The varying success rates in activating and inducing endogenous brown adipose tissue (BAT) to treat obesity, insulin resistance, and cardiovascular disease highlight some ongoing challenges. Rodent models have demonstrated the safety and efficacy of transplanting brown adipose tissue (BAT) from healthy donors as another strategy. In models of obesity and insulin resistance induced by diet, BAT transplants counteract obesity, augment insulin sensitivity, and enhance glucose homeostasis and whole-body energy metabolism. In mouse models of insulin-dependent diabetes, the sustained euglycemia following subcutaneous transplantation of healthy brown adipose tissue (BAT) obviates the need for insulin or immunosuppression. In the long-term management of metabolic diseases, transplantation of healthy brown adipose tissue (BAT), with its demonstrated immunomodulatory and anti-inflammatory properties, may prove to be a more efficacious approach. The technique of subcutaneous brown adipose tissue transplantation is presented in great detail.

White adipose tissue (WAT) transplantation, a technique often employed in research settings, is frequently utilized to understand the physiological role of adipocytes and their associated stromal vascular cells, such as macrophages, within the context of local and systemic metabolic processes. Within the context of animal models, the mouse is prominently used to study the transplantation of WAT, where the donor WAT is transferred either to the subcutaneous region of the same individual or the subcutaneous region of a different individual. Detailed procedures for heterologous fat transplantation are presented, incorporating survival surgery, perioperative and postoperative care, and the required histological confirmation of transplanted fat grafts.

Gene therapy strategies are significantly enhanced by the use of recombinant adeno-associated virus (AAV) vectors. Despite the aim, precisely targeting adipose tissue remains a complex undertaking. Gene delivery to brown and white fat tissues is strikingly efficient with the newly engineered hybrid serotype Rec2, as our recent research demonstrates. Besides this, the administration procedure has a direct impact on the tropism and effectiveness of the Rec2 vector; oral delivery results in transduction of interscapular brown fat, whereas intraperitoneal injection focuses on visceral fat and the liver. To mitigate off-target transgene expression in the liver, we developed a single recombinant adeno-associated virus (rAAV) vector containing two expression cassettes; one driven by the cytomegalovirus (CMV) promoter for the transgene, and another driven by the liver-specific albumin promoter to express a microRNA targeting the woodchuck post-transcriptional regulatory element (WPRE). In vivo research by our laboratory, and others, indicates that the Rec2/dual-cassette vector system is a significant tool for gaining insights into both gain-of-function and loss-of-function scenarios. We describe a refined approach to packaging and delivering AAV to brown adipose cells.

A danger sign for metabolic diseases is the over-accumulation of fatty tissues. Increasing energy expenditure and potentially reversing obesity-related metabolic dysfunctions are effects of activating non-shivering thermogenesis in adipose tissue. The metabolic activation and recruitment of brown/beige adipocytes in adipose tissue, crucial for non-shivering thermogenesis and catabolic lipid metabolism, can be spurred by thermogenic stimuli and pharmacological intervention. Thusly, adipocytes hold significant therapeutic potential for obesity treatment, and the need for effective screening strategies for thermogenic drugs is intensifying. Lipid-lowering medication As a well-established marker, cell death-inducing DNA fragmentation factor-like effector A (CIDEA) reflects the thermogenic potential of brown and beige adipocytes. A CIDEA reporter mouse model, newly generated in our lab, expresses multicistronic mRNAs for CIDEA, luciferase 2, and tdTomato proteins, under the regulatory control of the endogenous Cidea promoter. The CIDEA reporter system, utilized for screening drug candidates with thermogenic properties in both in vitro and in vivo settings, is presented, along with a detailed method for monitoring CIDEA reporter expression.

Brown adipose tissue (BAT), a key component in the process of thermogenesis, is closely related to the development of various diseases, notably type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and obesity. Employing molecular imaging technologies to track BAT activity can contribute to unraveling disease origins, improving diagnostic accuracy, and fostering the advancement of therapeutic strategies. As a promising biomarker for assessing brown adipose tissue (BAT) mass, the 18 kDa translocator protein (TSPO) is prominently situated on the outer mitochondrial membrane. The protocol for imaging BAT in mice with the [18F]-DPA TSPO PET tracer [18] is presented in detail below.

