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(The FASEB Journal. 2000;14:2158-2170.)
© 2000 FASEB

Transgenic study of energy homeostasis equation: implications and confounding influences

AKIO INUI1

Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan

1Correspondence: Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: inui{at}med.kobe-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
Recently novel molecular mediators and regulatory pathways for feeding and body weight regulation have been identified in the brain and the periphery. Mice lacking or overexpressing these mediators or receptors have been produced by molecular genetic techniques, and observations on mutant mice have shed new light on the role of each element in the homeostatic loop of body weight regulation. However, the interpretation of the phenotype is under the potential influence of developmental compensation and other genetic and environmental confounds. Specific alterations of the mediators and the consequences of the altered expression patterns are reviewed here and discussed in the context of their functions as suggested from conventional pharmacological studies. Advanced gene targeting strategies in which genes can be turned on or off at desired tissues and times would undoubtedly lead to a better understanding of the highly integrated and redundant systems for energy homeostasis equation.—Inui, A. Transgenic study of energy homeostasis equation: implications and confounding influences


Key Words: melanocortin • neuropeptide Y • arcuate nucleus • body mass


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
ANIMAL SURVIVAL DEPENDS on the ability to maintain a relatively constant body mass despite variable access to food. To regulate body mass, animals must monitor energy stored as fat and adjust food intake and energy expenditure appropriately (1 , 2) . Many factors contribute to the regulation of energy balance, including neurotransmitters/neuromodulators in the brain and proteins produced in peripheral fat tissues or muscles (Fig. 1 ). Leptin, a 16 kDa protein secreted by adipocytes in proportion to body fat, is a critical element of this system and acts via cell surface receptors in the hypothalamus (3 4 5) . In the absence of leptin signaling caused either by starvation or mutation (ob/ob and db/db mice that are lacking in functional leptin and leptin receptor, respectively), an adaptive response ensues with the animals eating more and burning fewer calories (6) . Conversely, rising levels of leptin signal the brain that excess energy is being stored, which results in decreased appetite and increased energy expenditure that resist obesity.



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  		Figure 1. Candidate molecules controlling energy intake, expenditure, and/or partitioning identified by conventional pharmacological and transgenic studies. Leptin and insulin act as part of a feedback loop to maintain constant stores of fat. A loss of body fat leads to a decrease in leptin that activates feeding stimulatory regulators in the hypothalamus, such as neuropeptide Y. Conversely, an increase in body fat leads to an increase in leptin, which activates feeding inhibitory regulators such as melanocortin and corticotropin-releasing factor. The feeding inhibitory molecules stimulate energy expenditure via the sympathetic nervous system-uncoupling protein axis, while the opposite applies to the feeding stimulatory molecules. An increase in sympathetic activity is associated with an increase in the levels of UCP-1 mRNA in brown adipose tissue that produces thermogenesis, leading to reduced storage of fat. Genetic manipulation identified previously unknown regulators of energy homeostasis, including ICAM-1, leukocyte integrin {alpha}Mß2 (Mac-1), metallothionein, and transcription factor Nhlh2, although fuller characterizations of these obese phenotypes need to be performed. Apparent discrepancies between conventional pharmacological and transgenic studies are observed in such cases as NPY and CRF, and remain to be clarified. Positive and negative regulators of body adiposity are expressed as red and blue, respectively. Transgenic studies for dopamine, TRH, IL-1, STAT5b, PTP-1B, FAT/CD36, C/EBPß and GPDH are from refs 179 180 181 182 183 184 185 186 187 188 189 . ASP, acylation-stimulating protein; AGRP, agouti-related protein; ARC, arcuate nucleus of the hypothalamus; ß1 R, ß1 adrenergic receptor; ß3 R, ß3 adrenergic receptor; CART, cocaine- and amphetamine-regulated transcript; CCK, cholecystokinin; C/EBPß, transcription factor CCAAT/enhancer-binding protein ß; DMH, dorsomedial nucleus of the hypothalamus; GLP-1, glucagon-like peptide 17–36 amide; GLUT-4, glucose transporter-4; GPDH, glycerol-3-phosphate dehydrogenase; IL-1, interleukin-1; LHA, lateral hypothalamic area; LPL, lipoprotein lipase; MCH, melanin-concentrating hormone; NE, norepinephrine; PP, pancreatic polypeptide; PTP-1B, protein tyrosine phosphate-1B; PVN, paraventricular nucleus of the hypothalamus; SREBP, sterol regulatory element-binding protein; STAT5b, signal transducer and activator of transcription 5b; TNF-{alpha}, tumor necrosis factor-alpha; VMH, ventromedial nucleus of the hypothalamus.

