Dysregulation of endocannabinoid concentrations in human subcutaneous adipose tissue in obesity and modulation by omega-3 polyunsaturated fatty acids

Obesity is believed to be associated with a dysregulated endocannabinoid system which may reflect enhanced inflammation. However, reports of this in human white adipose tissue (WAT) are limited and inconclusive. Marine long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and therefore may improve obesity-associated adipose tissue inflammation. Therefore, fatty acid concentrations, endocannabinoid concentrations, and gene expression were assessed in subcutaneous WAT biopsies from healthy normal weight individuals (BMI 18.5 to 25 kg/m 2 ) and individuals living with metabolically healthy obesity (BMI 30 to 40 kg/m 2 ) prior to and following a 12-week intervention with 3 g fish oil/day (1.1 g EPA + 0.8 g DHA) or 3 g corn oil/day (placebo). WAT from individuals living with metabolically healthy obesity had higher n-6 PUFAs and EPA, higher concentrations of two endocannabinoids (anandamide and eicosapentaenoyl ethanolamide), higher expression of PLA2G2D and PLA2G4A , and lower expression of CNR1 . In response to fish oil intervention, WAT EPA increased to a similar extent in both BMI groups, and WAT DHA increased by a greater extent in normal weight individuals. WAT eicosapentaenoyl ethanolamide and docosahexaenoyl ethanolamide increased in normal weight individuals only and WAT 2-arachidonyl glycerol decreased in individuals living with metabolically healthy obesity only. Altered WAT fatty acid, endocannabinoid, and gene expression profiles in metabolically healthy obesity at baseline may be linked. WAT incorporates n-3 PUFAs when their intake is increased which affects the endocannabinoid system; however, effects appear greater in normal weight individuals than in those living with metabolically healthy obesity.


