals associated with DAGLalpha and FAAH. Although the latter enzyme co-localized with insulin-expressing beta-cells, DAGLalpha was restricted to glucagon-expressing cells of the islet external layer (alpha-cells). MAGL, which could be stained only with DAB, appeared like FAAH, to be expressed instead mostly in insulin-expressing beta-cells. Using DAB staining for NAPE-PLD and MAGL and immunofluorescence staining for glucagon or insulin, respectively, on consecutive sections, we could confirm what very much seems to be a lack of co-localization of NAPE-PLD with insulin and of MAGL with glucagon (Figure 3a,b and g,h). In all the islets examined (see Methods and Procedures), exactly the same phenotype of EC enzyme staining was found.
Figure 3.
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Endocannabinoid metabolic enzymes in the mouse pancreas. Representative light photomomicrographs illustrating NAPE-PLD (a, immunoperoxidase staining) immunoreactivity (ir) in alpha-cells and the complementary expression of insulin (b, immunofluorescence) on consecutive sections. Double immunofluorescence micrographs of DAGLalpha with beta- and alpha-cell markers, demonstrating its expression complementary to that of insulin (c) and matching with that of glucagon (d). Representatives images of FAAH-ir within the pancreatic islet obtained by the means of double immunofluorescence with insulin (e) and glucagon (f) labeling are also shown. Microphotographs of consecutive sections of MAGL-ir (g, immunoperoxidase staining) and glucagon-ir (h, immunofluorescence). All scale bars correspond to 40 mum (h corresponds to a—c, e—h, and d only to itself). Asterisks in (b, g) indicate examples of NAPE-PLD-ir cells that are not insulin-ir, and of glucagon-ir cells that are not MAGL-ir cells, respectively. Images are representative of the islets obtained from three different mice. FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; NAPE-PLD,N-acyl-phosphatidyl-ethanolamine-selective phospholipase D.
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Figure 4.
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Expression of CB1 receptors and endocannabinoid metabolic enzymes in mouse islets of Langerhans following 3, 8, and 14 weeks of a high-fat diet (HFD). CB1 receptor-immunoreactivity (ir) in alpha-cells as demonstrated by means of immunofluorescence (a—f); note the similar expression pattern between lean (a—c) and HFD (d—f) at all time-points tested. NAPE-PLD-ir localizes mostly with some alpha-cells (g—i); note its remarkable ir increase within the islet as demonstrated by the use of immunoperoxidase staining (j—l). Representative light microscope images of DAGLalpha-ir restricted to outer alpha-cell layer (m—o). Note subsequent increase in both alpha- and non-alpha-cells at 3, 8, and 14 weeks of HFD (p—s). Immunofluorescence microphotographs of FAAH-ir inside the Langerhans islet of standard diet (t—v) and HFD (w—y) mice; note the decreased ir starting from 8 weeks of HFD (x, y). All scale bars correspond to 40 mum. Images are representative of the islets obtained from three different mice. FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; NAPE-PLD,N-acyl-phosphatidyl-ethanolamine-selective phospholipase D.
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Changes in pancreatic EC receptor and enzyme expression following a HFD

No changes were observed in the intensity of the staining of either receptor in mouse pancreatic islets following 3, 8, and 14 weeks of HFD (Figures 4 and 5). On the other hand, already 3 weeks, and also 8 and 14 weeks following a HFD, DAGLalpha became induced in all beta-cells, and NAPE-PLD became expressed in the vast majority of beta-cells, i.e., in 62.2 plusminus 12.8% vs. 4.3 plusminus 1.3%, 56.4 plusminus 10.4% vs. 6.1 plusminus 2.2%, and 59.5 plusminus 11.4% vs. 4.9 plusminus 0.9% in HFD vs. STD mice, respectively. FAAH expression in beta-cells significantly decreased, starting with 8 weeks of HFD vs. STD (Figures 4 and 5). In all HFD islets examined, the same phenotype was found.
Figure 5.
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Densitometric analysis in CB1-, FAAH-, DAGLalpha-, and NAPE-PLD-immunoreactivity (ir) cells in islets of pancreas at different time-points of standard diet (STD) and high-fat diet (HFD). For each bar, data are expressed as means of optical density plusminus s.d. of n = 60 cells per animal for N = 3 animals at each time and type of diet. Means were compared by ANOVA followed by Bonferroni's post-hoc analysis. All significant differences (HFD vs. STD) were found in beta-cells: *P < 0.05 for HFD vs. the respective STD control at the same time point. #P < 0.05 for HFD vs. corresponding value at 3 weeks. Please note that the optical density units are expressed in the log scale, and that the densitometric analyses refer to the intensity of expression per cell type in STD and HFD groups. Hence, there is no apparent increase in N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) optical density values, because this enzyme was maximally expressed in the analyzed beta-cells but the number of NAPE-PLD-ir beta-cells increased considerably when passing from the STD to the HFD. FAAH, fatty acid amide hydrolase.
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Effect of HFD on islet number and phenotype