The effects of leptin are mediated by a growing list of hypothalamic mediators (7 8 9 10 11 12 13) , including orexigenic (feeding stimulatory) substances such as neuropeptide Y (NPY), orexin/hypocretin, agouti-related protein (AGRP), and melanin-concentrating hormone (MCH) as well as anorexigenic (feeding inhibitory) substances such as melanocortin (MC), corticotropin-releasing factor (CRF), glucagon-like peptide 17–36 amide (GLP-1), cocaine- and amphetamine-regulated transcript, and interleukin 1 (Fig. 1) . Regulation of energy expenditure involves changes in sympathetic tone (mediated by ß-adrenergic signaling), thyroid hormone, and physical activity (2 , 14) . Treatment of the ob/ob mice with recombinant leptin decreases food intake and body weight and increases locomotor activity and metabolic rate, correcting all known phenotypic defects (15 16 17) . Several excellent reviews have recently covered these topics in detail (1 , 5 6 7 8 9 10 11 , 13 , 14 , 18 , 19) .

The mouse genome can be manipulated in two ways: overexpression, which typically involves the DNA construct into mouse zygotes to create gain-of-function mutations; and gene targeting, which creates loss-of-function mutations in which genes are deleted. Both approaches have been used extensively to investigate the in vivo functions of genes, including energy homeostasis equation (20 21 22 23 24 25 26 27 28 29) .


   IDENTIFICATION OF THE REGULATORS OF BODY MASS
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
The MC system involves peptides processed from the precursor proopiomelanocortin (POMC) that is produced in the arcuate nucleus (ARC) of the hypothalamus and is induced in response to leptin (12 , 30) . Several of the peptide products such as {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) have been implicated in the regulation of feeding and skin pigmentation. Mice lacking the POMC-derived peptides developed obesity, especially when raised on a high-fat diet, as well as altered pigmentation and defective adrenal development (31) . Heterozygous mice had an intermediate phenotype between homozygotes and control littermates, including leptin and corticosterone levels, suggesting a ‘gene dosage’ effect (31) .

The agouti gene normally controls pigmentation in the skin of the mouse by antagonizing the {alpha}-MSH and MC-1 receptor (32) , but agouti mutation is one of the extant single-gene mutations resulting in obesity in rodents (2 , 18 , 20 21 22) . Agouti causes obesity and diabetes when ectopically expressed as observed in obese yellow mice (Ay) or by transgenic technology using ubiquitous promoters such as ß-actin (33 , 34) . This is due to the inhibition of MC receptors in the brain, and the obese phenotype is associated with hyperphagia, decreased thermogenesis, and increased caloric efficiency (34 , 35) . Knocking out the MC-4 receptor gene produced mice that developed maturity-onset obesity, with hyperphagia and increased linear growth resembling that associated with agouti overproduction (36) . These results suggest that defective melanocortin signaling through MC-4 receptor in the hypothalamus is responsible for the weight gain, which is mediated either by changes in agonists ({alpha}-MSH) or antagonists (agouti or AGRP). MC-4 receptor is not an exclusive target of leptin action because double-mutant studies demonstrated that Ay has an additive effect on weight gain in ob/ob mice (37) . An AGRP has recently been isolated that shares some sequence homology with agouti (38 39 40) . The peptide is expressed within the hypothalamus, is regulated by leptin, and is a potent antagonist of MC-3 and MC-4 receptors. Overexpression of AGRP recapitulated unique features of the obese yellow and MC-4 receptor-deficient mice, suggesting that AGRP is an endogenous regulator of melanocortinergic neurons in the brain (39) . Humans also have an agouti gene, which (unlike in mice) is normally expressed in adipose tissue. To model human agouti expression, transgenic mice were generated that express murine agouti at high levels in adipose tissue under the control of the adipose lipid binding protein gene (aP2) promoter (41) . The mice were not obese or diabetic, but combined insulin treatment promoted obesity, suggesting an implication for human obesity.