Introduction 1
Obesity is characterised by a state of chronic low grade inflammation (1), contributed to by 2 altered levels of many fatty acid (FA) metabolites in white adipose tissue (WAT) and the circulation, 3 and loss of sensitivity to hormones and lipid signalling molecules (2). A family of FA-containing 4 metabolites called endocannabinoids may become dysregulated during obesity, but current evidence 5 of this in human WAT is limited and inconsistent (3,4). Endocannabinoids play a role in adipose 6 tissue expansion and inflammation, both of which are observed to be upregulated in obesity (5)(6)(7). 7 There is some evidence of alterations in endocannabinoids based on measurements in human plasma, 8 where the arachidonic acid (AA; 20:4n-6) containing endocannabinoids, anandamide (AEA) and 2-9 arachidonyl-glycerol (2-AG), are elevated in obesity and positively correlate with BMI and intra-10 abdominal adiposity (8)(9)(10)(11). Mechanisms behind these elevated concentrations are under-reported and 11 more comprehensive investigation of endocannabinoid concentrations and the expression of genes 12 encoding enzymes involved in the metabolism of these metabolites may provide greater insight into 13 the regulation of adipose tissue during the onset of obesity-associated inflammation and may identify 14 targets for intervention. An overview of endocannabinoid synthesis, degradation, actions, 15 observations in metabolically unhealthy obesity, and potential modulation by long chain omega- 3 16 polyunsaturated fatty acids (LC n-3 PUFAs) is detailed in Figure 1. 17 The endocannabinoid system has been targeted for therapeutic modulation in obesity, as it is involved 18 in the regulation of food intake and energy homeostasis, glucose and lipid metabolism, and 19 inflammation (12)(13)(14). Endocannabinoids act predominantly via cannabinoid receptors but they also 20 bind to peroxisome proliferator activated receptors (PPARs) and a range of G-protein coupled 21 receptors (6). Cannabinoid receptor-1 antagonists improve adipocyte insulin sensitivity and glucose 22 homeostasis through altered cytokine production from WAT macrophages (15), and the 23 downregulation of cannabinoid receptor-1 led to weight loss and normalisation of lipid homeostasis in 24 individuals living with obesity and type-2 diabetes (16,17). Activation of PPARs and G-protein 25 coupled receptors by endocannabinoids has anti-inflammatory actions and regulates FA metabolism 26 and energy balance (6). 27 In addition to AEA and 2-AG, some lesser reported endocannabinoids which are present in lower 28 concentrations contain the n-3 PUFAs eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic 29 acid (DHA; 22:6n-3); these include eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl 30 ethanolamide (DHEA) (18). LC n-3 PUFAs have been shown to decrease inflammation, a key factor 31 in the development of obesity-related metabolic dysfunction and co-morbidities (2,(19)(20)(21). This anti-32 inflammatory effect was linked to increases in LC n-3 PUFA containing endocannabinoids and 33 decreases in the AA containing "classical" endocannabinoids in WAT in experimental animals (10, 34 22-24) and in plasma from humans with obesity (25). In addition, EPA and DHA can act as signalling 35 molecules by binding to PPARs (26) to regulate the expression of genes associated with adipose 36 functions including expansion, lipid accumulation and energy homeostasis, and inflammation (27, 37 28). However, there is limited exploration of these effects in human WAT. 38 Modulation of the endocannabinoid system and response to dietary FAs have also been shown to 39 differ by metabolic status (4,29). Concentrations of circulating endocannabinoids, which may reflect 40 spill over from peripheral tissues such as the WAT, have been shown to decrease postprandially or 41 following an oral glucose load and euglycemic clamp in normal weight individuals (30,31), but not in 42 those individuals living with obesity accompanied by insulin resistance (31). These individuals, in 43 addition to those living with diagnosed type 2 diabetes, have higher fasting concentrations of 44 circulating endocannabinoids (4,12,30,(32)(33)(34). The endocannabinoid system is under the control of 45 leptin, insulin, and PPAR- agonists including glitozones used in the treatment of type-2 diabetes and 46 LC n-3 PUFAs (12,30,31,35). The use of LC n-3 PUFAs to modulate the endocannabinoid system 47 in the context of obesity with accompanying type-2 diabetes is of interest due to improvements seen 48 in WAT inflammation and energy homeostasis (17). However, influencing individuals earlier in the 49 causal pathway is desirable in order to potentially prevent the onset of serious non-communicable 50 conditions like diabetes. Despite this, the effects of LC n-3 PUFAs on WAT in individuals living with 51 metabolically healthy obesity (MHO) have not been studied. This study aimed to identify any 52 differences in the WAT endocannabinoid system, and the potential contribution of differences in FA 53 composition and expression of endocannabinoid related genes, in individuals living with 54 metabolically healthy obesity compared to individuals of normal weight to investigate the onset of 55 dysregulation in the WAT. Furthermore, the study aimed to investigate whether intervention with LC 56 n-3 PUFAs could modify the endocannabinoid system in WAT which may have potential to slow or 57 even reverse the onset of obesity-associated inflammation in the tissue. 58

Methods 59
All procedures involving human participants were approved by the National Research Ethics Service 60 or chronic gastrointestinal problems (e.g. inflammatory bowel disease, celiac disease, and cancer); the use of prescribed medicine to control inflammation, blood lipids or blood pressure; consumption of 71 more than one portion of oily fish per week (140 g cooked); use of fish oil or other oil supplements; 72 being pregnant or planning to become pregnant during the study period; or participation in another 73 clinical trial. 74

Study design 75
Fasted blood and an abdominal subcutaneous white adipose tissue (scWAT) biopsy (~1 g) were 76 collected at baseline (week-0) and following a 12-week intervention (week-12) during which 77 participants were randomised to consume either 3 g of fish oil (1.1 g EPA + 0.8 g DHA) or 3 g of corn 78 oil (1.65 g linoleic acid (LA) and 0.81 g oleic acid) per day (full composition of intervention oils are 79 detailed in Supplementary Table 1). Blinding, randomization, and supplement packaging were 80 completed by the Research Pharmacy at Southampton General Hospital, Southampton, United 81 Kingdom, by individuals independent of the researchers involved in the study. Treatment group 82 blinding was maintained until completion of statistical analysis of all data. 83

Sample preparation 84
Abdominal scWAT biopsies were collected by surgical removal under local anaesthetic (1% 85 lidocaine) to provide ~1 g of intact tissue and were directly stored on ice. Tissue was divided into 5 x 86 ~200 mg aliquots. scWAT designated for FA and lipid metabolite analyses was wrapped in foil, 87

