Transgenic animal models for the study of adipose tissue biology

Best Practice & Research Clinical Endocrinology & Metabolism Vol. 19, No. 4, pp. 605–623, 2005 doi:10.1016/j.beem.2005.07.006 available online at http://www.sciencedirect.com

9 Transgenic animal models for the study of adipose tissue biology Matthias Blu¨her* Department of Internal Medicine II, University of Ko¨ln, Ko¨ln, Germany

Abstract The traditional view of adipose tissue as a passive energy reservoir has changed. Adipose tissue is a complex, highly active metabolic and endocrine organ. With obesity as an increasingly important public health threat, a major development in the understanding of adipose tissue biology has come with observations in different biological spheres including whole-body physiology and application of transgenic animal models. Scientific progress has been made with the identification of several genes in spontaneous monogenic animal models of obesity, and in understanding the molecular mechanisms underlying phenotypes of altered body weight, adiposity and fat distribution by creating transgenic and knockout animal models. Mouse phenotypes resulting from inactivation or overexpression of molecules responsible for the regulation of adipose tissue metabolism have led to novel concepts in the understanding of adipocyte biology and development of obesity. This review presents an overview of transgenic animal models for the study of adipose tissue biology Keywords: Adipose tissue; type 2 diabetes; Obesity; knockout mice; FIRKO; Insulin; IGF-1; Animal models; Metabolism.

Adipose tissue is a complex, essential and highly active metabolic and endocrine organ.1 Both excess and deficiency of adipose tissue have severe metabolic consequences and represent significant medical and socio-economic burdens. In adipose tissue, adipocytes, connective tissue matrix, nerve tissue, stromal vascular cells and immune cells function as an integrated unit.1 Excess of adipose tissue mass, or obesity, is a major health problem conferring a higher risk of cardiovascular and metabolic disorders including type 2 diabetes, hypertension and coronary heart disease.2,3 Moreover, the rising prevalence of type 2 diabetes worldwide is mainly the consequence of the drastically increased prevalence of obesity.4 Obesity is further associated with insulin resistance, hyperglycaemia, dyslipidaemia, and prothrombotic and pro-inflammatory * Corresponding author. Address: Department of Internal Medicine II, University of Cologne, Kerpener Str. 62, 50937 Ko¨ln, Germany. Tel.:C49 221 478 4176; Fax: C49 221 478 3107. E-mail address: [email protected]

1521-690X/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved.

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states.5 The adipose organ serves as the site of triglyceride storage and free fatty acid and glycerol release in response to changing energy demands, and participates in the regulation of energy homeostasis.2 In addition, adipose tissue expresses and secretes a variety of bioactive molecules, known as adipokines, that act in a para-, auto- and endocrine manner. These adipokines include leptin, interleukin 6 (IL-6), other cytokines, adiponectin, complement components, adipsin, plasminogen activator inhibitor-1 (PAI-1), proteins of the renin-angiotensin system (RAS) and others. As several of these adipokines influence insulin sensitivity and glucose metabolism profoundly, they may provide a molecular link between increased adiposity and impaired insulin sensitivity. However, the role of adipose tissue in whole-body glucose homeostasis is not clear. Although some studies suggest that adipose tissue in humans may metabolize up to 20% of an orally-administered glucose load,6,7 euglycaemic hyperinsulinaemic clamp studies in rats indicate that adipose tissue is responsible for only 3–5% of glucose uptake.8 Excess of adipose tissue mass is the result of genetic predisposition and environmental factors including hypercaloric diet and inactivity. Due to considerable intra-individual variations of the human genetic pool and the variable effects of environmental factors, human studies of the pathophysiology of obesity and altered adipose tissue biology are complicated and fraught with uncertainties.9 In order to define the complex genetics and physiology of adipose tissue, investigators have generated a large number of transgenic and knockout mouse models.10,11 The number of transgenic animal models is continuing to increase. To date, more than 500 animal models involving the study of adipose tissue have been generated. Since it is impossible to cover all of these models in this review, a very subjective selection of animal models relevant to the study of adipose tissue is presented. The author is aware that numerous valuable transgenic animal models are not mentioned here; a more complete overview of current animal models created to investigate adipose tissue can be found in other recently published reviews.10,11 The impact of the central nervous system on the control of food intake and adipose tissue development, as well as specific aspects of the endocrine response and signal transduction in adipocytes, including adrenergic receptors, GTP-binding proteins, protein kinases, growth hormone and IGF-1 signalling, the role of oestrogens, glucocorticoids and other hormones, have been reviewed elsewhere and are therefore not discussed here.10–13

