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Method Article

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

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DOI:

10.3791/68759

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September 9th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Albert Einstein Research and Education Institute, Hospital Israelita Albert Einstein, 2School of Medicine, Faculdade Israelita de Ciências da Saúde Albert Einstein, 3Nephrology Division, Federal University of São Paulo

* These authors contributed equally

In This Article

Summary

Here we present a white and brown adipose tissue transplantation protocol that offers a promising strategy to reverse subfertility, obesity, and hyperglycemia, while improving kidney function in diabetic and obese BTBR mice.

Abstract

Transplantation of white adipose tissue (WAT) and brown adipose tissue (BAT) has emerged as a promising therapeutic strategy for reversing metabolic disorders without the need for sustained administration of exogenous agents. WAT functions as a key endocrine organ involved in energy homeostasis, while BAT is a thermogenic tissue critical for thermoregulation. The Black and Tan Brachyuric (BTBR) obese mouse model closely mimics key features of obesity, type 2 diabetes mellitus (T2DM), and diabetic kidney disease (DKD), including hyperglycemia, insulin resistance, hypometabolism, and subfertility.

Using the protocol presented below, combined transplantation of WAT and BAT derived from BTBR wild-type and heterozygous donors into BTBR obese recipients resulted in the reversal of subfertility, attenuation of weight gain, normalization of glycemic control, and improvement of renal parameters. Notably, renal hypertrophy and apoptosis were reduced, while podocyte numbers were preserved. These findings suggest that combined WAT and BAT transplantation may represent an alternative therapeutic strategy for the treatment of T2DM, with concurrent benefits in mitigating DKD and obesity. Furthermore, this approach may aid in increasing the number of viable BTBR obese mice within vivarium colonies. Further investigation into the underlying mechanisms may facilitate the clinical translation of this intervention.

Introduction

Diabetic kidney disease (DKD), type 2 diabetes mellitus (T2DM), and obesity represent interrelated, multifactorial conditions that pose significant and growing global health challenges. DKD, defined as the coexistence of diabetes mellitus (DM) and chronic kidney disease in the absence of other identifiable causes of kidney damage, affects approximately 20-40% of individuals with DM1,2. T2DM is a systemic metabolic disorder characterized by chronic hyperglycemia and disruptions in glucose, lipid, and insulin homeostasis. It ranks among the top ten leading causes of mortality worldwide, currently impacting over 500 million individuals3. Obesity, driven by a chronic imbalance between energy intake and expenditure, now affects one in eight people globally and is a major contributor to both morbidity and mortality4. These three conditions are tightly interconnected: obesity markedly increases the risk of developing T2DM, which, in turn, significantly elevates the risk of DKD1,4. The escalating global prevalence of obesity, T2DM, and DKD underscores the urgent need for a deeper understanding of their pathophysiological mechanisms and the development of effective therapeutic strategies.

BAT transplantation has emerged as a promising therapeutic strategy to combat obesity and T2DM. This intervention has been associated with reductions of body weight, improvements of insulin resistance and hepatic steatosis, and increases in circulating adiponectin, β3-adrenergic receptor expression, and genes involved in fatty acid oxidation5. To note, mammalian adipose tissue can be classified into three main types based on color: white, brown, and beige6. WAT begins expanding shortly after birth and is widely distributed throughout the body. It functions as a key endocrine organ, storing excess energy in the form of triglycerides by sequestering circulating glucose and free fatty acids6. In contrast, BAT plays a critical role in thermoregulation by dissipating energy as heat via non-shivering thermogenesis rather than storing it as ATP6. BAT activity decreases with age, obesity, and DM. In adults, BAT comprises a small fraction of total adipose tissue and is primarily located in the supraclavicular and cervical regions in humans, while in mice, it is predominantly located in the interscapular region6. Rich in mitochondria, BAT consumes substantial amounts of glucose and fatty acids and is associated with resistance to weight gain7. Emerging evidence supports the role of BAT in promoting a leaner and more metabolically favorable phenotype, generating growing interest in its therapeutic use through transplantation.

