Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes

Minimizing variation due to genotype and environment

Mouse models are increasingly popular tools to study and characterize the molecular and physiological bases for human diseases. Due to the readily available genetic tools to construct mouse models of complex diseases, the flow of efficient and vigilant mouse phenotyping has now become the bottleneck in mouse functional genomics. This unit addresses the importance of minimizing and defining confounding genetic and environmental sources of variabilit

Collection of blood and plasma from the mouse

There are many elements in plasma that can act as surrogate markers of the physiological well-being of a mouse, thus making the collection of blood and plasma a general technique with many applications in mouse phenotyping. For example, the presence of certain enzymes in plasma can serve as markers of tissue toxicity (AST, ALT) and general function, and the more sophisticated lipid and lipoprotein profile tests (cholesterol, LDL) can point to dyslipidemias. As many of the tests available to measure these parameters have been adapted to automated systems in a high-throughput fashion, they have become part of the first line of screening protocols in mouse phenotyping. In this section, general techniques associated with collection and processing of blood are describe

Uses of forward and reverse genetics in mice to study gene function

As the focus of human genetics shifts from Mendelian traits to complex diseases, a sophisticated genetic tool kit—with space for genetics (classical, molecular, statistical, and quantitative), metabolics, proteomics, bioinformatics, and mathematics—is required to elucidate their multifactorial traits and regulatory processes. Importantly, mouse resources optimized to study the actions of isolated genetic loci on a fixed background are insufficient on their own for studying intact polygenic networks and genetic interactions, and researchers must work in the context of experimental model systems that optimally mimic the genetic structure of human populations. The success of such phenogenomic approaches depend on the efficacy by which specific mutations (gene targeting) and variability (recombinant inbreeding) can be introduced into the mouse genome, and on the optimization of phenotyping analyses of the mutant mouse lines. This unit describes the basic genetic approaches used to in the study of mouse model system

Evaluation of energy homeostasis

Body mass and composition reflect the combined effects of three processes: energy intake, energy partitioning (storage), and energy expenditure. Energy is released from food as it is combusted to carbon dioxide and water, and is expended as heat and work within a cell. The energy stores, mainly in adipose tissue, represent the net balance between intake and expenditure. The methods outlined in this unit evaluate these three processes by measuring food intake and lipid absorption, body fat composition, and energy expenditure. Evaluation of food intake and fat mass is a useful first-line phenotyping test indicating altered energy homeostasis. Evaluation of energy expenditure in this unit addresses obligatory basal energy expenditure (for performance of cellular and organ functions), as measured by indirect calorimetry. The combined results of these tests provide indications of the metabolic defects in a mouse model and help to identify molecular targets that cause these abnormalitie

Lipid and bile acid analysis

Lipids are important body constituents that are vital for cellular, tissue, and whole-body homeostasis. Lipids serve as crucial membrane components, constitute the body's main energy reservoir, and are important signaling molecules. As a consequence of these pleiotropic functions, many common diseases, including atherosclerosis, chronic inflammatory disorders, and obesity, have been associated with altered lipid homeostasis. Lipid abnormalities are hence increasingly analyzed in mouse models. This unit describes commonly used methods to analyze mouse lipid metabolism, with techniques that evaluate lipids both in blood and in tissues. Despite the similarities between men and mice in many aspects of metabolism, important differences also exist in the area of lipid homeostasis. These differences are discussed and should be taken into account when extrapolating lipid data from mouse to me

Evaluation of glucose homeostasis

Obesity and dyslipidemia are often found in association with insulin resistance (IR). These components combined with hypertension characterize the most common endocrine disorder in humans, the metabolic syndrome. Thus, in addition to profiling body weight evolution and lipid metabolites, glucose tolerance (a reflection of IR) and insulin sensitivity should also be considered as part of any metabolic phenotyping protocol. The ability to measure IR and glucose tolerance is important not only in the quest to fully understand the pathogenesis of the metabolic syndrome in the mouse, but also to test the effects of potential interventions. This unit presents a variety of tests used for this purpose, including direct blood glucose measurements, insulin measurement by ELISA, the homeostatic model assessment, glucose tolerance and insulin sensitivity tests, and the euglycemic clam

Histopathology in mouse metabolic investigations

Due to the small size of the mouse, evaluating its clinical phenotype is sometimes problematic. In contrast, mouse models are readily accessible to post-mortem analyses at any time during the course of a disease and prior to its clinical onset. RNA, protein, and histological analyses following sacrifice represent a powerful means to identify affected cell types and molecular events underlying the altered phenotype, and therefore to understanding the signaling or metabolic pathways involved. In this unit, an overview of post-mortem analyses is provided with a strong emphasis on the principles of routine histology, including tissue fixation, processing, embedding, and staining with hematoxylin and eosin. There are also several protocols for staining with specialized histological stains used in the metabolic field to detect intracellular lipids, intracellular lipid “ghosts”, cholesterol esters, polysaccharides, mitochondria, pathological collagen deposits, and atherosclerotic plaque