Role and regulation of acylethanolamides in energy balance: focus on adipocytes and beta-cells

Abstract

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator-activated receptor (PPAR)-alpha ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti-inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic beta-cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography-mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin-induced differentiation, 2) rat insulinoma RIN m5F beta-cells kept in 'low' or 'high' glucose, 3) adipose tissue and pancreas of mice with high fat diet-induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR-gamma. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma beta-cells, OEA and PEA levels are inhibited by 'very high' glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in 'high' glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity-related pro-inflammatory states. In beta-cells and human blood, OEA and PEA are down- or up-regulated under conditions of transient or chronic hyperglycaemia, respectively.

Figure 1

Schematic representation of the anabolic and catabolic pathways so far proposed for the acylethanolamides and 2-AG. Adapted from Di Marzo and Petrosino (2007). Abbreviations used: Abh4, alpha/beta-hydrolase 4; 2-AG, 2-arachidonoylglycerol; DAGL, sn-1-selective diacylglycerol lipase; AMT, putative endocannabinoid membrane transporter; FAAH, fatty acid amide hydrolase; lyso-PLD, lysophospholipase D; MAGLs, monoacylglycerol lipases; NAPE-PLD, N-acylphosphatidyl-ethanolamine-selective phospholipase D; PLA2, phospholipase A2; PLC, phospholipase C; PTPN22, protein tyrosine phosphatase N22.

Figure 2

Regulation of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels during adipocyte differentiation. (a) Differentiation of mouse 3T3F442A pre-adipocytes into adipocytes induced by insulin (0.9 μM) was monitored by measuring peroxisome proliferator-activated receptor (PPAR)-γ expression by real-time reverse transcription (RT)–PCR (lower panel), or Oil Red-O staining under a microscope (upper panel; adapted from Matias et al., 2006). RNA expression is expressed as fold-enhancement over day 0. Error bars are not shown and they were always <10%. s.d. values for cycle threshold were always <1%. (b) Levels of OEA and PEA during adipocyte differentiation induced with insulin (0.9 μM). OEA and PEA levels were measured by isotope-dilution LC-MS (see Methods). Data are means±s.e.mean of n=3–6 separate experiments. ^*=P<0.01 vs day 0, respectively, as assessed by ANOVA followed by the Bonferroni's test.

Figure 3

Regulation of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in adipocytes. (a) Effect on OEA and PEA levels of a 24 h stimulation with leptin (20 nM) of differentiated adipocytes (8 days of treatment with 0.9 μM insulin). (b) Effect on OEA and PEA levels of prolonged 12 h stimulation with WY14643 (a peroxisome proliferator-activated receptor (PPAR)-α agonist, 20 μM) and ciglitazone (a PPAR-γ agonist, 20 μM) of differentiated adipocytes. Data are means±s.e.mean of n=4–6 separate experiments. ^*=P<0.05 vs vehicle, respectively, as assessed by ANOVA followed by Bonferroni's test.

Figure 4

Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in the visceral adipose tissue of normoweight and overweight/obese humans and in the subcutaneous fat of obese patients (Table 1). ^**=P<0.003 vs visceral fat from the corresponding obese patients, as assessed by paired Student's t-test.

Figure 5

Levels of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in RIN m5F β-cells kept on ‘low' (13 mM, G13) and ‘high' (25 mM, G25) glucose for 24 h before stimulation with either glucose (33 mM, 2 h, G33), insulin (100 nM, 2 h) or glucose+insulin (2 h). Data are means±s.e.mean of n=6 separate experiments. ^*=P<0.05 vs vehicle as assessed by ANOVA followed by the Bonferroni's test. Note that ‘low', ‘high' and ‘very high' glucose do not refer to, nor do they reflect, the fasting concentrations of glucose occurring in humans during normo- or hyperglycaemia, respectively. They refer instead to the optimal culturing conditions of RIN-m5F β-cells, which the manufacturer advises to grow in 25 mM glucose.

Figure 6

(a) Blood oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in overweight type 2 diabetes (T2D) vs healthy volunteers. Data are means±s.e.mean of n=8 healthy volunteers and n=10 T2D patients. (b) Blood OEA and PEA levels in postprandial vs preprandial healthy normoweight volunteers. Blood sampling was carried out 1 h before and after the meal, respectively. Data are means±s.e.mean of n=12 subjects. ^*=P<0.05 vs controls, respectively, as assessed by the Kruskal–Wallis nonparametric test (a), or the two-tailed paired Student's t-test (b).