Leptin, a hormone primarily secreted by WAT, plays a central role in regulating energy balance, metabolism, and fertility8. It acts on the central nervous system to stimulate gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus, which subsequently promotes the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary9. Leptin also acts directly on ovarian theca and granulosa cells to support follicular development9.

The BTBRob/ob (BTBR obese) mouse model, genetically designated as BTBR.Cg-Lep ob/WiscJ (black and tan, obese, tufted; see Table of Materials), is homozygous for a spontaneous loss-of-function in the leptin gene (Lep) and displays a range of metabolic and reproductive abnormalities, including hyperglycemia, glucose intolerance, hyperinsulinemia, hypertriglyceridemia, impaired wound healing, hypometabolism, hypothermia, and subfertility due to hypogonadotropic hypogonadism. Males typically develop T2DM by six weeks of age, while females do so by eight weeks10. Hyperglycemia is severe and progressive, with fasting blood glucose levels exceeding 400 mg/dL by ten weeks. Additionally, BTBR obese males exhibit time-dependent albuminuria by week eight, progressing to histologically evident DKD, including mesangial expansion and podocyturia. These features establish BTBR obese mice as a robust preclinical model that closely recapitulates human DKD10.

Given the profound metabolic consequences of leptin deficiency, several strategies have been investigated to reverse the ob/ob phenotype, including leptin replacement therapy and adipose tissue transplantation. While most studies have used ob/ob mice on the C57BL/6 background, relatively few have investigated these interventions in the more metabolically compromised BTBR strain. In C57BL/6 ob/ob mice, WAT transplantation has been shown to normalize insulin levels, improve insulin sensitivity, and restore fertility8. Moreover, BAT transplantation into lean C57BL/6 mice has been demonstrated to prevent body weight gain, improve glucose tolerance and insulin sensitivity, while reducing insulin resistance, particularly in the context of high-fat diet-induced obesity11. Additionally, BAT transplantation was shown to improve cardiac hemodynamics and metabolic health12. Despite encouraging results, these studies present several important limitations. Most investigations have been conducted in C57BL/6 ob/ob mice, which exhibit a relatively mild metabolic phenotype compared to the more severely affected BTBR ob/ob strain. Consequently, the generalizability of these findings to models with more pronounced insulin resistance, hyperglycemia, obesity, and kidney disease is limited. Moreover, the effects of leptin replacement and adipose tissue transplantation have been insufficiently studied in the BTBR background, representing a critical gap in the literature. Many of these interventions have also been evaluated in lean animals or over short durations, which may not adequately capture the complexity and chronic nature of human metabolic disease. Although improvements in insulin sensitivity, glucose tolerance, and fertility have been reported, the underlying mechanisms remain poorly understood, and the translational relevance of these findings to clinical settings is still uncertain. Notably, the combined metabolic effects of white and brown adipose tissue transplantation in BTBR ob/ob mice remain largely unexplored.

The aim of this study is to standardize a protocol for combined WAT and BAT transplantation in BTBR obese mice and to evaluate its effects on fertility restoration, kidney function, and histological changes. A secondary objective is to facilitate the expansion of the BTBR obese colony, while reducing the loss of heterozygous (BTBR ob/+) and wild-type (BTBR +/+) mice in the vivarium.

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Protocol

All experiments were approved by the Institutional Animal Care and Use Committees of Hospital Israelita Albert Einstein (HIAE) and registered with the Jewish Institute of Research and Education, São Paulo, SP, Brazil (N°. 3309-18 and N°. 4900-21). The details of the reagents and equipment used are listed in the Table of Materials.

NOTE: BTBR obese mice (leptin gene knockout, homozygous, ob/ob) were used as a model for T2DM, DKD, and obesity. Female BTBR obese mice aged 4-5 weeks served as recipients for adipose tissue transplantation. Donor animals were female BTBR ob/+ (heterozygous) or +/+ (wild-type) mice (genotype not confirmed, but phenotypically lean). At the time of transplantation, recipient mice had an average body weight ranging from 25 to 32 g. All animals were housed in individually ventilated cages under standard conditions, with ad libitum access to food and water, a 12 h light/dark cycle, and ambient temperature control. A maximum of five animals were housed per cage.