HFD did not significantly change the average number of islets per section (18 plusminus 3, mean plusminus s.d. n = 10), and did not appear to change islet full diameter (mean diameter 150 plusminus 7.4 mum) or alpha- or beta-cell morphology and number (Figure 4 and data not shown). HFD, therefore, did not influence the percentage of CB1-, CB2-, NAPE-PLD-, FAAH-, DAGLalpha-, and MAGL-stained islets or cells during the time course study simply because it was affecting the number and/or morphology of or alpha- or beta-cells. With all the antibodies tested, the number of positive islets in HFD mice were identical with respect to STD mice. Thus, HFD affected only the number and/or type of receptor- and enzyme-expressing cells in each islet.
Expression of CB1 and CB2 receptors and EC metabolic enzymes in the visceral adipose tissue

With both techniques used, we found that both CB1 and CB2 receptors are expressed in the mesenteric visceral fat (Figure 6). No significant change was observed in the staining of either receptor following 3, 8, and 14 weeks of HFD (data not shown). After 14 weeks of STD or HFD, staining of CB2 was not found to coincide uniquely with staining of the macrophage marker CD68, thus suggesting that CB2 receptor-immunoreactivity in both STD and HFD mice is mostly not due to macrophage infiltration of this tissue. We also found that a NAPE-PLD-specific signal could be observed using DAB staining only, and that DAGLalpha, FAAH, and MAGL, stained with both techniques, are expressed in the visceral fat (Figure 6). No significant differences were observed among the staining of any of the enzymes in HFD vs. STD following 3, 8, and 14 weeks of diet (data not shown).
Figure 6.
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Detection of CB1, CB2, and endocannabinoid (EC) metabolic enzymes in the mouse visceral adipose tissue. CB1- and CB2-immunoreactivity (ir) was observed in the plasma membrane of visceral adipocytes (immunofluorescence: (a, b) and immunoperoxidase staining at respective insets). CB2-ir was observed in some tissue macrophages as demonstrated by CD68 immunofluorescence staining in standard diet and high-fat diet (HFD) (14 weeks) mice (c, d, respectively). (e) The presence of N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) was detected by immunoperoxidase staining in visceral adipocytes. The presence of other EC system components, i.e., DAGLalpha, fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MAGL), was also detected by means of both immunofluorescence and immunoperoxidase staining (f—h and respective insets). All scale bars correspond to 100 mum. Images are representative of the tissues obtained from three different mice.
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Expression of CB1 and CB2 receptors and EC metabolic enzymes in the subcutaneous adipose tissue

