White and Brown Adipose Grafts: An Approach to Correct Reproductive, Metabolic, and Renal Deficits in Black and Tan Brachyury (BTBR) Obese Mice

This research investigated whether combining white and brown fat transplantation in leptin-deficient BTBR obese mice can reverse subfertility, obesity, hyperglycemia and kidney dysfunction, thereby improving reproductive metabolic and renal outcomes. We standardize the combined wide adipose tissue and brown adipose tissue transplantation protocol, defining optimal animal age and fat quantity to address metabolic and reproductive dysfunctions in the BTBR obese model. Compared to leptin therapy or single fat transplants, our protocol offers a physiological sustaining hormone release with broader improvements in metabolism, fertility and kidney function using a reproducible and scalable approach.

By standardizing this protocol, we enable broader use of the BTBR model to study cardiorenal metabolic disease and diabetic complications, while aligning with the three Rs in animal research. We aim to systematically refine and replicate this protocol, enhance transplantation efficacy, integrate novel biomedical platforms, and identify additional fat implantation sites to optimize therapeutic outcomes in BTBR obese mice. To begin, position the euthanized mouse in dorsal recumbency with its belly facing upward.

Disinfect the lower abdomen using 0.5%alcoholic chlorhexidine digluconate. Using surgical scissors, make a midline abdominal incision approximately two to three centimeters long to expose and harvest the inguinal white adipose tissue. Place the excised tissue into a three-milliliter tube and keep it on ice.

Next, reposition the mouse in ventral recumbency with its belly facing downward. Make a midline dorsal incision of one to two centimeters between the scapulae using surgical scissors to access and excise the interscapular brown adipose tissue. Using a three-milliliter syringe fitted with a 16-gauge needle, homogenize the white and brown adipose tissue separately.

Then measure the final weight of the fat using an analytical balance. And keep the samples on ice until transplantation. Next, take the anesthetized recipient BTBR obese mice.

Once its respiration slows to one breath every three seconds or less, position the mouse in ventral recumbency. Then disinfect the dorsal area using 2%non-alcoholic chlorhexidine digluconate. Using a 16-gauge needle, inject 100 to 200 microliters of homogenized fat per site across six to 26 locations on the dorsal surface.

Use forceps to gently assist in evenly distributing the fat at each site. Following transplantation, apply 0.5%chlorhexidine solution to each injection site. Administer tramadol intramuscularly for analgesia, and house each animal individually to prevent injury during recovery.

On the following day, clean each injection site with 0.5%chlorhexidine solution, and administer a second intramuscular dose of tramadol for continued analgesia. Two weeks after transplantation, co-house the female BTBR obese mice with male BTBR heterozygous mice for mating. Measure the body weight of the mice weekly, using an analytical balance.

Assess fasting blood glucose levels using a glucometer. The transplantation protocol using 10%to 15%donor fat resulted in a 2.2 fold higher effectiveness in achieving viable offspring compared to the 5%to-9.9%fat group. A progressive increase in protocol effectiveness was associated with higher proportions of brown adipose tissue in the injected fat, reaching 100%effectiveness when brown adipose tissue made up 25%to 30%of the total fat.

Crosses involving transplanted BTBR obese females and heterozygous males yielded 1.6 times more obese offspring compared to heterozygous-only crosses. Ovaries from transplanted mice showed restored follicular genesis, evidenced by increased corpora albicantia and balanced follicle populations compared to obese controls. Quantification confirmed higher numbers of pre-antral and antral follicles in obese mice, with partial normalization in the transplanted group.

Cleaved caspase-3 staining showed reduced ovarian apoptosis in transplanted mice to levels similar to lean controls, suggesting a protective effect of transplantation. Transplanted mice had consistently lower body weight from 10 to 24 weeks compared to obese controls, with significantly reduced percentage weight gain over the same period. Fasting blood glucose levels were significantly lower in transplanted mice than in obese controls between weeks eight and 20.