Serotonin (5-HT) is an important amine involved in the regulation of appetite and emotion, which originates from the midbrain dorsal raphe nucleus and projects to the hypothalamus, including the paraventricular nucleus (PVN) and the ventromedial nucleus (42 43 44 45) . Various serotonergic agonists/antagonists were developed and used to examine the effect of serotonin system on food intake and macronutrient selection (42 , 44 , 45) . Stimulants of this monoamine reduce food intake and weight gain and increase energy expenditure in both animals and humans. Based on the studies in which relatively selective agonists and antagonists were used, it was suggested that serotonin-induced satiety was mediated by postsynaptic 5-HT1B receptor (46) . However, since serotonin interacts with a larger number (probably up to 20) of receptors, it was difficult to assign specific functions to specific receptors by this pharmacological approach alone (47) . Recently, mice lacking serotonin receptor subtype IIc (5-HT2C) were generated (48 , 49) . The mutant mice had elevated body weight (and epilepsy) and were resistant to the anorectic effect of m-chlorophenylpiperazine, an agonist at 5-HT1B and 5-HT2C receptors, indicating that 5-HT2C receptor contributes substantially to the serotonergic regulation of body weight. The obesity syndrome was characterized by hyperphagia preceding the development of obesity and reduced sensitivity to leptin and by the absence of decreased energy expenditure (49) . The mice thus differ from other rodent models of obesity such as ob/ob and db/db mice, which are characterized primarily by metabolic dysfunction. The mutant mice were more sensitive to high-fat diet-induced obesity, and later they developed insulin resistance and hyperglycemia. These results suggest a dissociation of serotonin and leptin in the regulation of feeding and indicate that a perturbation of brain serotonin systems may predispose to diabetes. In contrast, mice lacking the 5-HT1B receptor developed normally and had normal food intake although they exhibited motor impulsivity, enhanced aggressive behavior, elevated alcohol consumption, and increased sensitivity to cocaine (47 , 50 , 51) .

MCH is a cyclic polypeptide originally discovered in vertebrate fish where it mediates color changes (52 , 53) . MCH is present in the lateral hypothalamus of mammalian brain, and both stimulation (54) and inhibition (55) of food intake are reported after administration into the brain. However, ablation of the gene led to a thin phenotype that was associated with reduced food intake and inappropriately increased metabolic rate (56) . VGF is a secreted polypeptide of unknown function that is synthesized by neurons and is abundant in the hypothalamus (57 , 58) . Mice lacking VGF displayed dramatically decreased body weight and body fat, the major defect in which is due to excess energy expenditure and not to decreased food intake (59) . The mice had increased oxygen consumption at rest and increased locomotor activity despite normal sympathetic tone and somewhat reduced levels of thyroid hormone, suggesting that VGF may play a novel role in energy expenditure regulation.

The brain controls energy expenditure, at least in part, via the sympathetic nervous system, which innervates brown adipose tissue (BAT) (2 , 14) . BAT is a specialized form of adipose tissue that functions as a thermogenic organ in rodents (60) . The role of BAT in body weight regulation has been demonstrated in mice in which expression of a transgene encoding for a diphtheria toxin under the control of a brown fat-specific promoter (uncoupling protein, UCP) was used to specifically ablate BAT(61–64). The mutant mice developed obesity due to decreased thermogenesis and lowered body temperature. The mice also had increased susceptibility to diet-induced obesity and diabetes (62) , providing compelling evidence that BAT protects against obesity caused by calorically dense diets.