With both techniques used, we found that both CB1 and CB2 receptors are expressed in the subcutaneous fat (Figure 7). No significant changes were observed in the staining of either receptor following 3, 8, and 14 weeks of HFD (Figures 8 and 9 and data not shown for CB2). We also found that a NAPE-PLD-specific signal could be observed using DAB staining only, and that DAGLalpha, FAAH, and MAGL, stained with both techniques, are expressed in the subcutaneous fat (Figure 7). No significant changes were observed in the staining of any of the enzymes following 3 weeks of HFD (data not shown). However, starting from 8 weeks of HFD, NAPE-PLD and DAGLalpha staining was significantly lower, and FAAH higher, in HFD vs. STD mice (Figures 8 and 9).
Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Expression of CB1 and CB2 receptors and endocannabinoid (EC) metabolic enzymes in the mouse subcutaneous adipose tissue. CB1 and CB2 receptors were identified by immunofluorescence as well as by single immunoperoxidase staining (representative images in (a) and (b) with corresponding insets). Immunoperoxidase and/or immunofluorescence reactions demonstrated the expression of (c) N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) and (d) DAGLalpha as well as (e) fatty acid amide hydrolase (FAAH) and (f) monoacylglycerol lipase (MAGL) in subcutaneous adipocytes. All scale bars correspond to 65 mum. Images are representative of the tissues obtained from three different mice.
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Figure 8.
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Changes in some endocannabinoid (EC) metabolic enzymes in mouse subcutaneous fat as a consequence of 8 and 14 weeks of high-fat diet (HFD). CB1 receptor-immunoreactivity (ir) in subcutaneous adipocytes demonstrated by means of immunofluorescence (a—d); note the similar expression pattern of standard diet (lean) (a, b) and HFD (c, d) mice at all time-points tested. Immunoperoxidase staining indicates a higher NAPE-PLD-ir in the subcutaneous fat of lean controls (e, f) mice with respect to HFD mice (g, h). Representative microphotographs indicate that DAGLalpha-ir decreases in the subcutaneous adipose tissue of HFD mice (i, j vs. k, l). Lower FAAH-ir at 8 and 14 weeks of lean controls (m, n) with respect to HFD tissues (o, p) was also observed. All scale bars correspond to 65 mum. Images are representative of the tissues obtained from three different mice. FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; NAPE-PLD,N-acyl-phosphatidyl-ethanolamine-selective phospholipase D.
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Figure 9.
Figure 9 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Densitometric analysis in CB1-, DAGLalpha, FAAH-, and NAPE-PLD-immunoreactivity (-ir) cells in subcutaneous fat at different time-points of standard diet (STD) and high-fat diet (HFD) mice. For each bar, data are expressed as means of optical density plusminus s.d. of n = 60 cells per animal for N = 3 animals at each time and type of diet. Means were compared by ANOVA followed by Bonferroni's post-hoc analysis. Please note that the optical density units are expressed in the log scale. Significant differences were found in HFD vs. the respective STD for DAGLalpha-ir, FAAH-ir, and NAPE-PLD-ir at the same time point (*P < 0.05) and not for CB1-ir in HFD vs. the respective STD control. FAAH, fatty acid amide hydrolase; NAPE-PLD,N-acyl-phosphatidyl-ethanolamine-selective phospholipase D.
Full figure and legend (33K)

Controls for immunostaining

No immunoreactivity was observed in any experiment in the presence of the corresponding blocking peptides, and in the absence of the primary or secondary antibody (data not shown).
Effect of HFD and age on EC levels in the mouse pancreas

As shown in Table 1, both AEA and 2-AG levels were higher in the pancreas of mice fed with a HFD, starting already 3 weeks after the beginning of the diet. Although after 8 weeks of HFD the levels of AEA and 2-AG remained elevated with respect to STD mice, they became undistinguishable from those of these controls after 14 weeks. Some of the differences, or lack thereof, found between STD and HFD mice might have been determined also by the fact that AEA, but not 2-AG, levels increased significantly with age. Pancreatic EC levels were also measured after 1 week from the start of the experiment, and no difference between STD and HFD mice was observed in this case (data not shown).
Table 1 - Endocannabinoid levels in the pancreas of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks.
Table 1 - Endocannabinoid levels in the pancreas of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table (25K)

Effect of HFD and age on EC levels in the mouse visceral and subcutaneous adipose tissue, and on the amounts of these depots

As shown in Table 2, age appeared to influence in different ways the levels in mesenteric visceral fat of the two compounds analyzed. In fact, AEA levels, normalized to grams of wet tissue, decreased with the passing weeks in STD and, more rapidly but to the same final extent, HFD mice; whereas 2-AG levels, even if normalized in the same way, increased under both dietary conditions. Because of these differences, the age-dependent changes in AEA and 2-AG levels cannot be accounted for only by the age-dependent increase of fat in the visceral depot (Figure 10). Despite these changes, no significant difference in EC levels normalized per gram tissue were observed in the visceral fat of mice fed with a HFD as compared to STD mice at any of the periods analyzed. Regarding total EC levels (which can be easily calculated from the data in Table 2 and Figure 10), a non-statistically significant trend toward increased AEA and no significant changes in 2-AG were observed at 14 weeks, whereas no change in the levels of either EC was found at 3 and 8 weeks (data not shown).
Figure 10.
Figure 10 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Changes in the amounts of fat in the visceral and subcutaneous fat of mice fed with a standard diet (STD) or high-fat diet (HFD) for 1—14 weeks. Data are means plusminus s.e.m. of N = 4—10. Means were compared by ANOVA followed by Bonferroni's post-hoc analysis. *P < 0.05 vs. the respective STD control at the same time point. **P < 0.05 vs. corresponding value at 8 weeks. In the inset, the fold-increase of fat amount in each group calculated vs. the amounts in week 1 are shown, and s.e.m. bars are not shown for the sake of clarity.
Full figure and legend (34K)