TRANSGENIC AND KNOCKOUT TECHNIQUES IN THE STUDY OF ADIPOSE TISSUE BIOLOGY The mouse remains the most appropriate species to create transgenic animals and to analyse the consequences of overexpressed genes and modifications introduced into the genome.10 Rats are also used for transgenic studies; however, practical and technical disadvantages are the reasons for favouring mice for transgenic studies. Although rabbits are more relevant for the study of some human diseases,14 this animal has not been used frequently for transgenic studies because the success rate for generating transgenic rabbits is very low (1%) and the costs of maintaining a rabbit colony are high.10 The main focus of transgenic animal models in the study of adipose tissue biology has been the expression or knockout of selected genes specifically in adipose tissue. Adipose-tissue-specific expression/knockout of genes became possible with

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the identification and characterization of promoter regions conferring adipose-tissuespecific expression. The promoters for the adipocyte lipid binding protein aP215 and for phosphoenolpyruvate carboxykinase (PEPCK)16 are commonly used to target both white and brown adipose tissue (WAT/BAT), whereas the UCP-1 promoter only targets BAT.17 Using the recombinase activity of the cyclization recombination (Cre) gene from the P1 bacteriophage, conditional transgenesis and knockout of specific genes became a commonly used method to create transgenic animal models. The Cre recombinase, a 38-kDa site-specific DNA recombinase, catalyses the recombination of two 34-bp-long loxP (locus of X-over of P1) sites, making it possible to excise loxPflanked DNA segments.10,18 The major application of the Cre/LoxP system is the generation of spatially or temporally regulated gene knockouts.19 Cre/LoxP-mediated recombination requires two steps. First, the endogenous gene to be targeted is modified by homologous recombination in murine embryonic stem (ES) cells so that LoxP sites flank a portion of the gene, and mice are generated from these recombinant ES cells to obtain flox (flanked by lox) mice. Second, the heterozygous flox mice are bred with mice expressing a Cre transgene to generate double heterozygous mice. These double heterozygous mice are bred with single heterozygous flox mice to generate conditional knockout mice.20

CONSEQUENCES OF GENETIC ABLATION OF ADIPOSE TISSUE The consequences of transgenic lipoatrophy have been studied in several mouse models (Table 1). In the first studies, the diphtheria toxin A (DT-A) chain gene was placed under the control of the aP2 promoter to achieve the ablation of WAT and, to some extent, BAT.21 aP2-DT-A mice, the first mouse model of lipoatrophy, die shortly after birth Table 1. Animal models for transgenic ablation of adipose tissue. Modified from Fru¨hbeck G & GomezAmbrosi J. Diabetologia 2003; 46: 143-17211. Model

Gene

Phenotype

Reference

aP2-DT-A Tg

–

21

aP2-SREBP-1c Tg

nSREBP-1c

A-ZIP/ F-1 Tg

B-ZIP

UCP-1-DT-A Tg

UCP-1

PEPCK-TGF-b1 Tg

TGF-b1

Transgenic skinny

Leptin

Neonatal lethal (high transgene expression) Lower Tg expression: atrophy, necrosis of fat at 5 months, hyperphagia, development of fatty liver and diabetes Decreased white fat mass, Insulin resistance, Hyperglycaemia, Hypertrophy of brown fat Complete ablation of white adipose tissue Reduced brown fat mass, Decreased longevity, Fatty liver, Hypoleptinaemia, Diabetes, hyperglycaemia, Hyperinsulinaemia, Hyperlipidaemia Complete ablation of brown adipose tissue Decreased energy expenditure, Hyperphagia Diabetes, hypertriglyceridaemia (with aging) Reduced white and brown fat depots Fibroplasia, Lipodystrophy-like syndrome Complete ablation of adipose tissue Increased glucose metabolism, Small liver

Tg, transgenic overexpression.