1. Surgical procedure

NOTE: All surgical instruments should be sterilized prior to surgery, by heating until 240-270 °C in a hot bead sterilizer, and then with alcohol 70%. All tubes should be sterilized by autoclaving prior to use.

  1. Euthanize donor mice using 100% v/v isoflurane overdose (2.5-5%), followed by cervical dislocation (Figure 1A).
  2. Position the animal in dorsal recumbency (belly up) and disinfect the lower abdomen with 0.5% alcoholic chlorhexidine digluconate. Make a 2-3 cm midline abdominal incision to harvest inguinal WAT (Figure 1B). Place the excised tissue in a 3 mL tube on ice.
  3. Reposition the animal in ventral recumbency (belly down) and disinfect the scapulae region with 0.5% alcoholic chlorhexidine digluconate. Make a 1-2 cm midline incision between the scapulae to harvest interscapular BAT  (Figure 1C). Pool adipose tissue from 10-14 donors. Ensure that the total WAT + BAT equals 10–15% of the recipient’s body weight.
  4. Carefully clean the fat to remove any hair that may have been collected. Homogenize WAT and BAT separately using a 16 G needle and a 3 mL syringe (Figure 1D).
  5. Measure the final fat weight using an analytical balance (Figure 1E) and keep samples on ice until transplantation.
  6. Anesthetize recipient BTBR obese mice in an 100% v/v isoflurane chamber (2.5-5% induction) and maintain anesthesia via facial mask (1.5-2.5%). Once respiration decreases to ≤1 breath every 3 s, confirm the depth of anesthesia with the withdrawal reflex test and place the animal in ventral recumbency (Figure 1F).
  7. Disinfect the dorsal area using 2.0% non-alcoholic chlorhexidine digluconate (Figure 1G).
  8. Inject 100-200 µL of homogenized fat per site using a 16 G needle across 6-26 sites on the dorsal surface. Use forceps to assist in distribution. Apply 0.5% chlorhexidine solution to each injection site post-transplantation (Figure 1H).
  9. Administer an intramuscular dose of tramadol (40 mg/kg; 50 mg/mL solution) for analgesia and house animals individually to prevent injury during recovery.
    NOTE: Observe the animal for 1-1.5 h every 15 min immediately after surgery, with a follow-up check 6-8 h post-surgery to assess clinical signs of pain and administer analgesia if necessary.
  10. On the following day, clean the injection sites with 0.5% chlorhexidine solution and administer a second dose of tramadol.

Rodent adipose tissue study process: euthanasia, tissue collection, weighing, anesthesia, injection.
Figure 1: Schematic of the fat transplantation procedure. (A) Donor euthanasia using isoflurane in a closed box. (B) Harvesting of inguinal WAT. (C) Harvesting of interscapular BAT. (D) Fat homogenization. (E) Fat weighing. (F) Recipient anesthesia. (G) Back asepsis. (H) Fat injection with forceps-assisted technique. Please click here to view a larger version of this figure.

2. Mating post-transplantation

  1. Two weeks after transplantation, co-house female BTBR obese mice with male BTBR ob/+ (heterozygous) mice for mating.

3. Functional assessments

  1. Measure body weight weekly using an analytical balance.
  2. Assess fasting blood glucose using a glucometer at five time points: weeks 4-7, 8-10, 14-16, 18-20, and 24-26.
  3. Place the animal in a metabolic cage with access to water for 6 hours, to collect urine sample.
  4. Measure urinary albumin levels using the ELISA method and determine urinary creatinine levels using the colorimetric picric acid method at 6-8 weeks. Calculate the urinary albumin-to-creatinine ratio (uACR) by dividing the albumin concentration (µg) by the creatinine concentration (mg).