Table 2 - Endocannabinoid levels in the visceral adipose tissue of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks.
Table 2 - Endocannabinoid levels in the visceral adipose tissue of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table (19K)

As shown in Table 3, the normalized levels of both AEA and 2-AG, expressed again as picomole or nanomole gram tissue, were, instead, approximately five- to sevenfold lower in the subcutaneous fat of mice fed with a HFD vs. a STD, starting 8 weeks after the beginning of the diet, and also after 14 weeks of the high-fat regimen. Also the total EC levels in subcutaneous fat were significantly lower (data not shown), as the HFD increased the amounts of this tissue only less than or equal to twofold more rapidly than STD (Figure 10, inset). AEA levels decreased with the passing weeks in HFD more than STD mice, whereas 2-AG levels only decreased in HFD mice. Subcutaneous and visceral fat EC levels were also measured after 1 week from the start of the experiment, and no difference between STD and HFD mice was observed in this case (data not shown).
Table 3 - Endocannabinoid levels in the subcutaneous adipose tissue of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks.
Table 3 - Endocannabinoid levels in the subcutaneous adipose tissue of mice fed with a standard diet (STD) or a high-fat diet (HFD) for 3, 8, and 14 weeks - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table (26K)

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Discussion

We have reported here, for the first time in the same study, the expression of EC receptors and metabolic enzymes in intact pancreatic islets of the mouse. In a previous study (20), using immunocytochemistry on cells isolated from mouse pancreatic islets, both CB1 and CB2 receptors had been described in non-insulin-secreting (and hence presumably alpha) cells, and only CB2 receptors were found in insulin-expressing (and hence presumably beta) cells, in agreement with our present immunohistochemistry data. These authors, however, did not investigate the presence of EC metabolizing enzymes. Although it was not possible in the present study to carry out double immunofluorescence studies for NAPE-PLD or MAGL with the alpha- and beta-cell markers in mouse pancreatic islets, the general scenario that emerges from the results obtained here using both DAB and immunofluorescence staining is that EC biosynthesizing enzymes are mostly localized in glucagon-secreting alpha-cells together with CB1 and CB2 receptors, whereas degrading enzymes appear to be mostly localized in insulin-secreting beta-cells, where staining of CB2 receptors was also localized. This distribution might suggest for both AEA and 2-AG a role as autocrine mediators in alpha-cells. The two compounds would be produced by and released from these cells to act on CB1 or CB2 receptors on the same cell, to be then inactivated mostly by beta-cells. In these latter cells, however, ECs, before being hydrolyzed, might also act at CB2 receptors and cause inhibition of insulin release, as demonstrated by Juan-Pico et al. (20), or, in the case of AEA, at vanilloid TRPV1 receptors, which are coupled to insulin release in beta-cells (37), and are known as an alternative target for this EC (ref. 38, for review). Furthermore, AEA can also activate peroxisome proliferator—activated receptor-gamma independently of cannabinoid receptors (39), and this nuclear receptor is expressed and functionally active in beta-cells (40,41,42). Finally, from our immunohistochemistry data in rat pancreatic islets, and in agreement with recent data from Bermudez-Silva et al. (43), it appears that CB1 receptors are also expressed in a small population of rat beta-cells. Therefore, a parsimonious conclusion that can be reached from our present findings is that, although ECs are produced from alpha-cells to act mostly on cannabinoid receptors on these cells, they might also act in a paracrine way on both cannabinoid and non-cannabinoid receptors expressed in beta-cells. In vivo studies in lean rats have shown that systemic activation of CB1 and CB2 receptors reduce or stimulate glucose tolerance, respectively (43,44). Although several organs apart from the pancreas, and changes in insulin sensitivity rather than insulin release, might be involved in these effects, our present data might help explaining these findings. In fact, autocrine stimulation of CB1 receptors in alpha-cells by ECs might inhibit glucagon release from alpha-cells and, hence, counteract the stimulatory effect of this hormone on insulin release (45,46,47). Future studies will have to investigate this hypothesis through specific studies of the effect of CB1/CB2agonists on alpha-cell hormone release.

