15 research outputs found
High Fat Diet Increases Circulating Endocannabinoids Accompanied by Increased Synthesis Enzymes in Adipose Tissue
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
Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomicmethods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system
Caloric restriction lowers endocannabinoid tonus and improves cardiac function in type 2 diabetes
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Pharmacokinetic Modeling of Paracetamol Uptake and Clearance in Zebrafish Larvae: Expanding the Allometric Scale in Vertebrates with Five Orders of Magnitude
Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 μL, this yields an absolute clearance of 2.96 × 10(7) L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration–effect relationships
High fat diet increases circulating endocannabinoids accompanied by increased synthesis enzymes in adipose tissue
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 (R 2 = 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
Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons
Diacylglycerol lipase
(DAGL)-α and -β are enzymes responsible
for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol
(2-AG). Selective and reversible inhibitors are required to study
the function of DAGLs in neuronal cells in an acute and temporal fashion,
but they are currently lacking. Here, we describe the identification
of a highly selective DAGL inhibitor using structure-guided and a
chemoproteomics strategy to characterize the selectivity of the inhibitor
in complex proteomes. Key to the success of this approach is the use
of comparative and competitive activity-based proteome profiling (ABPP),
in which broad-spectrum and tailor-made activity-based probes are
combined to report on the inhibition of a protein family in its native
environment. Competitive ABPP with broad-spectrum fluorophosphonate-based
probes and specific β-lactone-based probes led to the discovery
of α-ketoheterocycle LEI105 as a potent, highly selective, and
reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not
affect other enzymes involved in endocannabinoid metabolism including
abhydrolase domain-containing protein 6, abhydrolase domain-containing
protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase
and did not display affinity for the cannabinoid CB<sub>1</sub> receptor.
Targeted lipidomics revealed that LEI105 concentration-dependently
reduced 2-AG levels, but not anandamide levels, in Neuro2A cells.
We show that cannabinoid CB<sub>1</sub>-receptor-mediated short-term
synaptic plasticity in a mouse hippocampal slice model can be reduced
by LEI105. Thus, we have developed a highly selective DAGL inhibitor
and provide new pharmacological evidence to support the hypothesis
that “on demand biosynthesis” of 2-AG is responsible
for retrograde signaling