Genetic polymorphisms of the endocannabinoid system in obesity and diabetes

The endocannabinoid system (ECS) is involved in many physiological processes including fertility, pain and energy regulation. The aim of this systematic review was to examine the contribution of single nucleotide polymorphisms (SNPs) of the ECS to adiposity and glucose metabolism. Database searches identified 734 articles, of which 65 were included; these covered 70 SNPs in genes coding for cannabinoid receptors 1 and 2 (CB1, CB2), fatty acid amide hydrolase (FAAH) and N‐acyl phosphatidylethanolamine phospholipase D (NAPE‐PLD). No studies included SNPs relating to monoacylglycerol lipase or diacylglycerol lipase. The CB1 receptor SNP rs1049353 showed 17 associations with lower body mass index (BMI) and fat mass (five studies). It also showed three associations with lower insulin levels (one study). Conversely, the CB1 receptor SNP rs806368 was associated with increased BMI and waist circumference (two studies). The FAAH SNP rs324420 was associated with increased obesity (three studies). A haplotype of NAPE‐PLD was associated with decreased BMI (one study). A total of 60 SNPs showed no association with any measured outcome. This review suggests a complex but important role of ECS SNPs in energy and glucose metabolism.


| INTRODUCTION
The endocannabinoid system (ECS) consists of two G-protein coupled receptors (CB 1 and CB 2 ) and endogenously produced ligands, or endocannabinoids such as anandamide and 2-arachidonoyl glycerol, and the enzymes involved in their synthesis or degradation: fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), diacylglycerol lipase (DAGL) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). It is well established that CB 1 activation leads to increases in energy storage 1 which occur via increased motivation to consume food and to decreased satiety.
Single nucleotide polymorphisms (SNPs) are naturally occurring variations of a genetic sequence, which often affect protein structure.
To date, studies on the effects of endocannabinoid SNPs have focused on central nervous system disorders such as Parkinson's disease and Alzheimer's disease. 2 However, there is accumulating evidence for the role of endocannabinoid SNPs in adiposity 3 and glucose metabolism. 4 Therefore, the aim of this systematic review was to systematically collate the evidence relating to SNPs of the ECS in obese or diabetic phenotypes. By studying amino acid sequence alterations and any resultant residue changes, we hoped to identify important genetic changes that alter the normal physiology of adiposity and glucose metabolism.  Figure S1, and a full reference list is available in Appendix S1.

| MATERIALS AND METHODS
The SNP database dbSNP was used to gather information regarding nucleotide and amino acid changes. 5 Articles included were original studies relating to polymorphisms of the ECS affecting energy regulation, glucose homeostasis and adiposity. Demographic and clinical parameters included were: body mass index (BMI); waist circumference (WC); waist-to-hip ratio (WHR); body weight; adiposity; type II diabetes mellitus (T2DM); insulin and glucose levels; homeostatic model assessment for insulin resistance (HOMA IR ); adipokine levels (adiponectin, leptin and resistin); cardiovascular parameters (blood pressure, heart rate); inflammation (levels of interleukin 6 [IL-6], tumour necrosis factor alpha [TNFα] and C-reactive protein [CRP]); and lipid levels (triglycerides, HDL-C and LDL-C). Records excluded were review articles, articles on the ECS not relating to polymorphisms, studies regarding central disorders, studies in non-humans and studies in a language other than English.
Included articles were analysed for significant (P < 0.05) positive or negative associations between SNPs and relevant parameters. A "positive" association refers to a higher value of the measured outcome in the presence of the polymorphism, whereas a "negative" association refers to a lower value in the presence of the polymorphism. The absence of a significant association between the measured outcome and the polymorphism is described as a "neutral" association.
Risk of bias was assessed using the Cochrane Collaboration's tool for assessing risk of bias. 6