We have also shown here that, unlike CB1 and CB2 receptors, the expression of EC biosynthetic enzymes in pancreatic islets of HFD mice is higher than in STD mice following 3, 8, or 14 weeks of diet. In view of the present and previous (48) observation that a fat-enriched diet does not change the distribution of insulin- and glucagon-expressing cells in mouse pancreatic islets, our finding of a strong expression of NAPE-PLD and DAGLalpha inside the islets is strongly suggestive of the overexpression of these enzymes in the beta-cells of HFD mice. On the other hand, the expression of FAAH in these cells decreased starting at 8 weeks from the beginning of the HFD. These findings suggest that, particularly following a HFD, ECs are produced by beta-cells, whereupon they might act as autocrine mediators at CB2 (20), TRPV1 (37), and peroxisome proliferator—activated receptor-gamma (39,40,41) receptors, with subsequent inhibitory (via CB2 or peroxisome proliferator—activated receptor-gamma, (20,42)) or stimulatory (via TRPV1 (37)) effects on insulin secretion. Indeed, we observed here that these changes in EC metabolic enzyme expression are accompanied by higher pancreatic 2-AG and AEA levels in HFD vs. STD mice at both 3 and 8 weeks after the beginning of the diets, whereas, intriguingly, after 14 weeks EC levels were undistinguishable from those of mice fed with a standard chow. These observations suggest that the previously described higher EC levels in the pancreas of HFD mice (11) occur: (i) before the development of obesity; (ii) within pancreatic islets in particular; and (iii) at least in part because of increased biosynthesis and decreased degradation. Following prolonged HFD and development of overt obesity, pancreatic EC levels return to normal, despite the fact that dysregulation of biosynthetic and degradative enzyme persists. This might be due to the occurrence of compensatory and as yet unidentified degradation mechanisms (e.g., upregulation of cycloxygenase-2, which occurs during pancreatic islet dysfunction and following conditions of reduced insulin sensitivity (49)), or, in the case of AEA, to the fact that with the passing weeks the levels of this compound increase also in STD mouse pancreas. Alternatively, it is possible that HFD-induced enzyme up or downregulation at 14 weeks does not result in changes of enzyme activity at this time point, or that, if it does, enhanced biosynthetic enzyme activity is compensated by the lack of EC biosynthetic precursors whereupon these enzymes act to produce ECs (34). Finally, it is possible that changes in AEA and 2-AG levels reflect alterations in the metabolism of these two compounds occurring outside the islets and via different biosynthetic enzymes (13). In fact, although ECs are local mediators and not hormones, and their levels in a given tissue are regulated uniquely by the local availability of their biosynthetic precursors and the activity of anabolic and catabolic enzymes, we compared here EC levels in the whole pancreas (i.e., levels that reflect the ongoing metabolism also in parenchymal and non-endocrine cells), with the immunohistochemistry data that refer only to pancreatic islets. At any rate, our present data of an upregulation of EC levels and biosynthesizing enzymes already 3 weeks after a HFD, when obesity has not yet fully developed, confirm that an overactive EC system in the endocrine pancreas is a hallmark not only of obesity but also of hyperglycemia (which usually develops in HFD-fed mice before overt obesity (ref. 50 and present data)). This suggestion is in agreement with our previous findings, showing that upregulation of EC levels in insulinoma beta-cells occurs already after 2 h of exposure of cells to a high glucose concentration (11). These findings would suggest that an overactive EC system might be one of the causes, more than the consequence, of obesity. It is important to point out that we measured EC levels in tissues from mice killed after an overnight 12-h fasting period, in order to minimize a possible post-prandial decrease of EC levels, previously observed in the blood of human lean volunteers (11). However, fasting is also known to increase EC levels in the hypothalamus and duodenum of lean rats (51,52), and this might have influenced our results to some extent.