Brown adipose tissue (BAT) and beige adipocytes, which originate in subcutaneous white adipose tissue (WAT), are activated in response to cold induction, marking the process of WAT browning or beiging. The uptake and metabolism of glucose and fatty acids result in an augmentation of thermogenesis in adult humans and mice. By activating brown adipose tissue (BAT) or white adipose tissue (WAT) and subsequently generating heat, the body helps counteract the obesity effects of a poor diet. This protocol evaluates cold-induced thermogenesis in the active brown adipose tissue (BAT) (interscapular area) and browned/beige white adipose tissue (WAT) (subcutaneous region) of mice using 18F-fluorodeoxyglucose (FDG), a glucose analog radiotracer, coupled with positron emission tomography and computed tomography (PET/CT) scanning. PET/CT imaging capability extends beyond quantifying cold-induced glucose uptake in known brown and beige fat deposits to also showcasing the spatial location of previously unknown mouse brown and beige fat cells, which display heightened cold-induced glucose uptake. In order to ascertain the validity of the signals from delineated anatomical regions in PET/CT images as representative of mouse brown adipose tissue (BAT) or beige white adipose tissue (WAT) depots, histological analysis is further utilized.

Energy expenditure (EE) increases in response to food consumption, a process termed diet-induced thermogenesis (DIT). Raising DIT values could potentially lead to a reduction in weight, consequently predicting a decrease in BMI and body fat. biologic medicine Although a range of strategies have been applied to measure DIT in humans, there is no way to calculate absolute DIT values in mice. Accordingly, a technique for measuring DIT in mice was developed, adapting a procedure prevalent in human applications. To begin, we assess the energy metabolism of mice who are fasting. The procedure involves plotting EE on the vertical axis and the square root of the activity on the horizontal axis, followed by fitting a linear regression line. Then, we determined the energy metabolism of mice given free access to food, and the calculated EE was presented using the same graphing method. Establishing the DIT involves subtracting the anticipated EE value from the actual EE value observed in mice with the same activity count. This method facilitates not only the observation of the absolute value of DIT over time but also the calculation of the ratio of DIT to caloric intake and the ratio of DIT to EE.

Brown adipose tissue (BAT) and brown-like fat, through their mediation of thermogenesis, play a substantial role in maintaining metabolic homeostasis in mammals. Essential for characterizing thermogenic phenotypes in preclinical studies is the accurate measurement of metabolic responses to brown fat activation, including the generation of heat and increased energy expenditure. Lorlatinib cell line Below, we present two methods employed to assess thermogenic profiles in mice during non-basal metabolic states. We describe a protocol for continuous monitoring of body temperature in mice subjected to cold, utilizing implantable temperature transponders. Using indirect calorimetry, we describe a technique to assess how 3-adrenergic agonists impact oxygen consumption, a surrogate for the activation of thermogenic fat.

Carefully monitoring food consumption and metabolic rates is indispensable for grasping the influences on body weight regulation. The recording of these features is a function of modern indirect calorimetry systems. This paper elucidates our methodology for the reproducible analysis of energy balance studies performed with indirect calorimetry. CalR, a freely accessible online tool, calculates instantaneous and cumulative totals related to metabolic variables like food intake, energy expenditure, and energy balance, positioning it as a commendable starting point for the study of energy balance experiments. Among the metrics CalR calculates, energy balance stands out as a key indicator, revealing the metabolic patterns produced by experimental treatments. The sophisticated nature of indirect calorimetry apparatus, coupled with the occurrence of mechanical failures, underscores the crucial role of data refinement and visualization. Visualizations of energy intake and expenditure relative to body mass or physical activity levels can assist in determining whether the equipment is operating correctly. An important visualization for experimental quality control is introduced: a graph demonstrating the relationship between energy balance changes and body mass changes. This graph effectively represents many key components of indirect calorimetry. The investigator can make inferences about the quality control and validity of experimental results by employing these analyses and data visualizations.

Brown adipose tissue's efficiency in expending energy through non-shivering thermogenesis has been strongly correlated with its protective and therapeutic properties against obesity and metabolic diseases in numerous studies. Primary cultured brown adipose cells (BACs) are favored for their genetic malleability and tissue-like characteristics in the investigation of heat generation mechanisms.