Activity-based Photoacoustic Probes Reveal Elevated Intestinal MGL and FAAH Activity in a Murine Model of Obesity

Abstract

Obesity is a chronic health condition characterized by the accumulation of excessive body fat which can lead to and exacerbate cardiovascular disease, type-II diabetes, high blood pressure, and cancer through systemic inflammation. Unfortunately, visualizing key mediators of the inflammatory response, such as monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), in a selective manner is a profound challenge owing to an overlapping substrate scope that involves arachidonic acid (AA). Specifically, these enzymes work in concert to generate AA, which in the context of obesity, has been implicated to control appetite and energy metabolism. In this study, we developed the first selective activity-based sensing probes to detect MGL (PA-HD-MGL) and FAAH (PA-HD-FAAH) activity via photoacoustic imaging. Activation of PA-HD-MGL and PA-HD-FAAH by their target enzymes resulted in 1.74-fold and 1.59-fold signal enhancements, respectively. Due to their exceptional selectivity profiles and deep-tissue photoacoustic imaging capabilities, these probes were employed to measure MGL and FAAH activity in a murine model of obesity. Contrary to conflicting reports suggesting levels of MGL can be attenuated or elevated, our results support the latter. Indeed, we discovered a marked increase of both targets in the gastrointestinal tract. These key findings set the stage to uncover the role of the endocannabinoid pathway in obesity-mediated inflammation.

Keywords: Activity-Based Sensing; Endocannabinoid System; Inflammation; Obesity; Photoacoustic Imaging.

Figure 1

a) Synthesis of PA‐HD‐MGL and schematic illustrating MGL‐catalysed activation to compound 2. b) Synthesis of PA‐HD‐FAAH and schematic illustrating FAAH‐catalysed activation to compound 3.

Figure 2

a) Summary of photophysical properties for PA‐HD‐MGL, compound 2, PA‐HD‐FAAH, and compound 3. Absorbance and PA maxima determined in 7 : 3 PBS:MeCN pH 7.4. Molar absorptivity coefficients and fluorescence quantum yields determined in DMSO. pKa were determined in 0.1 % SDS in PBS (pH ranged from 2.7 to 11.8). b) PA spectra of compound 2 and PA‐HD‐MGL (10 μM, 7 : 3 PBS:MeCN, pH 7.4). c) PA spectra of compound 3 and PA‐HD‐FAAH (10 μM, 7 : 3 PBS:MeCN, pH 7.4). d) Fold absorbance turn‐on of PA‐HD‐MGL in the presence of MGL, FAAH, COX‐1, COX‐2, 5‐LOX, RLM, esterase, lipase or catalase after 15 minutes at optimal pH for respective enzymes in Tris buffer. e) Normalized absorbance turn‐on of PA‐HD‐FAAH in the presence of FAAH, MGL, COX‐1, COX‐2, 5‐LOX, RLM, esterase, lipase or catalase after 15 minutes at optimal pH for respective enzymes in Tris buffer.

Figure 3

Fluorescent images of PA‐HD‐MGL turn‐on in LNCaP cells treated with a) vehicle control, b) MGL inhibitor, or c) FAAH inhibitor using PA‐HD‐MGL. Fluorescent images of PA‐HD‐FAAH turn‐on in LNCaP cells treated with d) vehicle control, e) FAAH inhibitor, or f) MGL inhibitor using PA‐HD‐FAAH. MGL inhibitor=JZL184 (40 μM). FAAH inhibitor=PF‐3845 (40 μM). Scale bar represents 50 μm. g) Quantification of data in a), b), and c). h) Quantification of data in d), e), and f). Statistical analysis was performed using a two‐tailed Student's t‐test (α= 0.05), **P<0.01, *P<0.05, ns=not significant.

Figure 4

a) Schematic representing workflow for the generation of murine models to study the effect of diet on MGL and FAAH activity using in vivo photoacoustic imaging. b) Lateral schematic of a mouse where the field of view is indicated by the dotted box. c) Cross‐sectional schematic of a mouse to reference the positioning of the spinal cord, kidneys, and intestines.

Figure 5

Representative MSOT images showing the a) lateral and b) cross‐sectional views of mice fed low‐fat and high‐fat diets treated with PA‐HD‐MGL (200 μM, 200 μL DMSO in saline). c) Average PA fold turn‐on from MSOT imaging (n=3, independent animals). Representative MSOT images showing the d) lateral and e) cross‐sectional views of mice fed low‐fat and high‐fat diets treated with PA‐HD‐FAAH (200 μM, 200 μL DMSO in saline). f) Average PA fold turn‐on from MSOT imaging (n=3, independent animals). All images were acquired 0.5 h post‐injection of the probe. All images shown are spectrally unmixed to show probe activation (in cyan for MGL and orange for FAAH) and arrow indicates intestines. Scale bar represents 5 mm. Statistical analysis was performed using a two‐tailed t‐test (α=0.05, ** P<0.01).