Another outcome of our study is to have confirmed, by the use of immunohistochemistry, the presence of cannabinoid receptors and EC metabolic enzymes in visceral mesenteric and subcutaneous adipocytes. Previous immunofluorescence studies showed the presence of CB1 and CB2 receptors in adipocytes from human subcutaneous and omental adipose tissue (19), and of CB1, TRPV1, FAAH, MAGL, and NAPE-PLD in human subcutaneous fat (12), whereas neither the presence of DAGLalpha in adipocytes had been investigated before nor specific DAB staining of the proteins of the EC system had ever been reported in this tissue. Furthermore, evidence for the presence of these proteins in adipocytes and adipose tissue of the mouse was previously obtained only using different techniques (reverse transcriptase—PCR or western blot) (16,17). Importantly, although no differences in the expression of EC metabolic enzymes and EC normalized or total levels were found in the visceral fat of mice fed with a HFD as compared to STD, in the subcutaneous fat the high-fat regimen, starting 8 weeks from its beginning, resulted in the expression of EC metabolic enzymes and normalized or total levels of AEA and 2-AG that were lower than the corresponding ones in STD mice. Interestingly, whilst, in the subcutaneous fat, HFD-induced changes in anabolic and catabolic enzymes go in the same direction of HFD-induced changes in EC levels, the age-dependent decrease of AEA levels in both dietary groups, and of 2-AG levels in HFD mice (Table 3), cannot be explained by corresponding changes in FAAH, MAGL, DAGLalpha, and NAPE-PLD (Figure 9). This observation highlights again how the expression/activity of these enzymes cannot be considered the sole determinant of EC levels (see above).

The above data on EC levels in the adipose tissues do not entirely agree with the previous finding of increased and unchanged levels of 2-AG in the visceral and subcutaneous fat of obese patients, respectively, as compared with non-obese patients (11). Nevertheless, they still suggest that a different distribution between visceral and subcutaneous fat in individuals with the same BMI and overall adiposity might have a dramatic effect on the levels of ECs in this organ. In fact, it can be predicted that, in both humans and mice, a higher percentage of visceral vs. subcutaneous fat will result in higher peripheral EC levels, thus possibly accounting for the strong association between high circulating 2-AG levels and intra-abdominal adiposity observed in obese patients with the same BMI (28,29). As to the possible reasons for decreased EC (but not CB1 or CB2 receptor) levels in the subcutaneous fat of HFD-fed mice, in view of the inhibitory effects exerted by insulin on EC levels in insulinoma cells grown in low, but not high, glucose concentrations (11), one might speculate that the insulin resistance that occurs during obesity in visceral more than subcutaneous adipocytes (53,54,55) might result in the loss of the possible insulin inhibition of EC levels in visceral adipose tissue.

In a previous study, we also found that feeding mice for 8 weeks with a HFD with a similar caloric content but different fat composition, as compared to the one used here, did cause a twofold increase of 2-AG levels in the epididymal fat of mice (11). With the HFD used in this study, we did not find any significant increase in EC levels in the epididymal fat (data not shown), although, in full agreement with the previous study (11), we found here that a HFD for 8 weeks does cause an increase of pancreatic EC levels. These observations open the possibility that the fatty acid composition of the diet, rather than the presence of a high-fat intake itself, is one of the causes of the changes, or lack thereof, of EC levels in the adipose tissue. Indeed, Petersen et al. (34) recently suggested that the fatty acid composition of the biosynthetic precursor of AEA, which in turn depends on the intake of polyunsaturated fatty acids (PUFAs) and their precursors, rather than the activity of biosynthetic and degrading enzymes, underlies the increase of the small intestine levels of this EC observed after food deprivation in mice. Therefore, it is possible that, apart from the changes in the expression of biosynthetic and degrading enzymes, the amounts and composition of PUFAs influence the levels of ECs in peripheral tissues, also because they have been reported to do so in the brain where an increase of the dietary omega6/omega-3 PUFA ratio is accompanied by increased EC levels (56,57). Experiments comparing the different effect of different HFDs on EC levels in peripheral tissues are currently ongoing in our laboratory and might confirm or discard this hypothesis. However, if one compares the fatty acid compositions of the diets used in the previous (11) and present studies (Table 4), it is possible to speculate that the previously observed increases of 2-AG in epididymal fat (11) were not due to an increase of the dietary omega6/omega-3 PUFA ratio (which, in fact, was decreased in the HFD). Conversely, the decrease in EC levels observed here in the subcutaneous fat might be due to a decrease in the total amounts of omega6-PUFAs, whereas the lack of changes in visceral fat EC concentration might be due to the absence of any significant change in the dietary omega6/omega-3 PUFA ratios when passing from the STD to the HFD (Table 4).
Table 4 - Fatty acid compositions of standard (STD) or high-fat diet (HFD) used in the present and a previous (11) study on the effect of HFD on endocannabinoid levels in the adipose tissue.
Table 4 - Fatty acid compositions of standard (STD) or high-fat diet (HFD) used in the present and a previous (11) study on the effect of HFD on endocannabinoid levels in the adipose tissue - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table