24 22

30

32 33

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when the DT-A transgene is highly expressed.21 Mice expressing lower DT-A levels have normal WAT development until the age of 2 months. Extensive atrophy and necrosis of WAT start at 5 months of age. The phenotype of the young transgenic mice is characterized by hyperphagia, resistance to obesity and adipose tissue necrosis induced by monosodium glutamate.21 In addition, these mice develop fatty liver and diabetes.21 Another transgenic mouse model for lipoatrophy was created using the dominant negative protein A-ZIP/F under the control of the aP2 promoter.22 The transgene A-ZIP/F prevents DNA binding of b-ZIP transcription factors of the C/EBP and Jun families, causing early impairment of growth and differentiation in WAT.22 A-ZIP/F-1 mice are hyperphagic and hypermetabolic, and develop diabetes with marked insulin resistance and fatty liver (Table 1). Implantation of WAT reverses the metabolic phenotype including improvements of whole-body insulin sensitivity, hyperglycaemia and hyperinsulinaemia.23 These data demonstrate that a lack of adipose tissue causes diabetes and dyslipidaemia. Another valuable model for the study of metabolic alterations associated with the lack of WAT is mice that express a constitutively active form of human sterol responsive element binding protein-1c (nSREBP-1c) under the control of the aP2 promoter.24 Surprisingly, the expression of a constitutively active form of the adipogenic factor SREBP-1c leads to reduced WAT mass, most likely due to downregulated expression of essential genes for adipogenesis including peroxisomeproliferator activated receptors (PPARs) and CCAAT/enhancer-binding protein alpha (C/EBPa).24 aP2-nSREBP-1c mice show a similar but weaker phenotype as the A-ZIP/F1 mice (Table 1). Lipoatrophy is associated with low leptin levels that could, at least in part, contribute to the phenotype of lipoatrophy models. Indeed, in aP2-nSREBP-1c mice, infusion of recombinant leptin leads to a major improvement in insulin sensitivity, suggesting that leptin deficiency plays a central role in the development of insulin resistance associated with lipoatrophy.25 However, leptin infusion has only moderate effects on insulin sensitivity in A-ZIP/F-1 mice,26 whereas increased insulin sensitivity is observed when A-ZIP/F-1 mice are crossed with skinny mice that overexpress leptin in the liver.27 Severe depletion of adipose tissue is also observed in mice with a targeted disruption of the gene encoding lysosomal acid lipase.28 However, the link between the lack of lysosomal acid lipase and adipose tissue depletion is not yet known. In fatty liver dystrophy mutant mice, a mutation in the gene coding for lipin 1 causes lipodystrophy, suggesting a critical role of this protein in adipocyte differentiation.29 Furthermore, the depletion of BAT in UCP-1-DT-A mice causes decreased energy expenditure and hyperphagia-induced obesity.30 However, UCP-1-DT-A mice can not be considered as a model of human lipodystrophy, since brown fat is not of physiological relevance in adult humans.10 Taken together, data from mouse models for lipoatrophy suggest that adipose tissue is required for maintaining whole-body glucose homeostasis, lipid metabolism and insulin sensitivity.

OBESITY, LEANNESS AND RESISTANCE TO OBESITY With the identification of leptin in 1994,31 great progress has been made in understanding adipose tissue biology in spontaneous monogenic animal models of obesity, as well as in identifying the molecular mechanisms underlying phenotypes of altered body fat, adiposity and fat distribution in transgenic animal models. Regarding the regulation of adipose tissue mass, transgenic animal models can be subdivided into

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Table 2. Selected transgenic animal models with an obese phenotype. Modified from Fru¨hbeck G & Gomez-Ambrosi J. Diabetologia 2003; 46: 143-17211. Model

Gene

Phenotype

Reference

11bHSD-1 Tg

11bHSD-1

34

Agrt Tg

Agrt

FORKO (KO) aP2-GLUT4 Tg IL-6 KO

FSH-R GLUT4 IL-6

MC4-R KO

MC4R

POMC KO

POMC

aERKO (KO)