Other examples that genetic manipulation has been proved to be powerful are regulators expressed broadly or locally in the fat, muscle, or other tissues, including intracellular adhesion molecule-1 (ICAM-1), leukocyte integrin {alpha}Mß2 (Mac-1), metallothioneins, and transcription factor Nhlh2, as well as glucose transporter-4 (GLUT4) and lipoprotein lipase (LPL). ICAM-1 functions as a major cell–cell adhesion molecule in inflammatory and immune systems through binding and interacting with Mac-1 and other counter-receptors expressed on leukocytes (65) . Mice deficient in ICAM-1 or its counter-receptor Mac-1 developed late onset obesity without overeating, in addition to inflammatory and immune defects (66) . Both mice exhibited a phenotypically similar diet-induced obesity, suggesting that leukocyte functions may influence fat storage. Metallothioneins comprise a family of highly conserved metal binding proteins that have a role in the detoxification of heavy metals and other functions. The metallothionein null mice became obese at a young age and were hyperphagic in established obesity (67) . The family of basic helix-loop-helix genes comprises transcription factors involved in growth and development. Mice deficient for Nhlh2, a transcription factor made in the hypothalamus, displayed adult-onset obesity with impaired gonadal growth associated with puberty (68) . GLUT4 is a major facilitative glucose transporter isoform in skeletal muscle and adipose tissue (69) . The specific overexpression of human GLUT4 in white adipose tissue using the aP2 promoter developed mice that do not exhibit hyperphagia, but produce hyperplasia of adipocytes and increased fat mass (70) . Obesity may thus occur by preferential nutrient uptake into white adipose tissue. Conversely, GLUT4 null mice had severely depleted adipose tissue and growth retardation (71) . LPL is the rate-limiting enzyme for the import of triglyceride-derived fatty acids by muscle for utilization and by adipose tissue for storage. The targeted overexpression of LPL in skeletal muscle using the muscle creatine kinase promoter prevented diet-induced obesity by diverting fatty acids away from storage in adipose tissue to oxidation in muscle (72) . LPL-deficient mice were normal at birth, but developed lethal hypertriglyceridemia within the first day of life (73) . When these mice were rescued by mating to transgenics expressing muscle-specific LPL, LPL deficiency in adipose tissue was found to be compensated for by large increases in endogenous adipose tissue fatty acid synthesis (74) . However, when these genotypes were examined on the ob/ob background, significantly decreased body weight and fat mass were observed in the ob/ob mice rendered deficient in adipose tissue LPL.


   DISCREPANCIES BETWEEN CONVENTIONAL PHARMACOLOGICAL AND TRANSGENIC STUDIES
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
However, interpretation was less straightforward in such cases as NPY and opioid, as well as CRF, GLP-1, leptin, and tumor necrosis factor (TNF-{alpha}). NPY is a potent orexigenic signal that is widely expressed throughout the brain, including the ARC of the hypothalamus (7 8 9 10 11 12 13 , 75 76 77) . Central NPY administration increases food intake, decreases energy expenditure, and increases lipogenesis; chronic NPY administration produces obesity (75 , 78) . It is also known that hyperphagia and obesity in genetic and experimental models are associated with augmentation of NPYergic signaling in the hypothalamus, that is, either up-regulation of NPY in the ARC and other novel sites such as the dorsomedial nucleus or increased NPY receptor abundance and sensitivity (13 , 79) . It is surprising that NPY knockout mice showed normal food intake and body weight, and responded normally to fasting and with an increased sensitivity to leptin (80) . Subsequent experiments using mice with a deficiency of both leptin and NPY demonstrated that the double mutant mice were halfway between normal lean and ob/ob mice in body weight and fat mass as well as in reproductive and other neuroendocrine disturbances (81) . The absence of NPY, however, did not attenuate the development of obesity induced by a high-fat diet, chemical lesions of the hypothalamus, impaired BAT function due to a diphtheria toxin transgene, or the lethal agouti mutation (Ay) (82) . The response of NPY knockout mice to anorectic and orexigenic substances was reported to be unaltered including NPY, CRF, dexfenfluramine (an enhancer of serotonergic transmission), and MTII (a MC-4 receptor agonist), although the initial response to galanin might be lost(83). It remains to be determined whether NPY is involved in hyperphagia and obesity only under extreme conditions such as in ob/ob mice or whether the normal phenotype is due to compensation by other orexigenic signals that replace and maintain seemingly normal feeding and body weight regulation.

NPY activates at least six G-protein-coupled receptor subtypes, Y1–Y6, all of which have been cloned except for the Y3 receptor (79 , 84) . NPY analogs exhibit varying degrees of affinity and specificity for these Y receptors, as well as potency in stimulating feeding (79) . Mice lacking the Y1 or Y5 receptors, which are supposed NPY feeding receptors (79 , 84 , 85) , have been generated. The Y1 receptor knockout mice exhibited a markedly reduced feeding response to fasting and slightly reduced daily food intake and NPY-stimulated feeding (86) . The Y5 receptor knockout mice exhibited a normal response to fasting and leptin but markedly reduced NPY-stimulating feeding (87) . However, they developed unexpected late-onset obesity, which was associated with hyperphagia (87) (Y5-deficient mice), or lowered metabolic rate associated with reduced locomotor activity (86) or changes in UCP gene expression (88) (Y1-deficient mice). These results are in contrast to the potent inhibitory effects of Y1 or Y5 receptor-specific antagonists on feeding and body weight gain in rodent models of obesity (79 , 89 , 90) . Although the Y2 receptor agonists have no or only minor effects on feeding after administration into the brain (79) , mice lacking the Y2 receptor developed mild obesity caused by hyperphagia and displayed an attenuated feeding response to leptin (91) .

