White adipose tissue (WAT) stores excess energy as triglycerides, and brown adipose tissue (BAT) is specialized in dissipating energy as heat. The endocannabinoid system (ECS) is involved in a broad range of physiological processes and is increasingly recognized as a key player in adipose tissue metabolism. High ECS tonus in the fed state is associated with a disadvantageous metabolic phenotype, and this has led to a search for pharmacological strategies to inhibit the ECS. In this review we present recent developments that cast light on the regulation of adipose tissue metabolism by the ECS, and we discuss novel treatment options including the modulation of endocannabinoid synthesis and breakdown enzymes.

The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity. Eight groups of age-matched male C57Bl/6J mice were randomized to receive a chow diet (control) or receive a high fat diet (HFD, 45% of calories derived from fat) ranging from 1 day up to 18 weeks before euthanasia. Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA), and related N-acylethanolamines, were quantified by UPLC-MS/MS and gene expression of components of the ECS was determined in liver, muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) during the course of diet-induced obesity development. HFD feeding gradually increased 2-AG (+132% within 4 weeks, P < 0.05), accompanied by upregulated expression of its synthesizing enzymes Daglα and β in WAT and BAT. HFD also rapidly increased AEA (+81% within 1 week, P < 0.01), accompanied by increased expression of its synthesizing enzyme Nape-pld, specifically in BAT. Interestingly, Nape-pld expression in BAT correlated with plasma AEA levels (R2 = 0.171, β = 0.276, P < 0.001). We conclude that a HFD rapidly activates adipose tissue depots to increase the synthesis pathways of endocannabinoids that may aggravate the development of HFD-induced obesity.

Careful design of experiments using living organisms (e.g. mice) is of critical importance from both an ethical and a scientific standpoint. Randomization should, whenever possible, be an integral part of such experimental design to reduce bias thereby increasing its reliability and reproducibility. To keep the sample size as low as possible, one might take randomization one step further by controlling for baseline variations in the dependent variable(s) and/or certain known covariates. To give an example, in animal experiments aimed to study atherosclerosis development, one would want to control for baseline characteristics such as plasma triglyceride and total cholesterol levels and body weight. This can be done by first defining blocks to create balance among groups in terms of group size and baseline characteristics, followed by random assignment of the blocks to the various control and intervention groups. In the current study we developed a novel, user-friendly tool that allows users to easily randomize animals into blocks and identify random block divisions that are well-balanced based on given baseline characteristics, making randomization time-efficient and easy-to-use. Here, we present the resulting software tool that we have named RandoMice.

Background: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3 , has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. Methods: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC -E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance–based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC -E97G overexpression. Results: Family members carrying the LIPC -E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC -E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC -E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC -E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. Conclusions: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3 , causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

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