Oestrogen Ra

b3-AR KO hGHRH Tg

b3-AR GHRH

Predominant visceral obesity, Insulin resistance Hyperlipidaemia, Hyperphagia, despite hyperleptinaemia Obesity, increased body length, Hyperinsulinaemia, Hyperglycaemia, pancreatic islet hyperplasia, Fatty liver Obesity, Skeletal abnormalities Increased fat mass, adipocyte hypertrophy Mature-onset obesity, Altered lipid and carbohydrate metabolism, Hyperleptinaemia Mature-onset obesity, Hyperphagia, Hyperinsulinaemia, Hyperglycaemia Obesity, Altered pigmentation Defective adrenal development Obesity, Adipocyte hypertrophy and hyperplasia Insulin resistance Obesity Increased abdominal fat mass, Hyperleptinaemia Hyperinsulinaemia

35

36 37 38 39 40 41 42 43

Tg, transgenic overexpression; KO, knockout.

animal models of obesity (Table 2), leanness (Table 3) and obesity-resistant phenotypes (Table 4).11 Transgenic and knockout animal models are important tools to investigate the physiological roles of individual genes; however, transgenic approaches have limitations, especially because of the complementarity of genes and the capacity of animals to develop adaptive mechanisms to preserve crucial physiological functions.11

ANIMAL MODELS FOR THE STUDY OF ADIPOSE TISSUE METABOLISM Glucose metabolism Glucose transporter 4 (GLUT4) is the major glucose transporter in WAT. Overexpression of GLUT4 selectively in adipose tissue results in increased body weight and total lipid content due to hyperplasia of adipocytes with an enhanced glucose disposal.37 Whole-body GLUT4K/K mice are characterized by decreased body weight due to a marked reduction in WAT mass, accompanied by normoglycaemia and a normal response to glucose load.49 However, selective inactivation of the GLUT4 gene in adipose tissue has no effect on growth, body weight and fat mass in vivo, but these mice exhibit impaired insulin action in muscle and liver leading to glucose intolerance and insulin resistance.64 This phenotype demonstrates that glucose transport in adipose tissue plays a critical role in whole-body glucose homeostasis, and the downregulation of GLUT4 in adipose tissue of obese humans may contribute to the development of insulin resistance and type 2 diabetes. Although leptin, tumour

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Table 3. Selected transgenic animal models with a lean phenotype. Modified from Fru¨hbeck G & GomezAmbrosi J. Diabetologia 2003; 46: 143-17211. Model

Gene

Phenotype

Reference

Acc2 KO C3 (-/-) KO

ACC2 ASP

44 45

Crebbp (C/K) KO FIRKO

Crebbp InsulinR

Foxc2 Tg GLUT4-null

FOXC2 GLUT4

MCK-CD36 (Tg in muscle) Mstn (K/K) KO Plin(K/K) KO

CD36

50% reduced fat content in adipose tissue Decreased body weight and fat mass, Low glucose, insulin and leptin concentrations Reduced white adipose tissue mass, Increased insulin sensitivity Reduced fat mass, Normal insulin sensitivity, Normal glucose metabolism, Increased longevity Reduced fat mass Growth retardation, decreased longevity, Reduced adipose tissue depots, Postprandial hyperinsulinaemia Reduced overall adipose tissue, Low triglycerides and free fatty acids Increased skeletal muscle mass, Slightly decreased metabolic rate, Decreased adipose tissue mass Increased lean body mass, Increased metabolic rate Elevated basal lipolysis Decreased body weight and fat mass, Decreased insulin, triglyceride and leptin levels

TNFa(K/K) KO

Myostatin Perilipin TNFa

46 47 48 49

50 51 52 53

Tg, transgenic overexpression; KO, knockout.