4. Morphological analysis

  1. Euthanize recipient mice at 24 weeks of age by administering 100% v/v isoflurane overdose (2.5-5%), followed by cervical dislocation to ensure death.
  2. Collect ovaries, kidneys, and pancreas at 24 weeks of age.
  3. Place the harvested organs in 10% neutral buffered formalin and fix at room temperature for 24-48 h.
  4. Following fixation, process tissues using an automatic tissue processor to remove water and embed in paraffin for histological analysis.
    1. Sequentially immerse the tissues in the following ethanol solutions for 1 h each: 70%, 85%, 95% (2x), and 100% (2x).
    2. Transfer the tissues to fresh xylene for 1 h, followed by a second xylene bath for an additional 1 h.
    3. Immerse the tissues in melted paraffin wax at 6 °C for 1 h, then transfer to fresh paraffin for another 1 h.
    4. Orient the tissues in a heated mold filled with molten paraffin and allow to solidify at room temperature.
    5. Cut four non-consecutive 3.5 µm-thick sections from each block using a microtome. Float the ribbons on a 4 °C water bath to flatten, then mount individual sections onto positively charged glass slides. Drain excess water and place slides on a 3 °C warming plate for 60 min.
  5. Assess follicle number in ovaries and pancreatic islet size using Hematoxylin and Eosin (H&E) staining.
    1. Incubate paraffin-embedded tissue slides in an oven at 6 °C for 1 h. Transfer the slides to a container with fresh xylene and incubate for 30 min to dissolve paraffin, replacing the xylene with a fresh solution after 15 min.
    2. Immerse slides sequentially for 30 s each in xylene:ethanol mixture, 100% ethanol, 85% ethanol, and 70% ethanol.
    3. Rinse in tap water (10 s), stain in Harris hematoxylin (30 s), and dip in 95% ethanol (3 s).
    4. Sequentially immerse the slides in 70%, 85%, 100% ethanol, and xylene:ethanol mixture for 30 s each.
    5. Immerse the slides in fresh xylene for 5 min, followed by a second xylene bath for an additional 5 min to ensure stain fixation.
    6. Mount slides with coverslips using a mounting medium.
  6. Evaluate glomerular size and mesangial matrix expansion using Periodic Acid-Schiff (PAS) staining of kidney sections.
    1. Incubate slides at 6 °C for 1 h and immerse in fresh xylene for 5 min to deparaffinize the tissue sections.
    2. Sequentially immerse slides in 99%, 95%, and 70% ethanol for 30 s each.
    3. Rinse in tap water for 10 s.
    4. Apply 10 drops of periodic acid and incubate for 10 min.
    5. Rinse in running water for 3 min and dry.
    6. Add 10 drops of Schiff reagent and incubate for 15 min in a dark chamber.
    7. Rinse in running water for 3 min and dry.
    8. Add 10 drops of Harris hematoxylin to counterstain and incubate for 3 min.
    9. Rinse slides in running water for 3 min and dry.
    10. Immerse the slides sequentially in 70%, 85%, 100% ethanol, and xylene:ethanol mixture for 30 s each.
    11. Immerse the slides in fresh xylene for 5 min, followed by a second xylene bath for an additional 5 min to fix the stain.
    12. Mount slides with coverslips using a mounting medium.
  7. Assess follicular apoptosis (cleaved caspase-3) in ovaries and kidneys, and podocyte number (WT-1+ cells) in kidneys by Immunohistochemistry (IHC).
    1. Incubate the slides at 6 °C for 1 h, followed by xylene treatment for 30 min (replace xylene after 15 min).
    2. Sequentially immerse the slides in 100%, 90%, and 70% ethanol, and water (30 s each).
    3. Perform antigen retrieval by heating the slides in citrate buffer (pH 6.0) at 95 °C for 12 min using a microwave to unmask antigen sites.
    4. Rinse in 1x wash buffer for 15 min, changing buffer every 5 min.
    5. Block the endogenous peroxidase with hydrogen peroxide to avoid background staining and prevent non-specific binding using bovine serum albumin (BSA).
      NOTE: For cleaved caspase-3 staining, block endogenous peroxidase activity for 7 min and 30 s; for WT-1 staining, block for 5 min.
    6. Repeat the wash as in 4.7.4.