Opioid peptides such as ß-endorphin and dynorphin, an endogenous mu and kappa opioid receptor ligand, respectively, are involved in the regulation of feeding as part of the orexigenic network that may center on NPY (12 , 13) . ß-Endorphin, derived from precursor POMC, and dynorphin from prodynorphin stimulate feeding after central administration (12 , 13) . Unexpected phenotypic changes were observed in the feeding stimulatory opioid system, in which larger litter size and increased body weight occurred in kappa opioid receptor and ß-endorphin knockout mice, respectively (23 , 92 , 93) .

CRF is a major catabolic peptide in the PVN of the hypothalamus (1 , 9 , 12 , 13 , 43) . In addition to its role as controller of the hypothalamic-pituitary-adrenal axis, CRF inhibits food intake, increases energy expenditure, and produces sustained weight loss. Central pharmacological blockade using CRF antagonists or antisense oligonucleotide, immunoneutralization, or immunotoxin targeting of CRF in the hypothalamus enhances basal and NPY-stimulating feeding, suggesting that CRF may tonically restrain the actions of orexigenic signals (1 , 43) . Over the past year, there has been a rapid increase in the availability of CRF peptide, CRF postsynaptic receptor, and CRF binding protein (CRF-BP) overexpressing and knockout mouse models. Transgenic mice overexpressing the CRF gene expressed elevated levels of activity of the hypothalamic-pituitary-adrenal axis and the mice became obese, resembling Cushing’s syndrome in humans (94 , 95) . CRF-deficient mice exhibited a fatal glucocorticoid requirement for lung maturation and an abnormal corticosterone response to stress after birth (96 , 97) . However, they appeared to be healthy without glucocorticoid replacement and were equal in size to their normal littermates. They did not exhibit smaller decreases in feeding after adrenalectomy, which is known to up-regulate CRF production and release (98) . These results, together with normal emotional response and learning task performance in mutant mice in which CRF is also involved (25) , suggest compensatory actions of other endogenous CRF family agonists, such as urocortin (99 , 100) . CRF and urocortin are bound by CRF-BP, a 37 kDa protein that binds both peptides with an affinity equal to or greater than the CRF receptors (101) . CRF-BP overexpressing mice developed by the pituitary-specific glycoprotein hormone {alpha}-subunit promoter or the ubiquitous metallothionein-1 promoter, produced an altered circadian pattern of food intake and a sexually dimorphic body weight gain, respectively (102 , 103) . CRF-BP-deficient mice, in contrast, exhibited a decrease of body weight gain in male mice, providing genetic evidence for the involvement of endogenous CRF/urocortin in body weight regulation (104) . Elucidation of the phenotype of knockout mice of CRF2 receptor had been awaited since this receptor was thought to be involved primarily in the feeding suppressive and thermogenic response of CRF and related peptides (105 , 106) . CRF2 receptor-deficient mice were recently developed (107 108 109) . Mutant mice exhibited normal basal feeding and weight gain, although they had no urocortin-induced feeding suppression except in the initial phase, a likely mediation by CRF1 receptor (107) .

The mice in which type II corticosteroid receptor antisense RNA construct was incorporated and expressed primarily to neural tissue by using a human neurofilament gene promoter developed obesity despite clear evidence for reduced glucocorticoid receptor activity in the hypothalamus, cerebral cortex, and liver (110) . The type of obesity produced was associated with reduced food intake and oxygen consumption during the dark phase and thus an increased energetic efficiency (111) . The result diverges from other evidence indicating the dependence of almost all obesities in the presence of adrenal glucocorticoids and an overactivity of type II corticosteroid receptor (22 , 112) . Tissue-specific mutation of this gene was recently performed using the Cre/loxP-recombination system (113 ; see Conditional Knockout section). This made it possible to generate viable adult mice with loss of glucocorticoid receptor function in the nervous system (114) . The mutant mice were reduced in length and weight, although altered fat distribution characteristic of Cushing’s syndrome was observed.