necrosis factor alpha (TNFa) and free fatty acids have been excluded as potential insulin-resistance-inducing factors, other adipocyte-derived molecules could be involved in the impaired insulin action in skeletal muscle and liver of adipose-selective GLUT4 knockout mice.64 The hexosamine pathway has been hypothesized to mediate some of the toxic effects of hyperglycaemia. Overexpression of the rate-limiting enzyme of this pathway, glutamine:fructose-6-phosphate amidotransferase, in skeletal muscle and adipose tissue results in insulin resistance with a defect in GLUT4 translocation, suggesting that the hexosamine pathway may serve as a glucose sensor linked to glucose disposal.65 Consequences of adipose-tissue-specific insulin resistance In the mouse, genetic disruption of insulin receptors (IRs) or proteins involved in insulin signalling, either in the whole body or in specific organs, usually leads to insulin resistance and the tendency to develop diabetes.66 Whole-body knockout of IRs leads to neonatal lethality due to diabetic ketoacidosis.67 IR null mice have a markedly decreased amount of fat due to a reduced fat cell volume.67 Surprisingly, adipocytespecific inactivation of the IR gene in adipose-specific IR knockout (FIRKO) mice results in normal whole-body insulin sensitivity and glucose homeostasis, reduced fat mass and protection from age- and hyperphagia-induced obesity.47 Moreover, this phenotype is associated with increased longevity in FIRKO mice.68 The FIRKO mouse model clearly shows that reduced adiposity, even in the presence of normal or increased food intake, can extend life span, most likely by mimicking some of the effects of caloric restriction. The inactivation of IRs specifically in adipose tissue further revealed a heterogeneity of

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Table 4. Selected transgenic animal models with resistance to obesity. Modified from Fru¨hbeck G & Gomez-Ambrosi J. Diabetologia 2003; 46: 143-17211. Model

Gene

Phenotype

Reference

Agt (K/K) KO

AGT

54

Cav-1 (K/K) KO

Caveolin-1

DGAT(K/K) KO

DGAT

Gipr (K/K) KO

GIPr

GLP-1R (K/K) KO Mch1r (K/K) PPARg (C/-) KO

GLP-1R MCH1R PPARg

PTP-1B (K/K) KO

PTP-1B

VLDLR (-/-) KO b1-AR Tg

VLDL-R b1-AR

Less weight gain under high-fat diet, Decreased lipogenesis, Increased locomotor activity Resistance to high-fat-diet-induced obesity despite hyperphagia Resistance to diet-induced obesity by increasing energy expenditure Protection from obesity and insulin resistance induced by high-fat diet Resistance to high-fat-diet-induced obesity Resistance to diet-induced obesity Resistance to increased fat mass and fatty liver Protection from insulin resistance induced by high-fat diet Resistance to weight gain, Normal insulin sensitivity Resistance to high-fat-diet-induced obesity Resistant to increased fat mass, Decreased lipid accumulation in adipose tissue

55 56 57 58 59 60

61 62 63

Tg, transgenic overexpression; KO, knockout.

adipose tissue that is unrecognized to date.47,69,70 While adipocytes of control mice exhibit a bell-shaped size distribution, adipocytes of FIRKO mice demerge into groups of small and large cells. Interestingly, these different adipocyte subgroups show different expression of fatty acid synthase, the transcription factors C/EBPa and SREBP-1c, and other key genes and proteins of adipocyte biology.69,70 Lipid metabolism Numerous mouse models with disruptions or overexpression in pathways relevant to fatty acid and triglyceride metabolism have been generated to clarify the role of different proteins involved in lipid metabolism. Transgenic mouse models have contributed to establish links between fatty acid transport in lipoprotein particles and adipose tissue development. More complete overviews of such transgenic models have been published recently.10,11 For example, the absence of the very-low-density lipoprotein (VLDL) receptor leads to reduced triglyceride storage, as determined by a decreased average fat cell size, and to resistance to both genetic- and diet-induced obesity, suggesting that fatty acid delivery to adipose tissue is impaired in VLDL-RK/K mice.62 In the capillaries of adipose tissue, lipoprotein lipase (LPL) catalyses the rate-limiting step in the hydrolysis of triglycerides from circulating VLDLs and chylomicrons. The knockout of LPL leads to neonatal death due to marked hypoglycaemia, hypertriglyceridaemia and minimal amounts of tissue lipids.71 Reduction of LDL in the whole body of heterozygous LPL knockout mice only causes mild hypertriglyceridaemia and hyperinsulinaemia accompanied by 20% decreased fasting glucose concentrations.71 LPL deficiency exclusively in adipose tissue renders a normal growth and body composition, suggesting that despite the LPL control of fatty acid entry into