    7. Apply 10 µL of primary antibodies against cleaved caspase-3 (apoptosis) and WT-1 (podocytes). Incubate overnight at  °C in a dark, humidified chamber.
    8. Repeat the wash as in 4.7.4.
    9. Apply 10 µL of Horseradish Peroxidase (HRP)-conjugated secondary antibody for 1.5 h at room temperature in the dark.
    10. Repeat the wash as in 4.7.4.
    11. Apply 10 µL of DAB (3,3'-Diaminobenzidine) as a chromogen to visualize HRP activity, producing a brown precipitate.
      NOTE: For cleaved caspase-3 staining, incubate with DAB for 1 min and 30 s; for WT-1 staining, incubate for 45 s.
    12. Repeat the wash as in 4.7.4.
    13. Counterstain sections with Harris hematoxylin for 30 s to 1 min, depending on desired intensity.
    14. Rinse in tap water for 1-2 min or until water runs clear.
    15. Sequentially immerse the slides in 70%, 85%, 95%, and 100% ethanol (30 s each), and xylene:ethanol mixture (30 s).
    16. Place the slides in fresh xylene solution for 5 min, and transfer to a second xylene bath for an additional 5 min.
    17. Mount slides with coverslips using a mounting medium.
  8. Analyze stained sections using an inverted light microscope at 40x magnification. Use automated image analysis software to select regions of interest (ROIs) for each organ.
    1. Ovaries (H&E): Count and classify follicle types per section.
    2. Pancreas (H&E): Select islets using the ROI tool to manually trace the outline of each pancreatic islet. Once the ROI is defined, the software will automatically calculate the area in square micrometers (µm2).
    3. Kidney (H&E): Quantify glomerular area by selecting the ROI tool to manually trace the outer edge of each glomerular tuft. Use the Measure Area function to obtain its area in µm2. After tracing, the software will automatically calculate the area in µm2.
    4. Kidneys (PAS)
      1. To quantify mesangial matrix, use the Threshold tool to identify and select PAS-positive (magenta/pink) regions within the glomerulus. Adjust the color and intensity thresholds as needed to accurately select PAS-stained areas, using the preview window to refine selection. Define the selected PAS-region as a ROI. After tracing, the software will automatically calculate the area in µm2.
      2. Normalize mesangial matrix expansion by calculating the percentage of PAS-positive area relative to the total glomerular area.
    5. Ovaries and kidneys (IHC for cleaved caspase-3)
      1. Select ROIs corresponding to follicular or glomerular structures. Apply a color mask to identify DAB-positive (brown) staining by adjusting the hue, saturation, and intensity thresholds. Use the preview function to ensure accurate selection of DAB signal and refine the threshold settings to exclude hematoxylin (blue) counterstaining and nonspecific background.
      2. Once the desired threshold is defined, set the selection as an ROI and measure the DAB-positive area (µm2). Calculate the percentage of stained area by dividing the DAB-positive area by the total ROI area and multiplying by 100.
    6. Kidneys (IHC for WT-1+): Count brown-stained nuclei per glomerulus to estimate podocyte number.
      NOTE: Follicle counts per section (mean ± SD) were: BTBR obese (7.7 ± 3.11), Transplanted (5.8 ± 3.24), BTBR lean (4.6 ± 5.62). Islet counts per section (mean ± SD) were: BTBR obese (18.3 ± 8.42), Transplanted (9.7 ± 5.45), BTBR lean (6.8 ± 1.72). Assess 30 glomeruli per animal for glomerular area and mesangial matrix expansion measurement. Data were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), as appropriate. Normality was assessed using the Shapiro-Wilk test. One-way ANOVA with Tukey's post hoc test was applied for comparisons among normally distributed groups. Repeated measures were analyzed using two-way ANOVA or mixed-effects models, with Geisser-Greenhouse correction where necessary. For non-normally distributed data, the Kruskal-Wallis test with Sidak's multiple comparisons was used. Perform all analyses using an appropriate statistical software, with statistical significance set at p < 0.05 (see Table of Materials for details).