Anthropometry 101
Height was measured using a Seca stadiometer (Seca, Hamburg, Germany) with shoes 102 removed, and weight and body composition measurements were made using digital bioelectric impedance apparatus (TANITA BC-418) with shoes and socks, personal items, and heavy clothing 104 removed. Waist and hip circumference measurements were made using a tape measure whilst 105 participants wore loose clothing. The tape was placed flat around their waist between the ribs and hip 106 bone, one centimetre under the umbilicus for waist measurement, and around the greatest area of 107 protrusion of the buttocks for hip measurement. 108

Obesity is associated with an altered scWAT fatty acid composition, higher concentrations of 207 scWAT endocannabinoids, and altered expression of fatty acid metabolite pathway genes 208
The most abundant FA present in scWAT was oleic acid (18:1n-9) followed by palmitic acid 209 Individuals living with metabolically healthy obesity had altered scWAT gene expression with 226 significantly lower expression of SLC27A2 (P < 0.001) and CNR1 (data obtained by qRT-PCR, P < 227 0.05), and significantly higher expression of PLA2G2D (phospholipase A 2 Group IID -secretory), 228 PLA2G4A (phospholipase A 2 Group IVA -cytosolic) and PLA2G7 (lipoprotein-associated 229 phospholipase A 2 Group VII; data obtained by RNA-Seq, P < 0.01) ( Figure 6). In addition to this, 230 there was > 50% lower expression of DAGLA (diacylglycerol-α lipase), MGLL (monoacylglycerol 231 lipase), FAAH (fatty acid amide hydrolase), SLC27A1 (long chain fatty acid transport protein-1), and 232 SLC27A2 (very long chain fatty acid Co-A synthetase) in scWAT from individuals living with 233 metabolically healthy obesity, although the differences were not statistically significant (data obtained 234 by qRT-PCR, Figure 6). 235 FAAH is involved in the degradation of endocannabinoids and as there was a > 50% lower expression 236 of mRNA encoding this enzyme in scWAT from individuals living with metabolically healthy 237 obesity, which may result in greater concentrations of endocannabinoids in the tissue, the activity of 238 this enzyme in MHO individuals was of interest. However, FAAH activity was not detectable in WAT 239 from either normal weight or individuals living with metabolically healthy obesity. 240

LC n-3 PUFA containing endocannabinoids by fish oil 242
In response to 12-week fish oil intervention, the proportions of scWAT EPA, DPA and DHA 243 significantly increased (by 59%, 29% and 36% respectively) in normal weight individuals (P = 0.006, 244 <0.001 and <0.001 respectively) and the proportion of EPA significantly increased (by 56%) in 245 individuals living with metabolically healthy obesity (P <0.001) (Figure 7). The proportions of DPA 246 and DHA also increased in individuals living with metabolically healthy obesity (by 9% and 17%) but 247 this did not reach statistical significance (Figure 7). Despite a smaller increase in these FAs, DPA The effect of LC n-3 PUFA intervention on the endocannabinoid system appears to be limited in 292 individuals living with metabolically healthy obesity. 293 In normal weight individuals, LC n-3 PUFA significantly increased EPEA and DHEA without 294 affecting AEA or 2-AG, while in individuals living with metabolically healthy obesity, the biggest 295 effect was on 2-AG, which was decreased. There were no significant changes to scWAT AA in either 296 group of individuals following the intervention period resulting in a greater proportion of AA 297 remaining in the scWAT of individuals living with metabolically healthy obesity in comparison to 298 normal weight individuals. It may be that having a higher proportion of AA, as seen in those with 299 metabolically healthy obesity, hinders the synthesis of EPA and DHA containing endocannabinoids, 300 and a higher dose of LC n-3 PUFAs is required to alter the n-6: n-3 ratio and favour the synthesis of 301 these endocannabinoids in such individuals. Rossmeisl et al. (18) report no change to AA or to AA 302 containing endocannabinoids in scWAT of individuals living with obesity and diabetes following 303 intervention with a higher dose of LC n-3 PUFAs containing a greater proportion of DHA and over a 304 longer period time than used in the current study. Therefore, the metabolic health of the individual may also play a role in the handling and metabolic effects of these FAs and this may relate to insulin 306 sensitivity of the WAT. 307 In the current study LC n-3 PUFAs did not modulate the expression of scWAT genes encoding 308 enzymes involved in endocannabinoid synthesis suggesting that the increased concentrations of EPEA 309 and DHEA following intervention may more likely result from increased availability of EPA and 310 DHA within substrates for synthesis of these metabolites. In contrast, lack of significant change in 311 synthesis of LC n-3 PUFA containing endocannabinoids in individuals living with metabolically 312 healthy obesity may be due to altered expression of enzymes involved in the metabolism of LC n-3 313 PUFA, particularly DHA, observed at baseline. There was 92% lower expression of SLC27A2 (P 314 <0.001, Figure 5) in scWAT of individuals living with metabolically healthy obesity at study entry; 315 the enzyme encoded by this gene is responsible for activating DHA to its Co-A ester required for 316 further metabolism ultimately to DHEA (48). If there is a similar proportion of DHA in the scWAT of 317 normal weight individuals and those living with metabolically healthy obesity, but its activation is 318 lower in the latter, this could result in reduced ability to convert DHA into DHEA. This may 319 contribute to why an increase in EPEA (albeit not significant) but not DHEA is observed in these 320