GLP-1 is a brain-gut peptide that acts on the brain to decrease food intake and body weight and on the pancreas to potentiate glucose-stimulated insulin secretion (9 , 12 , 13 , 115 , 116) . The GLP-1 receptor antagonist exendin9–39 stimulated feeding in satiated animals, and daily administration of exendin9–39 augmented food intake and body weight gain (115 , 117) . Mice with a targeted disruption in the GLP-1 receptor gene were generated (118) . They did not exhibit disturbances in the regulation of feeding and body weight although they did exhibit fasting hyperglycemia and defective glucose-stimulated insulin secretion. Obesity failed to develop in these mice with aging or high-fat feeding (119) . Gastrin-releasing peptide (GRP) is a mammalian bombesin-like peptide that induces a dose-dependent reduction in food intake and body weight in rodents and humans (120 121 122) . Bombesin is a peptide originally isolated from frog skin and acts on the three receptor subtypes. Mice lacking GRP receptor (bombesin receptor subtype 1) exhibited loss of bombesin-induced feeding suppression, but showed no overt changes in body weight regulation (123) . A redundant mechanism was suggested in these mice that had an increased sensitivity to cholecystokinin (CCK), another peptide involved in regulating individual meal size (9 , 12) . Mice lacking bombesin receptor subtype 3 developed mild obesity associated with hypertension and impairment of glucose metabolism (124) . However, bombesin shows only low affinity for this receptor and its natural ligand remains to be determined. Overexpression of leptin in the liver by using the human serum amyloid P component promoter produced mice that had markedly decreased food intake and body weight with complete disappearance of the adipose tissue (125) . This is in sharp contrast to a state called leptin resistance in which high levels of circulating leptin fail to prevent obesity in most obese humans and animals (126 127 128) . TNF-{alpha} is a proinflammatory cytokine that has been studied as a potential mediator of insulin resistance in obesity, as well as anorexia and cachexia in wasting disorders (18 , 129) . Mice lacking TNF-{alpha} or its receptors, p55 (type 1) and p75 (type 2), showed no significant effect on the development of obesity induced by a high-fat or high-calorie diet, except for a small decrease in adiposity (130 , 131) .

BAT thermogenesis is due to unique mitochondrial protein, UCP, a protein translocator that uncouples respiration from ATP synthesis (60) . Mice lacking UCP-1 in BAT were cold sensitive (indicating a defect in thermoregulation) but not obese (132) . The mutant mice actually had a lean phenotype, even when fed a high-fat diet. However, they showed an increased compensatory expression of UCP-2, an uncoupling protein expressed in many tissues, including white adipose tissue, with high homologies to the brown fat UCP-1 (133) . UCP expression is physiologically stimulated by the sympathetic nervous system, and agonist activation of the ß3-adrenergic receptor leads to thermogenesis in BAT and lipolysis in white adipocytes (19 , 27 , 134) . Selective ß3-adrenergic receptor agonists are known to increase the metabolic rate, leading to weight loss and improvement in glucose tolerance in obese rodents (135) . However, mice lacking the ß3-adrenergic receptor showed only a modest tendency to become obese relative to normal controls (136 , 137) , and there was a compensatory increase in the level of ß1 adrenergic receptor in the knockout mice. The transgenic mice overexpressing ß1-adrenergic receptor in the adipose tissue by the aP2 promoter were resistant to obesity at least partly because of increased lipolytic activity (138) . Sympathetic stimulation of protein kinase A in BAT promotes energy expenditure through UCP. Protein kinase A has two regulatory and two catalytic subunits, and the RIIß regulatory subunit is abundant in brown and white adipose tissue (139) . Targeted disruption of the RIIß subunit, however, resulted in lean mice that were resistant to dietary-induced obesity due to chronic activation of BAT thermogenesis and elevated body temperature (140) . Mutant BAT exhibited a compensatory increase in RI{alpha}, almost entirely replacing lost RIIß.


   CONFOUNDING INFLUENCES
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
The attempt to interpret the phenotypic changes that arise in genetically engineered mice is subject to several caveats, including genetic background, compensation, and environmental factors (24 , 141 142 143 144 145 146 147 148 149 150) .


