New Animal Models for Study of Metabolism Minireview Series*

Richard W. Hanson {ddagger}

From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935

This minireview series will present several specific examples of the surprising new information that has been generated by the application of molecular genetics to the study of metabolism. The ability to manipulate the genome of mice has provided a major tool for the study of metabolism and its regulation in normal, physiological states and in disease. It is now possible, for example, to determine the metabolic function of a specific gene product in animals by ablating the expression of the gene in a tissue of interest and determining the resulting phenotype. As a result of this technology, the metabolic functions of many proteins that were once thought to be well characterized are being re-examined in these animal models. In addition, it is possible to alter the tissue-specific expression of a gene of metabolic interest during development to allow the determination of the impact of that change on the appearance of metabolic function. The recent literature on the biological function of the transcription factor C/EBP provides an excellent example of the potential of genetic manipulation to provide new insights into energy metabolism. It is known from a number of studies on the role of C/EBP that two members of this family of transcription factors, C/EBP{alpha} and C/EBP{beta}, are required for the expression of a number of genes involved in lipid and carbohydrate metabolism in the liver and adipose tissue of mammals. Deleting the gene for C/EBP{alpha} results in mice that die in the immediate perinatal period with profound hypoglycemia and a failure of the urea cycle to develop normally (1). The livers of these mice have no glycogen at birth and the genes for the enzymes that are involved in hepatic glucose synthesis, the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) and glucose 6-phosphatase, are not induced in the liver in the normal fashion (1). A deletion in the gene for C/EBP{beta}, on the other hand, produces litters of mice with two phenotypes; one dies within an hour after birth whereas the other lives until adulthood with major metabolic anomalies (2, 3). The genes for both C/EBP{alpha} and C/EBP{beta} are first expressed in the liver during the last trimester of pregnancy, and their appearance is linked to the development of critical metabolic pathways (4). This type of information could only be generated using animal models where the development of specific processes can be determined within the physiological context of normal metabolic development.

The work of Chen et al. (5) has greatly extended this story. They have generated a C/EBP{alpha} null mouse in which the structural gene for C/EBP{alpha} was replaced by the structural gene for C/EBP{beta}. These mice are healthy and fertile and demonstrate none of the abnormalities noted with the C/EBP{alpha}-deficient mouse. However, these animals fail to develop white adipose tissue in the normal manner, indicating that C/EBP{alpha} is required for adipose tissue development but not for the other metabolic processes in the liver and other tissues. Thus, C/EBP{beta} can replace C/EBP{alpha} in controlling hepatic gene transcription. This fact was not evident from a number of previous studies that stressed the importance of C/EBP{alpha} in the response of the gene for PEPCK-C to hormones such as glucagon (acting via cAMP) (6, 7). This finding underlines the importance of the timing and context of the expression of members of the C/EBP gene family, rather than a specific individual role for either isoform (except for the development of adipose tissue). What is clearly important is the control of the developmental and tissue-specific expression of the gene (either C/EBP{alpha} or C/EBP{beta}) that is regulated by the gene promoter. This important insight regarding the mechanisms by which transcription factors regulate the development of metabolic processes would not have been possible without the ability to substitute one gene for another and to preserve the genomic context in which the gene of interest is expressed.

This minireview series will present articles from a number of laboratories that have had important roles in generating animal models to study metabolism. These reviews range from studies of the function of glucose transport and the role of insulin receptor in this process to insights that these animal models provide for understanding the development of genes for critical enzymes in metabolism. The minireview by Haruka Okamoto and Domenico Accili entitled "In Vivo Mutagenesis of the Insulin Receptor" provides a new look at the role that the insulin receptor plays in tissues that are not normally considered acutely insulin sensitive (non-canonical insulin-responsive tissues) such as the liver, brain, and pancreas. The review also assesses the relative roles of the insulin receptor and the insulin-like growth factor receptor in controlling growth and metabolic processes in mice. As discussed above, the availability of powerful techniques for manipulating the genome permits the tissue-specific ablation of the insulin receptor gene in mice; from this has come a detailed physiological analysis of the response of the animals. This genetic mapping of the insulin receptor function in mice has a number of metabolic surprises and makes for fascinating reading.