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adipose tissue, fat mass is preserved by endogenous synthesis.72 Hormone-sensitive lipase (HSL) has been considered to be the key enzyme of lipolysis in adipose tissue. Surprisingly, HSL-deficient mice have normal body weights and fat content, despite adipocyte hypertrophy.73 Although catecholamine-induced lipolysis is markedly blunted in these mice, basal lipolytic activity remains unchanged, suggesting that other lipases may play a role in fat metabolism in adipose tissue.73 Activation of lipolysis could also depend on proteins interacting with HSL, including aP2, lipotransin or perilipins.11 Disruption of aP2 in aP2K/K mice leads to a decrease of approximately 40% in the basal and isoproterenol-stimulated lipolytic rates.74 aP2K/K mice develop diet-induced obesity, but show normoglycaemia and normal insulin levels, providing evidence for uncoupling of obesity from insulin resistance through aP2 deficiency.74 Perilipins allow access to the lipid droplet, thereby allowing lipases to interact with their substrates. In two independent studies, inactivation of perilipin (PlinK/K) resulted in mice with decreased fat and increased lean body mass.52,75 These mice were resistant to dietinduced obesity, and had no hepatic steatosis or alterations of the lipid profile. Basal lipolysis was increased in adipocytes of PlinK/K mice, confirming the role of perilipin as a suppressor of lipolysis. Martinez-Botas et al52 suggested that without perilipin, adipocytes have a permanent lipolytic activity, whereas Tansey et al75 reported a blunted increase in glycerol and free fatty acid release induced by b-adrenergic stimulation in PlinK/K adipocytes, indicating that perilipin is a necessary cofactor for full lipolytic stimulation. Another fatty acid transporter, the CD36/fatty acid transporter molecule, has been proposed as a transporter of long-chain NEFA because CD36 null mice have decreased capacity to incorporate long-chain NEFA into triglycerides.76 This defective fatty acid esterification is most likely to be due to a limited supply of acylCoA that impairs conversion of diglyceride to triglyceride at the level of diacylglycerolacyltransferase (DGAT), suggesting a regulatory role for this enzyme in vivo. Mice lacking DGAT-1 are capable of synthesizing triglycerides and have a normal body weight on a standard chow diet, despite a reduced fat mass and adipocyte size.56 DGAT-1-deficient mice are resistant to diet-induced obesity (Table 4) due to increased energy expenditure. Interestingly, the study also demonstrated that triglyceride synthesis can occur without DGAT-1, suggesting the existence of another enzyme with DGAT activity; this has recently been characterized as DGAT-2 and may compensate for the lack of DGAT-1.77 Increased fatty acid oxidation may also lead to reduced adipose tissue mass as demonstrated in acetyl CoA carboxylase (ACC2)-deficient mice.44 Disruption of ACC2 causes reduced malonylCoA levels in heart and skeletal muscle, with a parallel increase in fatty oxidation. ACC2 null mice have reduced fat pad sizes despite an increased food intake.44 Thermogenesis Uncoupling proteins, especially UCP-1, play a major role in thermoregulation and in the protection from diet-induced obesity in rodents by uncoupling oxidation of fuels from ATP production in mitochondria of BAT, resulting in heat generation instead of ATP synthesis. Genetic ablation of BAT causes obesity, lower body core temperature, decreased metabolic rate and an increase in total body lipid due to hyperphagia.30 Unexpectedly, mice lacking UCP-1 show no difference in resting metabolic rate and do not develop hyperphagia and obesity, but show blunted b-adrenergic-stimulated oxygen consumption.78 The discrepancies between mice lacking BAT and UCP-1 knockout

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mice provide evidence that additional BAT-derived factors are responsible for dietinduced thermogenesis. However, targeted disruption of UCP-2 and UCP-3 does not affect adipose tissue development or cold responsiveness.10 In contrast, the lack of the known b-adrenergic receptors in b-less mice demonstrates that b-adrenergic signalling is required for diet-induced thermogenesis, and that this efferent pathway plays a critical role in the body’s defence against diet-induced obesity.79 On a high-fat diet, b-less mice develop severe obesity due to a failure of diet-induced thermogenesis.79