   GENETIC BACKGROUND
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
Most gene targeting has been carried out in cultured embryonic stem cells derived from the mouse strain 129 (141) . It is unfortunately one of the most unique strains in terms of behavior (poor performance on memory tasks and low levels of anxiety-like behaviors) as well as neuroanatomy (absence of the corpus callosum) (142 , 143) . Null mutant mice of gene targeting studies are often hybrids of two mouse strains (strain 129 and, for example, C57BL/6) and are genetically different from their control littermates in a typical F2 analysis (142 , 144 , 145) . Mutations may have very different phenotypes in different backgrounds (144 145 146 147 148) , although this is poorly examined in the models of energy homeostasis equation. It will be difficult to compare the overexpression of a gene with its deletion without a common genetic background. The situation may even be complicated by new experimental strategies involving the derivation of compound mutant mice that result from crossing random insertion transgenics with targeted mutagenesis. The compensatory changes triggered by the disruption of the targeted gene will also depend on the background genotype, in addition to the targeted gene itself and its involvement in certain molecular pathways (see Compensation section).


   ENVIRONMENTAL BACKGROUND
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
Null mutants and overexpressing mice are often scarce and tend to be behaviorally characterized in a single laboratory with a limited array of available tests. It was recently reported that differences in investigators and unforeseen environmental factors from laboratory to laboratory can alter behavioral results, including anxiety level, alcohol consumption, and body size (151) . Stress can have significant effects on feeding (25 , 43) , and food composition, texture, and moisture can have substantial effects on growth, nutrient choice, and other behaviors (152) . Previous studies in diet-induced obesity models suggested that there may be some central ‘set-point’ for the regulation of body weight that was genetically predetermined but expressed only in the presence of appropriate dietary exposure (153 , 154) . The increased sensitivity to diet-induced obesity was reported in 5HT2C-deficient, POMC-deficient, or BAT-ablated mice (31 , 49 , 62) . Our recent study demonstrated that modest overexpression of NPY leads to obesity that was observed only in the presence of a highly palatable diet (ref 155 and unpublished data). The obesity phenotype in transgenic animals may thus be critically dependent on the dietary and other environmental exposures.


   COMPENSATION
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
A major issue is the compensation by other genes for the missing or overexpressed gene in knockouts or transgenics (143 , 144 , 149) . When a gene of interest is knocked out by the conventional method, it is absent throughout development and the entire organism. Energy homeostasis is known to be accomplished through a highly integrated and redundant neurohumoral system (9 , 12 , 13) . A compensatory process may take over the function of the missing gene and mask functional outcome of the mutation. Conversely, behavioral deficits may arise secondarily from the animal’s inability to execute its normal development program since many genes play a role not only in the functioning of a mature brain, but also the development. Typical examples of the compensation are the UCP-1 (132) , ß3-adrenergic receptor (136 , 137) , and RIIß subunit of protein kinase A (140) knockout mice in which UCP-2, ß1-adrenergic receptor and RI{alpha} subunit substituted, respectively. An increased sensitivity to CRF and CCK was also reported in MC-4 receptor- and GRP receptor-deficient mice, respectively (123 , 156) . These processes are a fascinating study unto themselves that could reveal genetic redundancy and alternative biochemical pathways. Experiments with replacement genes or replacement products of the missing gene will allow investigators to test the hypothesis that their experimental mutation is truly responsible for the altered phenotype. This was successfully shown in mice lacking the POMC derived peptide (31) or those overexpressing pancreatic polypeptide (PP) (157) in which melanocortin or anti-PP antiserum specifically reversed the obese or the lean phenotype. Also, mice and humans may differ in their mechanisms of compensation as exemplified in CRF, POMC, or leptin deficiency (31 , 96 , 158) . In human POMC-deficient patients, ACTH deficiency results in hypocortisolism and, if untreated, in death (159) . Despite undetectable serum corticosterone levels, POMC null mice are viable (31) , similar to mice lacking CRF (96) . The mutations for leptin and leptin receptor showed that these proteins have similar physiological roles, including regulation of appetite, reaction to starvation, and control of energy expenditure in humans and rodents (160 161 162 163) . However, differences include lack of decreased body temperature, of enhanced cortisol levels, and absence of diabetes, which so far has been seen in just one out of eight leptin or leptin receptor-deficient patients (158 , 163) .