A major area of metabolic research in obesity involves the role of thermogenesis in controlling the rate of energy expenditure in all mammals. Studies of human obesity have demonstrated that individuals reach a set point for body fat and that food intake and energy expenditure are balanced by a complex set of hormonal and neural signals that control this processes. A critical factor in the regulation of energy expenditure involves the control of thermogenesis. It was known for many years that brown adipose tissue contains an uncoupling protein (UCP1) that is activated by norepinephrine that is induced in mammals by exposure to the cold. This in turn causes an elevation of cAMP in the tissue and the subsequent induction of lipolysis. The fatty acids generated by this process activate UCP1, resulting in the insertion of this protein into the inner mitochondria membrane causing a partial collapse of the proton gradient and an uncoupling of respiration from phosphorylation. The end result of this metabolic alteration is a rise in body temperature; this process is often referred to as "non-shivering thermogenesis." More recently, isoforms of UCP1, termed UCP2 and UCP3, have been discovered in a number of tissues including muscle and the {beta}-cells of the pancreas. Interestingly, transcription for the gene for UCP2 in muscle can be induced by fatty acids (8, 9) suggesting that this protein could be responsible for an increased rate of fatty acid oxidation in muscle of individuals that express the gene at significant levels and could be a factor in weight regulation. The research of Bradford B. Lowell and his colleagues (10, 11) has provided a number of interesting clues regarding the importance of the regulation of thermogenesis in the control of energy expenditure. The minireview by Lowell and Eric S. Bachman entitled "{beta}-Adrenergic Receptors, Diet-induced Thermogenesis, and Obesity" reviews the literature regarding the metabolic consequence of ablating expression of the genes for members of the UCP family on energy metabolism in mice.

The critical factor, often ignored in our considerations of metabolic control, is the rate of futile cycling of metabolic fuels that takes place in all organisms (12). An example of futile cycling that occurs extensively in mammals, including humans, is the triglyceride/fatty acid cycle. During fasting, triglycerides in white adipose tissue are hydrolyzed by lipolysis to yield free fatty acids and glycerol. The glycerol cannot be re-used by the white adipose tissue and ends up in the liver where it is predominantly used for gluconeogenesis (13). About 40% of the fatty acids are re-esterified back to triglyceride by the white adipose tissue, and the rest go to either liver or muscle for further metabolism. The liver also reesterifies a considerable fraction of the fatty acids to triglyceride and exports them back to the white adipose tissue as very low density lipoprotein (VLDL). The overall rate of fatty acid recycling, about 60% in humans (14), is relatively constant during different physiological states. The metabolic pathways used to support this recycling in both white adipose tissue and liver are discussed in detail in the minireview by Lea Reshef and her colleagues entitled "Glyceroneogenesis and the Triglyceride/Fatty Acid Cycle." A major factor in controlling the level of fatty acid re-esterification in these tissues is a pathway termed glyceroneogenesis that involves the enzyme PEPCK-C. The role of glyceroneogenesis and its regulation in the liver and white adipose tissue are discussed, as is the coordinate control of the expression of the gene for PEPCK-C in these tissues. This regulation of PEPCK-C gene transcription by glucocorticoids is especially important for the proper function of glyceroneogenesis in the two tissues.