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Results

Amount of transplanted fat and protocol effectiveness
After protocol standardization, two fat transplantation volumes (5-9.9% and 10-15% relative to donor body weight) were evaluated for effectiveness, defined as achieving at least one pregnancy resulting in viable offspring. The 5-9.9% group exhibited a 23.8% success rate, whereas the 10-15% group achieved 52.2%, corresponding to a 2.2-fold increase in protocol effectiveness (Figure 2A)...

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Discussion

This study demonstrates that combined WAT and BAT transplantation is an effective therapeutic approach for controlling hyperglycemia, preventing body weight gain and islet cell hypertrophy, restoring female subfertility, and improving kidney function by reducing apoptosis and preserving podocyte numbers in a preclinical model of T2DM, DKD, and obesity.

Although previous studies have investigated WAT and BAT transplantation as strategies to regulate glucose homeostasis and insulin sensitivity

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

We thank the technical teams of the Experimental and Surgical Training Center (CETEC) and the Jewish Institute of Research and Education (IIEP) for their support. This work was supported by a masters research fellowship grant from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Coordination for the Improvement of Higher Education Personnel) to S.B, a Direct Doctorate research fellowship grant from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Coordination for the Improvement of Higher Education Personnel) to M.L.F, a masters research fellowship grant from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo/São Paulo Research Foundation; no. 2024/08173-6) to M.O.M., and a scientific initiation fellowship grant from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo/São Paulo Research Foundation; no. 2019/12636-3) to M.T.A.B-R. This work was also supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo/São Paulo Research Foundation; no. 2017/23195-2 and no. 2021/02216-7), and EFSD (European Foundation for the Study of Diabetes) to É.B.R. The authors declare that they have not used Artificial Intelligence (AI)-generated work in this manuscript.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.5% alcoholic chlorhexidine digluconate solutionRioquímicaSKU 1068253
16-gauge needleBecton DickinsonSKU 305198
2.0% non-alcoholic chlorhexidine digluconate solutionRioquímicaSKU 1113045
3 mL syringeBecton DickinsonSKU 309657
Accu-Check strips – PerformaRocheEAN 4015630981960
Alcian Blue PAS KitEasyPathSKU EP-11-20019
Bovine Serum Albumin (BSA)Sigma-AldrichA4612
CellSens softwareOlympusCELLSENS
Citrate Buffer pH 6.0: Citric acid monohydrate + Sodium hydroxideSigma-AldrichC1909 + 28-3010
Cleaved Caspase-3 antibodyCell Signaling Technology9661
Cobas C 111 analyzerRoche4528778001
Creatinine KLabtest96-300
EnVision FLEX DAB+ ChromogenAgilent-DakoK3468
EnVision FLEX /HRP detection reagentAgilent-DakoSM802
Envision FLEX Peroxidase-Blocking ReagentAgilent-DakoSM801
EnVision FLEX Substrate BufferAgilent-DakoSM803
EnVision FLEX Was Buffer 20xAgilent-DakoK800721-2
EosinAgilent-DakoCS701
GloMax Discover Microplate ReaderPromegaGM3000
Glucometer Accu-Check - PerformaRocheEAN 4015630980512
GraphPad Prism 8 software (statistical software)GraphPad by Dotmatics-
100% v/v Isoforine (Isoflurane)Cristália-
HematoxylinAgilent-DakoCS700
IX51 light microscopeOlympusIX51
Metabolic Cage for single mouseTecniplast3600M021
Mounting medium - EntellanSigma-Aldrich100869
Mouse Albumin ELISA KitAbcamAb207620
Mouse strain: BTBR.Cg-Lepob/WiscJJAX Laboratories4824
Periodic Acid Schiff (PAS) KitEasyPathEP-11-20014
RM255 MicrotomeLeica Biosystems14050237960
TP1020 Tissue processorLeica BiosystemsTP-1020
Tramadol 50 mg/mLGrünenthal4022444
WT-1 antibodySanta CruzSC-192

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Adipose Tissue TransplantationBrown Adipose TissueWhite Adipose TissueMetabolic DysfunctionReproductive DeficitsRenal DysfunctionFasting Blood GlucoseOvarian ApoptosisFollicular Genesis
White and Brown Adipose Grafts: An Approach to Correct Reproductive, Metabolic, and Renal Deficits in Black and Tan Brachyury (BTBR) Obese Mice
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