Beige adipocytes mediate the neuroprotective and anti-inflammatory effects of subcutaneous fat in obese mice

Visceral obesity increases risk of cognitive decline in humans, but subcutaneous adiposity does not. Here, we report that beige adipocytes are indispensable for the neuroprotective and anti-inflammatory effects of subcutaneous fat. Mice lacking functional beige fat exhibit accelerated cognitive dysfunction and microglial activation with dietary obesity. Subcutaneous fat transplantation also protects against chronic obesity in wildtype mice via beige fat-dependent mechanisms. Beige adipocytes restore hippocampal synaptic plasticity following transplantation, and these effects require the anti-inflammatory cytokine interleukin-4 (IL4). After observing beige fat-mediated induction of IL4 in meningeal T-cells, we investigated the contributions of peripheral lymphocytes in donor fat. There was no sign of donor-derived lymphocyte trafficking between fat and brain, but recipient-derived lymphocytes were required for the effects of transplantation on cognition and microglial morphology. These findings indicate that beige adipocytes oppose obesity-induced cognitive impairment, with a potential role for IL4 in the relationship between beige fat and brain function.


Behavioral tests
Novel object preference testing began with 10min video analysis of locomotor behavior in the arena without objects, followed by 10min video tracking of interaction with two identical objects. After a 30min delay, mice were reintroduced to the arena with one For the Barnes maze, mice were tested on a white circular platform (122cm diameter) with twelve holes (9cm diameter) evenly spaced around the perimeter (each hole 5cm from edge). The circular platform was surrounded by a curtain to prevent the use of distal cues. Each trial began with 30sec under the start box in the center of the maze, followed by 2min free exploration. Mice that did not locate the escape box within

Intraperitoneal glucose tolerance testing
Intraperitoneal glucose tolerance testing was carried out after an overnight fast, as reported previously. 1 To summarize, baseline samples were collected from the tail vein using a handheld glucometer immediately before intraperitoneal administration of glucose in sterile saline (1.0g/kg; Sigma-Aldrich). Subsequent samples were collected 30min, 60min, and 120min after glucose administration. Tail-nicks were cleaned with Betadine and sealed with styptic powder before returning the mouse to its home cage.

Electrophysiology
Acute slices (350 micron thickness) were prepared on a Vibratome and allowed to recover for at least 1hr in carboxygenated artificial cerebrospinal fluid (ACSF) before recording, as reported previously. was discarded, as was data from slices that exhibited >10% variability during baseline recording.

Adipose transplantation surgery and transplant viability
For subcutaneous fat transplantation, recipients and donors (6-8wk old lean males) were maintained under Isoflurane anesthetic in side-by-side aseptic surgical stations.
Each inguinal fat pad was unilaterally excised from the donor, starting from the dorsolumbar region to just above the femoral artery. The excised fat was trimmed to 250mg and secured against the inner surface of the peritoneal wall with a dissolving suture, as reported previously for VAT transplantation.   Fig.1, Supplementary Fig.3

In vivo capture and quantification of interleukin-4
Mice received bilateral injections of azide-free, low endotoxin biotinylated rat anti- Hamilton Company, Reno, NV USA). For IP administration, non-overlapping groups of mice were injected with 10 micrograms of the capture antibody in 0.2mL sterile saline.
One day after administration of the capture antibody, mice were anesthetized for CSF collection via the cisterna magna or decapitated for blood collection and serum separation. Samples were snap-frozen and stored at -80 in preparation for ELISA. Plates were coated with unlabeled rat anti-mouse IL-4 (clone BVD6-24G2.3; Southern Biotech, Birmingham, AL USA), and recombinant mouse IL-4 (Shenandoah Biotechnologies) was used to generate the standard curve, as described. 8

Isolation of stromal-vascular fraction from adipose tissue
For collagenase digestion and isolation of stromal-vascular fraction (SVF) from adipose tissue (Fig.6c), transplanted SAT, resident SAT, and VAT were dissected, weighed, and washed in DMEM to remove hair and debris. Visible blood vessels were removed and discarded, then fat pads were minced and transferred to conical tubes.
Dissociation buffer (1xdPBS with 2.0mg/mL collagenase IV and 20mg/mL BSA; 3x wt/vol) was added to each tube and samples dissociated for 30-40min at 37C on a shaker inside of a hybridization oven. After dissociation, 3x (vol/vol) DMEM+2%FBS was added to each tube. Cell suspensions were passed through a 100 micron strainer and pelleted by centrifugation at 250xg for 8min. The lipid layer and media were aspirated and discarded, and the SVF pellet was resuspended in 5mL DMEM+FBS. The SVF pellet was filtered through a 40 micron strainer, pelleted by centrifugation at 400xg for 10min, then washed 2x by repeated resuspension and centrifugation before antibody labeling and flow cytometry. Yield was determined by hemocytometer and cells were immediately processed for antibody labeling and flow cytometry.
For ex vivo stimulation (Supplemental Fig.7), FMCs and astrocytes were separated from one hemisphere by centrifugation on a 4-step gradient of isotonic Percoll (75%/50%/30%/0%). FMCs were collected from the 75%/50% interphase, astrocytes were collected from the 50%/30% interphase, as described. 2,9 BVECs were isolated from the opposite hemisphere by dextran gradient centrifugation followed by Percoll gradient ultracentrifugation according to published protocols 2,9 and as detailed in the Reporting Summary.

Immunofluorescence and imaging in decalcified skull preparations
For immunofluorescence detection of meningeal T cells, mice were transcardially perfused with PFA and denuded heads were postfixed for 24hr prior to mechanical thinning of the skull using a dremel tool and grinding wheel. Thinned-skull preparations were then decalcified in 0.3M EDTA for 6d at RT with shaking before dehydration in 30% sucrose in phosphate buffer. Dehydrated skulls were frozen in OCT in preparation for sectioning on the transverse plane at 20 micron thickness using a cryostat. Horizontal

Immunofluorescence and 3D reconstruction of microglia
For immunofluorescence visualization of microglia, 40 micron coronal sections were cut throughout the rostrocaudal extent of the hippocampus as a 1:6 series using a freezing microtome (Leica), as described.