The genes involved in regulating the development of metabolic pathways, such as the glycolytic pathway or the citric acid cycle, are difficult to study in detail because of the critical role these pathways play in supporting the metabolic requirements of the developing embryo and the small size and rapid growth of the embryo. However, newer genetic approaches using the mouse as a model system have provided an increasing body of knowledge regarding the details of this process. In their minireview entitled "Intermediary Metabolism and Energetics during Murine Early Embryogenesis," Mulchand S. Patel and colleagues review the available data on the developmental control of the pathways of energy metabolism, using as a centerpiece their own studies on the development of the pyruvate dehydrogenase complex (PDC). This multienzyme complex is composed of multiple copies of three catalytic proteins and several regulatory proteins involved in phosphorylation of pyruvate dehydrogenase, the first enzyme in the complex. PDC is a critical step in energy metabolism because it links the metabolism of glucose via glycolysis to the generation of energy via the citric acid cycle. Deletion of the genes for two catalytic proteins of PDC, pyruvate dehydrogenase {alpha}1 and dihydrolipoamide dehydrogenase, in the mouse results in animals that die between 7 and 9 days of embryonic life. The implications of studies in this area for understanding the metabolic requirement of the early embryo are especially important as in vitro fertilization of embryos from humans and domestic animals becomes more routine.

The metabolism of carbohydrate and lipid metabolism is remarkably interrelated in mammals and controlled by hormones and other regulatory compounds in a tissue-specific manner as part of an exquisitely regulated metabolic network. An excellent example of the complexity of this control can be found in the recent research involving the enzymes PEPCK-C and glucokinase, both present in the liver and both critical for the metabolism of glucose. These enzymes are present in different areas of the liver and are generally thought to be involved in the regulation of two different processes, glycolysis (glucokinase) and gluconeogenesis (PEPCK-C). The tissue-specific ablation of expression of the genes for these two enzymes has provided a number of surprises concerning the predicted function of both proteins in the liver and other tissues. For example, She et al. (15) have used the cre recombinase system to ablate the expression of PEPCK-C specifically in the livers of mice. Surprisingly, these animals have normal glucose homeostasis, despite the lack of hepatic gluconeogenesis, but develop a profound fatty liver! The physiological rationale for this metabolic situation and the insights it provides for understanding the integration of lipid and carbohydrate metabolism are discussed in the minireview by Mark A. Magnuson and colleagues entitled "Gene-altered Mice and Metabolic Flux Control."

The transport of glucose into all tissues is controlled by a family of transport proteins termed GLUTs (glucose transporters). To date, 12 GLUTs have been identified; they function in specific tissues and have different biological properties, such as differing Km values for glucose and tissue distribution. Of special significance for understanding the etiology of obesity and diabetes in humans is the biological properties of GLUT4, a glucose transporter that is present in white adipose tissue and muscle. GLUT4 is dependent on insulin and/or exercise for translocation to the cell surface for glucose transport and is of special importance in Type 2 diabetes because insulin resistance, a metabolic indicator of this disease, is caused in part by the inability of GLUT4 in muscle to transport glucose from the blood in a normal manner. Recent studies in which the genes for GLUT4 and the insulin receptor have been ablated in specific tissues of the mouse have challenged our understanding of the metabolic role of specific target tissues in the development of insulin resistance and Type 2 diabetes. In the minireview by Yasuhiko Minokoshi and colleagues entitled "Tissue-specific Ablation of the GLUT4 Glucose Transporter or the Insulin Receptor Challenges Assumptions about Insulin Action and Glucose Homeostasis," the roles of each of these factors in muscle and adipose tissue are critically evaluated from work with genetically modified mouse models. The results of this research have generated a number of new insights into the biology of glucose transport in mammals and promise to provide a better understanding of the etiology of human obesity and diabetes.

The selection of articles included in this minireview series reflects a combination of my personal metabolic interests as well as the willingness of individual scientists to participate in the project. Clearly, there are many other, equally interesting examples of the use of genetic techniques for the study of metabolic regulation that could have been included in this series. However, this is a rapidly moving field of research and depending on the reception of the current minireview series among our readers, more on the same subject will be forthcoming. Please read and enjoy these reviews and let me know your opinion both on the current series of articles and whether you would like to see a continuation of this, or a related metabolic theme, in future minireview series in the Journal of Biological Chemistry. Your feedback and suggestions would be most welcome by the Editors.

FOOTNOTES

* These minireviews will be reprinted in the 2003 Minireview Compendium, which will be available in January, 2004. Back

{ddagger} To whom correspondence should be addressed. E-mail: rwh{at}po.cwru.edu.


REFERENCES

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