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Alcohol predisposes obese mice to acute pancreatitis via adipose triglyceride lipase-dependent visceral adipocyte lipolysis

We read with great interest articles by Wang et al and Hegyi et al, in which the authors reported that alcohol increased the risk of hereditary susceptibility to chronic pancreatitis. These results indicated an interaction effect between environmental and genetic risk factors on the development of pancreatitis. Epidemiological data suggested that alcohol abuse increased the risk of acute pancreatitis (AP) in people with type 2 diabetes mellitus (adjusted HR 86.3 (65.3–111.0)). However, no study investigated the synergistic effect between obesity and alcohol excess on AP development. Here we report the combination of acute alcohol intake and obesity causes AP with multiorgan injury (MOI) in mice, mediated by visceral adipocyte tissue (VAT) lipolysis. The schedule of highfat feeding and ethanol administration is shown in figure 1A. Body weight was significantly higher in the highfat (obese) than the chow (lean) group after 12 weeks (figure 1B). Acute ethanol administration in obese mice induced significant increases in pancreatic histopathology scores (oedema, inflammation and necrosis; figure 1C), elevated circulating pancreatic enzymes (figure 1D), pancreatic and lung myeloperoxidase, and serum interleukin6 levels (figure 1E). Timecourse changes in this obese alcoholic acute pancreatitis (OAAP) model showed pancreatic injury parameters were significantly elevated from 3 to 6 hours after the first ethanol injection, with rises in MOI indices (online supplemental figure 1A–F); almost all parameters peaked at 12 hours. In contrast, acute ethanol administration in lean mice caused only mild pancreatic oedema without discernable pancreatic necrosis or elevations of MOI indices. We speculated that lipolysis from excess abdominal fat is critical to OAAP, releasing free fatty acids (FFAs) from ethanolinduced VAT lipolysis. Indeed, fat saponification was seen in the peritoneal cavity and around the pancreas of ethanoltreated obese mice (online supplemental figure 2A,B). Circulating baseline FFA levels were higher in obese mice than in lean mice, which were further increased after acute ethanol administration (figure 1F). FFA and glycerol release over 3 hours from freshly isolated epididymal VAT of ethanoltreated obese mice was higher than that of lean mice (figure 1G). While pancreatic amylase or pancreatic triglyceride lipase (PNLIP) were comparable (figure 1H), adipose triglyceride lipase (ATGL) of ethanoltreated epididymal VAT taken from obese mice was, however, significantly higher than from lean mice (figure 1I). To confirm our hypothesis, we injected ethanol and adipocytes simultaneously Letter

Shanghai China) was dissolved in sterile normal saline at 37.5% concentration (v/v). Control mice were given equal-volume saline injections. Animals were sacrificed at 3, 6, 12, 24, and 48 h after the first injection.

Adipocytes and alcohol-induced acute pancreatitis
Adipocytes were freshly isolated from HFD obese mouse as previously described with minor modifications. 1 Briefly, mouse white adipose tissue fat pads were dissected, weighted, and minced thoroughly until no obvious tissue pieces were visible. The minced tissue was incubated at 37°C with shaking at ~220 rpm for 1 h with collagenase solution (Krebs Ringer Bicarbonate HEPES [KRBH] buffer containing 2% bovine serum albumin [BSA], 500 nM adenosine, and 3 mg/ml Collagenase I; 3 ml per gram of tissue). After digestion, the mixture was filtered through a 300 mm nylon mesh strainer, where after adipocytes were washed 3 times by free floating using a total of 40 ml wash buffer (2% BSA, 500 nM adenosine in KRBH) at room temperature. For each round of washing cells were allowed to float for > 5 min and the infranatant removed with a long needle and syringe. After the final wash, about 1.5-2 x 10 7 in 0.3 ml volume was intraperitoneally injected into 8-10 weeks-old mice using an 18gauge needle. After 1 h, alcohol stimulation was conducted as above described.
Animals were sacrificed at 12 h after the first alcohol injection.

Caerulein-induced acute pancreatitis
CD or HFD mice were given seven intraperitoneal injections of caerulein (50 μg/kg, Tocris, Shanghai) at hourly intervals as described previously. 2, 3 Control mice were given equal-volume saline injections. Animals were sacrificed at 12 h after the first injection. Animals were sacrificed at 12 h after induction of acute pancreatitis.

Histology
The tissues were collected and fixed in 10% formalin for 24 hours, embedded with paraffin, and cut into slices before haematoxylin and eosin (H&E) staining.
The severity of pancreatic injury was evaluated by two independent, blinded investigators grading oedema, inflammatory cell infiltration, and acinar necrosis (scale, 0-4) on × 10 high-power fields/slide/mouse. 5

Myeloperoxidase activity
Myeloperoxidase (MPO) activity was measured as described. 6 Pancreatic or lung tissue was homogenised, resuspended in 100 mM potassium phosphate buffer (pH 5.4) containing 0.5% hexadecyltrimethyl ammonium bromide, 10 mM ethylene diamine tetraacetic acid and protease inhibitors, freeze-thawed three times, sonicated for 30 sec and centrifuged for 15 min at 16,000 × g. MPO activity was measured in supernatants mixed with 3,3,5,5-tetramethylbenzidine as substrate (1.6 mM, final) with freshly added H2O2 (3 mM, final). Absorbance was measured at 655 nm and MPO was calculated as the difference between absorbance at 0 and 3 minutes. The activity was calculated by standard curve and expressed as units/mg protein or normalised to control.

Ex vivo lipolysis measurement
Lipolysis of adipose tissue was measured as the release of glycerol and FFA into the culture medium ex vivo. 7 Briefly, the fresh epididymal adipose tissue explants were isolated from mice. Approximately 30 mg of adipose tissue was transferred in 1 ml KRBH medium containing 2% FFA-free BSA, minced into small pieces, and then incubated at 37°C for 3 h. The contents of glycerol and FFA (Abcam) in incubation media were quantified according to the manufacturer's protocols.

Western blot analysis
After collection, the adipose tissues were centrifuged at 250 × g for 5 min, followed by removal of supernatant and addition of RIPA lysis buffer with protein extraction cocktail (100 μl). Protein was extracted by ultrasonication and centrifuged at 12000 × g for 15 min 4°C. The protein concentration was quantified by a bicinchoninic acid assay. Proteins (20 μg) were subjected to 8% or 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat milk for 1 h at room temperature. Then the membranes were further incubated overnight at 4°C with respective primary antibodies: adipose triglyceride lipase (ATGL; Santa Cruz Biotechnology, Shanghai, China), and β-actin (Proteintech, Wuhan, China). After primary antibody incubation, membranes were washed with phosphate-buffered saline containing 0.1% Tween 20 and followed by the incubation of secondary goat anti-mouse IgG-HRP antibody (Proteintech) for 1 h at room temperature.
Protein bands were visualised using a Chemiluminescent Detection System (Bio-Rad, Hercules, CA). β-actin was used as the internal reference control.

Statistical analysis
Data were presented as mean ± SEM and analysed using GraphPad Prism 8.4.1 (San Diego, CA). For two-group comparisons, mean differences were analyzed by 2-tailed Student's t test. For multi-group comparisons, mean differences were analysed by one-way ANOVA analysis of variance with the BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)