   OTHER FACTORS
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 
Other possible explanations for differences in phenotype involve differences in the methods of gene manipulations. The most widely used strategy for gene disruption in the mouse involves the deletion of part or all of the target gene together with concomitant insertion of a drug selection cassette. This may disrupt the other genes located near the intended target and produce different phenotypes, ranging for example from complete viability of homozygotes to complete lethality in mice lacking the myogenic basic-helix-loop-helix gene MRF4 (164) . Another such example may be dopamine D1 receptor knockout mice, which showed quite different severity of growth retardation with feeding deficits on the similar 129-C57BL/6 background (29 , 165 , 166) . The disruption of the gene of leukocyte antigen-related protein tyrosine phosphatase, which may affect the insulin signaling cascade, also produced mice with normal body weight(167) or half that of the control mice (168) . Other factors include gender differences in fat storage in mutant mice (86 , 88 , 103 , 104 , 136 , 157) , as well as incomplete null alleles that may occur as a post-transcriptional event and influence the ultimate phenotype of a knockout model (144) . It was recently reported that a heterozygous mutation in a stimulatory G-protein {alpha} subunit led to opposite effects on body adiposity and energy expenditure depending on parental inheritance (169) . In random insertion transgenics, a problem is that the site of integration of introduced DNA into the genome is frequently nonspecific and position effects are not uncommon (29 , 170 , 171) . Such position effects influence transgene expression patterns and can alter the expression of genes near the site of integration.


   CONDITIONAL KNOCKOUT
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
CONCLUSION
 REFERENCES
 
The best current hope for overcoming these obstacles is conditional knockout or overexpression, in which genes can be turned on or off at specific times and in particular regions or cell types. Obesity and associated physiological derangements such as diabetes are complex traits, developing later in life and involving interactions between genetic factors and acquired factors such as diminished exercise. It is necessary to refine gene targeting and gain control over the timing, locale, and degree of genetic manipulation (29 , 142 , 149) . The regional and temporal specificity can be achieved by new genetic strategies such as Cre/lox P recombination system and a tetracycline-controlled gene induction system (149 , 172 , 173) , which could resolve the major drawbacks of the conventional transgenic technology and allow identification of causal relationships between mechanisms at behavioral levels. Although the technique has not been applied to a component of the feeding regulatory cascade in the brain (except for type II corticosteroid receptor; ref 114 ), Cre/lox P mediated, tissue-specific targeting was successfully achieved to reveal the role of insulin receptor in muscle (174) and pancreatic ß cells (175) for fat metabolism and insulin secretion, respectively. Mice lacking the muscle insulin receptor displayed elevated fat mass, serum triglycerides, and free fatty acids (some aspects of the metabolic syndrome), but no evidence for impairment of glucose homeostasis (174) . Mice lacking the ß cell insulin receptor showed an insulin secretory defect similar to that in type 2 diabetes, but no evidence for impairment of body weight regulation (175) . An additional advantage of this Cre/lox P-mediated system is that recombination does not occur until the third postnatal week, thereby reducing developmental concerns (149 , 176) . A caveat of both the tissue-specific and inducible system is the penetrance of the knockout, which may affect experimental results (29) . This is because both systems rely on the Cre expression, and targeted excision may not occur in all cells with the same efficiency.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
REFERENCES
 
The ability to manipulate the expression patterns of specific gene products in vivo has led to significant advances in our understanding of energy homeostasis equation. In many cases, gene manipulation studies have provided valuable information that could not be achieved by conventional pharmacological techniques alone. The molecular genetic techniques have been used to effectively model human disease processes affecting body adiposity, and the promise of such models for potential therapies is ever-expanding (20 21 22 23 24 25 26 27 28 29 , 177 , 178) .

Several examples of the different types of methodological problems that have been described in this review might be encountered when gene targeting or, more generally, molecular genetic techniques are used. It is absolutely necessary to control for as many potential confounding factors as possible since molecular genetic techniques are not uniquely different from other scientific methods. Gene disruption or gene expression may perturb the organism and force it to respond in a way that is inherent to its biological organization. In some cases with unexpected phenotypic changes it may be difficult to assign, or not to assign, specific functions to specific genes because of redundancy and plasticity of the regulatory machinery (12 , 21) . However, with the inducible, reversible, and cell-specific gene targeting on the horizon, molecular genetic techniques will yield unprecedented insights into the biological mechanisms underlying energy homeostasis equation.


   ACKNOWLEDGMENTS
 
I am indebted to Prof. Masato Kasuga and Prof. Shigeaki Baba (Kobe University) for many stimulating discussions. The work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF THE REGULATORS...
 DISCREPANCIES BETWEEN...
 CONFOUNDING INFLUENCES
 GENETIC BACKGROUND
 ENVIRONMENTAL BACKGROUND
 COMPENSATION
 OTHER FACTORS
 CONDITIONAL KNOCKOUT
 CONCLUSION
 REFERENCES
 

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