individuals. 321
EPA and DHA induced regulation of the endocannabinoid system in human adipose tissue has been 322 reported previously in only one study. In that study, scWAT EPEA and DHEA increased with 323 increased n-3 PUFA intake and positively correlated with the n-3 index in the serum (18) The authors 324 studied 16 individuals with obesity and medication-controlled type 2 diabetes in response to a 24-325 week fish oil intervention (2.8 g EPA + DHA/day). Although EPEA and DHEA increased, there was 326 no change in AA containing endocannabinoids (18). The current study also observed an increase in 327 EPEA in individuals living with metabolically healthy obesity, but this was less than that observed in 328 normal weight individuals and was not significant. Furthermore, the current study reports no increase 329 in the concentration of DHEA in these individuals. The composition of the fish oil used in the current 330 study differs from that used by Rossemeisl et al. in that it is richer in EPA than DHA. It may be that 331 by using an oil with a higher proportion of DHA than EPA is required in obese individuals to raise the 332 concentration of DHEA. 333 Why LC n-3 PUFAs affect the endocannabinoid system differently in normal weight individuals and 334 in individuals living with metabolically healthy obesity is not clear but may relate to the specific 335 metabolic changes induced by the FA or to differences in expression of genes at baseline between the 336 two groups. These data question the use of LC n-3 PUFAs in individuals living with metabolically 337 healthy obesity for the amelioration of scWAT inflammation and improvement of adipose function 338 via the endocannabinoid system. The greatest effects were limited to normal weight individuals, 339 suggesting a potential role for LC n-3 PUFAs in the prevention of adipose tissue dysfunction through 340 modulation of the endocannabinoid system in more healthy individuals. It seems that even in those 341 with metabolically healthy obesity, lipid and endocannabinoid metabolism may already be 342 dysregulated. Further exploration of the mechanisms resulting in these differences in individuals 343 living with metabolically healthy obesity and the consequences of these observations on WAT 344 functionality is required. 345 One limitation of this study is that dietary FA intake, which may be a possible explanation for 346 baseline differences in FA composition (and lipid metabolite profile), was not measured. Despite this, 347 individuals were recruited on the basis that they consumed no more than one portion of oily fish per 348 week, limiting EPA and DHA intake. As full dietary data were not collected, intake of n-6 PUFA 349 could not be examined and the contribution of these FAs to changes in FA composition (and 350 contribution to endocannabinoid concentrations) could not be assessed. The current study has many 351 strengths which are its sample size, its phenotyping of the individuals, the use of an intervention to 352 potentially modify the WAT endocannabinoid profile, and compliance to this intervention which was 353 > 90%. We have shown that 12-weeks of 1.9 g of EPA + DHA daily was adequate to increase EPA 354 and DHA in human scWAT and to alter endocannabinoid concentrations in both normal weight and 355 individuals living with metabolically healthy obesity. This dose of EPA + DHA is lower than 356 previously used in a study of WAT endocannabinoids humans (18) and could be achievable by diet or 357 a combination of diet and supplementation amongst the general public. Furthermore, we show that 12 358 weeks is sufficient time to increase scWAT LC n-3 PUFAs and to modulate the endocannabinoid 359 system in normal weight individuals but we suggest that a longer intervention period or a higher LC