| RESULTS
A total of 65 studies were identified from among 733 full-text articles.
Risk of bias in these studies was low overall and is summarized in Supporting Information Figure S2. In total, 38 CB 1 , 18 CB 2 , 13 FAAH and one NAPE-PLD SNPs were studied. No studies relating to MAGL or DAGL SNPs were found. The most commonly studied SNPs, and those that showed the most significant associations, were CB 1 SNPs rs1049353 and rs806368, and FAAH SNP rs324420. Their associations with body weight and glucose metabolism parameters are presented in Table 1 The rs1049353 mutant allele was associated with lower BMI in six European populations [15][16][17]19,37 and with decreased fat mass in a Danish population (n = 783). 38 Conversely, homozygosity for the rs1049353 mutant allele was associated with higher WHR and WC in obese men (P < 0.01; n = 1064) 7 and with increased childhood obesity in a European population (P = 0.01; n = 200). 39 The majority of associations with rs1049353 were neutral (90%) ( Table 1). However, negative associations were more common than positive associations ( Figure 1), suggesting that this SNP plays a part in a more complex genetic susceptibility to increased adiposity. Male carriers of the rs806368 mutant allele showed greater BMI values in a Japanese cohort (P = 0.001) and were more likely to be obese (P = 0.01; n = 1452) 25 (Supporting Information Table S1).

| FAAH
FAAH polymorphism rs324420 was positively associated with obesity in four cohorts (n = 18 987). 27-30 The mutant allele of CB 2 SNP rs3123554 was associated with lower total body fat in women but not men, in a European cohort (P = 0.001), with lower BMI in individuals at risk of T2DM (P < 0.01) and with reduced weight loss (P < 0.01; n = 2006). 40

| NAPE-PLD
In a Norwegian cohort, a haplotype of NAPE-PLD showed an association with increased BMI (P < 0.05; n = 5011). 12 The mutant allele of CB 1 polymorphism rs1049353 was associated with lower insulin, glucose and HOMA IR levels in Spanish obese women 22 and with lower insulin in two other European cohorts (n = 983). 20,21 CB 1 SNP rs806365 was associated with decreased HOMA IR values and with incidence of T2DM in a North American cohort (P ≤ 0.05; n = 2411). 4

| CB 2
The mutant allele of CB 2 polymorphism rs3123554 was associated with raised insulin levels and with HOMA IR values in an obese population (n = 1027) 41 (Figure 1).

| FAAH
The mutant allele of FAAH polymorphism rs324420 was associated with lower insulin levels in two obese populations (P < 0.05; n = 165), 32,34 and was also associated with lower HOMA IR levels in obese Spanish females (P < 0.05; n = 143). 32

| Lipids
Overall, 22 positive associations with lipid levels were seen. The mutant allele of CB 1 SNP rs1049353 was associated with higher HDL and lower TGs in three cohorts, 18,22,23 as well as with lower TGs in two populations (n = 808) 19,22 (Table 1).

| DISCUSSION
The aim of this study was to collate evidence relating to SNPs of the ECS and obese or diabetic phenotypes to identify important genetic  overactivity. Subsequent CB 1 activation leads to adipogenesis and reduced expenditure, all of which contribute to obesity-related phenotypes. Our analysis showed that rs324420 was associated with higher anandamide levels, 42 increased BMI and obesity, 27,30,31 which suggests cannabinoid over-activation and subsequent adiposity and that this SNP, therefore, reduces FAAH activity ( Table 1).
The potential contribution of CNR2 polymorphisms to human metabolism is less clear. Fewer studies investigated these SNPs, and the two polymorphisms studied (rs3123554 and rs35761398) showed conflicting associations with body weight parameters and glucose metabolism. As CB 2 receptors are found primarily in the central nervous system and on immune cells, it is likely that they are less involved in the regulation of body fat and, therefore, any alterations in their genetic structure are less relevant here. As no studies were found relating to SNPs of DAGL or MAGL, their contribution to obesity and glucose metabolism remains unclear.
Increasing age may determine the impact of the polymorphism.
For instance, associations between SNPs rs2023239 and rs806381 and increased anthropometric measurements were found only in adult subjects. 46,47 Ageing leads to reductions in ligand binding 48 and coupling between the CB 1 receptor and its G-protein, 49 which may account for the delayed onset of increases in body weight parameters in some populations. There may also be an impact of gender on these data. Male carriers of the mutant alleles of CNR1 polymorphisms rs1049353 and rs806368 have an increased likelihood of obesity. 7,25 Similarly, the associations between the CNR2 polymorphism rs3123554 and lower BMI, weight and body fat percentage were reported in women. 40 Gender differences in feeding behaviour have been observed previously in animal models. 13 This may be explained by the action of oestrogen, which uncouples CB receptors from their effector systems in synaptic terminals, thus reducing the effect of cannabinoids. 50 Higher oestrogen levels in non-pregnant females may therefore contribute to these gender-specific findings.
In conclusion, associations between the mutant allele of the