ANIMAL MODELS FOR THE STUDY OF SECRETORY FUNCTION OF ADIPOSE TISSUE Various adipocyte-secreted factors have been described that affect insulin sensitivity profoundly and may potentially link obesity, insulin resistance and cardiovascular disease. Among those, adiponectin appears as an insulin-sensitizing adipocytokine, whereas TN Fa, IL-6, resistin, PAI-1 and others induce insulin resistance. Moreover, leptin is a fat-derived key regulator of appetite and energy expenditure. Leptin Defects in leptin signalling either due to leptin deficiency or to mutant and dysfunctional leptin receptors lead to extreme obesity, hyperphagia, decreased energy expenditure, lower body temperature, defective thermogenesis, infertility, hyperinsulinaemia, dyslipidaemia and other metabolic alterations.11,31 In transgenic ob/ob mice expressing leptin under the control of the aP2 promoter, some phenotypic alterations in ob/ob mice are reversed but the animals continue to show moderate obesity.80 Adiponectin Adiponectin/Acrp30 is an adipocyte-secreted hormone with insulin-sensitizing effects. Serum adiponectin concentrations are inversely associated with obesity, insulin resistance and type 2 diabetes in rodents and humans, whereas increased serum adiponectin concentrations are associated with improved insulin sensitivity.81 In accordance with its insulin-sensitizing role, transgenic mice lacking adiponectin show impaired insulin sensitivity.82 Moreover, heterozygous PPARg knockout mice60 as well as FIRKO mice47 are protected from high-fat-diet-induced obesity, adipocyte hypertrophy and insulin resistance. Systematic protein and gene expression analysis of these mice revealed that adiponectin was overexpressed, suggesting that increased adiponectin secretion could help to compensate for the adipocyte-specific insulin resistance in FIRKO mice and could therefore explain, at least in part, the phenotype of FIRKO mice with normal insulin sensitivity and normal glucose homeostasis. IL-6 IL-6 is secreted from adipose tissue even during non-inflammatory conditions, with pleiotropic effects on different tissues, including stimulation of acute-phase proteins, increased thermogenesis and downregulation of adipocyte LPL.10,11 Mice deficient in IL6 develop mature-onset obesity with a predominant increase in subcutaneous fat mass accompanied by increased triglyceride levels, decreased leptin sensitivity and decreased

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glucose tolerance.38 The phenotype of the IL-6-deficient mice suggests an anti-obesity effect of IL-6, most likely exerted via effects in the central nervous system.10 TNFa TNFa stimulates lipid mobilization in mature adipocytes and suppresses the expression of key regulator proteins in adipocyte differentiation, C/EBPa and PPARg, subsequently causing downregulation of many adipocyte-specific genes including LPL, aP2, fatty acid synthase, GLUT4 and others.11 To investigate the role of the TNFa system, several transgenic animal models have been generated, such as TNFa-deficient mice and mice lacking the expression of either or both TNFa receptor (p55 and p75) subtypes. TNFa is a potential candidate for obesity-induced insulin resistance because TNFa-knockout mice are protected from insulin resistance associated with obesity.53,83 However, disruption of TNFa receptors has no effect on body weight or glucose homeostasis under a normal chow diet.84 On a high-fat diet, transgenic animals lacking the p75 receptor show a lower body weight, whereas absence of both receptor subtypes (p55 and p75) results in severe hyperinsulinaemia.84 Resistin The role of the adipocyte-secreted molecule resistin in obesity and diabetes has been controversial. Recently generated resistin knockout mice show normal glucose and insulin sensitivity with lower fasting glucose levels.85 Upon feeding with a high-fat diet, the knockout mice exhibit increased glucose tolerance with decreased hepatic glucose output, possibly due to phosphorylation and activation of AMP-activated protein kinase and suppression of gluconeogenic genes. In comparison, transgenic mice overexpressing a dominant negative form of resistin show increased adiposity with elevated leptin and adiponectin levels accompanying enhanced glucose tolerance and insulin sensitivity on both a chow and a high-fat diet.85 Although its underlying mechanisms need further elucidation, the in-vivo studies demonstrate a role of resistin obesity and insulin resistance.85 Angiotensinogen In addition to its importance in the regulation of blood pressure, the RAS contributes to adipose tissue development.10 Adipocytes secrete both angiotensinogen and angiotensin-converting enzyme. Inactivation of angiotensinogen in AGTK/K mice leads to lower fat mass and body weight, less weight gain under a high-fat diet, decreased lipogenesis and increased physical activity. 54 In contrast, overexpression of angiotensinogen causes an increase in body weight.86 The presence or absence of angiotensinogen correlates with the modification of adipocyte size, which is in accordance with in-vitro studies suggesting the control of fatty acid synthase by the RAS.10,54,86 Other adipocyte-secreted molecules and mediators of the endocrine response in fat Several other molecules may play a role in the metabolic response of adipose tissue including lipids, prostaglandins, leukocyte adhesion molecules and their receptors,