Also methodological and species differences might account for the different effects of HFD reported so far not only on EC levels but also on the expression of CB1 receptors. Here, we did not observe higher or lower CB1 or CB2 expression in subcutaneous and visceral adipose tissue of HFD as compared to STD mice. On the other hand, Yan et al. (30) showed that in rats fed with a HFD the expression of CB1 in the adipose tissue is increased. Conversely, a decrease of CB1 receptor expression in the visceral fat of obese vs. normoweight women and a negative correlation between CB1 mRNA levels and the amounts of visceral fat in obese patients were reported by another group (10,28), whereas two other studies showed no changes in CB1 expression (11,58). Nevertheless, a study carried out in human subcutaneous gluteal fat and visceral adipose tissue from normoweight and obese patients, and published during the revision of this manuscript (23), indicates that our present finding of decreased EC biosynthesis and levels in the subcutaneous fat of HFD mice might have a correlate in human obesity (see below).

In conclusion, this study, by showing that EC levels and metabolic enzymes are dysregulated following a prolonged HFD, provides support to the hypothesis that hyperglycemia and obesity are accompanied by higher EC signaling in the endocrine pancreas, as well as in visceral as compared to subcutaneous adipose tissue (11). Our findings in the mouse endocrine pancreas support an important role for the ECs in the control of insulin release (11,20,25), and suggest a possible regulatory action also on the release of other pancreatic hormones. On the other hand, our findings in the adipose tissue, together with the previous reports of the stimulatory effect of CB1 receptors on adipocyte proliferation and differentiation, and its negative effect on adiponectin expression (see Introduction and ref. 13, for review), might explain the recent positive association found between 2-AG levels and intra-abdominal adiposity as well as other cardiovascular risk factors, such as low adiponectin levels, hyperglycemia, and glucose intolerance (28,29), whose occurrence seems to depend particularly on the presence and amounts of this type of fat (59). The HFD-induced lower levels of ECs in the subcutaneous fat might eventually result in ever decreasing amounts of this fat depot and, subsequently, in uncontrolled visceral and ectopic fat accumulation, with subsequent increase of cardiometabolic risk (59). In fact, the subcutaneous adipose tissue is viewed as a buffer preventing fat accumulation into visceral adipose tissue or outside the adipose organ (59). Unfortunately, it was not possible to gain support to this hypothesis solely based on our present data. In fact, although in Figure 10 it is possible to observe how, in HFD mice, the net amount of visceral mesenteric fat is still significantly increasing when passing from 8 to 14 weeks of the diet, whereas the amount of subcutaneous fat reaches a plateau already 8 weeks after the diet, it is also clear that this phenomenon occurs in STD mice too (even though HFD-subcutaneous fat, i.e., the only depot in which the decrease of EC levels observed with passing weeks was also dependent on the diet, was the only one to almost stop increasing after 8 weeks). Specific studies need to be performed in order to assess the pathological consequences of the decreased EC signaling found here in the subcutaneous fat following a HFD. Such studies have become all the more necessary following the finding of the EC system in human adipocytes and adipose tissue (11,12,19,60), and the report, appeared during the revision of this manuscript, that a downregulation of EC signaling analogous to the one described here is found in the gluteal subcutaneous vs. visceral fat of obese patients (23).

We thank Marco Allarà, Endocannabinoid Research Group, CNR, Italy, for technical assistance. This study was partly supported by a research grant from Sanofi-Aventis, France (to V.D.M.).
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Disclosure

The authors declared no conflict of interest.
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References
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Source: Obesity - Endocannabinoid Dysregulation in the Pancreas and Adipose Tissue of Mice Fed With a High-fat Diet