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

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 DM^1,2. 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 ....

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....

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).......

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% alcoholic chlorhexidine digluconate solution	Rioquímica	SKU 1068253
16-gauge needle	Becton Dickinson	SKU 305198
2.0% non-alcoholic chlorhexidine digluconate solution	Rioquímica	SKU 1113045
3 mL syringe	Becton Dickinson	SKU 309657
Accu-Check strips – Performa	Roche	EAN 4015630981960
Alcian Blue PAS Kit	EasyPath	SKU EP-11-20019
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4612
CellSens software	Olympus	CELLSENS
Citrate Buffer pH 6.0: Citric acid monohydrate + Sodium hydroxide	Sigma-Aldrich	C1909 + 28-3010
Cleaved Caspase-3 antibody	Cell Signaling Technology	9661
Cobas C 111 analyzer	Roche	4528778001
Creatinine K	Labtest	96-300
EnVision FLEX DAB+ Chromogen	Agilent-Dako	K3468
EnVision FLEX /HRP detection reagent	Agilent-Dako	SM802
Envision FLEX Peroxidase-Blocking Reagent	Agilent-Dako	SM801
EnVision FLEX Substrate Buffer	Agilent-Dako	SM803
EnVision FLEX Was Buffer 20x	Agilent-Dako	K800721-2
Eosin	Agilent-Dako	CS701
GloMax Discover Microplate Reader	Promega	GM3000
Glucometer Accu-Check - Performa	Roche	EAN 4015630980512
GraphPad Prism 8 software (statistical software)	GraphPad by Dotmatics	-
100% v/v Isoforine (Isoflurane)	Cristália	-
Hematoxylin	Agilent-Dako	CS700
IX51 light microscope	Olympus	IX51
Metabolic Cage for single mouse	Tecniplast	3600M021
Mounting medium - Entellan	Sigma-Aldrich	100869
Mouse Albumin ELISA Kit	Abcam	Ab207620
Mouse strain: BTBR.Cg-Lepob/WiscJ	JAX Laboratories	4824
Periodic Acid Schiff (PAS) Kit	EasyPath	EP-11-20014
RM255 Microtome	Leica Biosystems	14050237960
TP1020 Tissue processor	Leica Biosystems	TP-1020
Tramadol 50 mg/mL	Grünenthal	4022444
WT-1 antibody	Santa Cruz	SC-192