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vasoactive factors such as PAI-1, tissue factor, transforming growth factor b, IL-1b, metallothionins and other factors. The underlying mechanisms linking these molecules to adipose tissue biology have been reviewed elsewhere.10,11

INVESTIGATION OF TRANSCRIPTIONAL REGULATION OF ADIPOGENESIS IN TRANSGENIC ANIMALS Several classes of transcription factors and nuclear factors control adipocyte proliferation and differentiation (Figure 1). For in-vitro adipogenesis, C/EBPs and PPARs appear to be essential.10 However, in vivo, the regulation and control of adipocyte differentiation involves a more complex transcriptional network including other proteins and factors. Further studies, including transgenic animal models, are necessary to fully elucidate regulatory mechanisms of adipogenesis in vivo. C/EBPs Six isoforms of C/EBPs that play a role in the differentiation of several cell types including adipocytes have been characterized. In cell culture systems of adipogenesis (Figure 1), C/EBPb and d are transiently expressed at early stages of adipocyte A

Proliferation

Differentiation

Adipocyte

RXRα

B

PPARγ

C/EBPβ/ δ

C/EBPα

E2F Hormonal stimulation C

SREBP-1c

Leptin Day 0

D

FOXC2

Day 8

Adiponectin Control

Figure 1. The transcriptional control of proliferation and differentiation in adipogenesis. Different stages of in-vitro adipogenesis in 3T3L1 adipocytes are associated with morphological changes (A). (B) In parallel, the network of transcription factors controls the induction of gene expression of genes that characterize the terminal differentiated mature adipocyte including leptin (C) and adiponectin (D). Results of reversetranscriptase polymerase chain reaction for leptin and adiponectin mRNA expression on different days of adipocyte differentiation are compared with mature adipocytes (control). Modified from A. Bo¨ttnes.

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differentiation and have been shown to transactivate C/EBPa and PPARg genes, which are both required for the differentiation of pre-adipocytes to mature adipocytes.10,11 The inactivation of C/EBPb and d is lethal in most knockout mice shortly after birth; in surviving mice, it causes decreased fat pad weights with reduced adipocyte numbers due to the incompetence of embryonic fibroblasts to differentiate into adipocytes.87 Surprisingly, the inactivation of C/EBPb and d has no effect on fat cell size, and C/EBPa and PPARg gene expression. However, embryonic fibroblasts derived from C/EBPb and d knockout mice do not differentiate into adipocytes, most likely due to a lack of expression of C/EBPa and PPARg, suggesting that alternative pathways exist in vivo which may compensate for the lack of C/EBPb and d.10 The knockout of C/EBPa also causes neonatal death and is associated with hypoglycaemia due to decreased expression of glucose-6-phosphatase and PEPCK in liver.88 C/EBPa null mice do not accumulate lipid in BATand WAT, demonstrating that C/EBPa is required for adipogenesis. Insulin-stimulated glucose transport is absent in these mice which is, at least in part, explained by a decreased expression of IRs and insulin receptor substrate-1. As further evidence for cross-regulation between C/EBPa and PPARg, no endogenous induction of PPARg is observed in fibroblasts derived from C/EBPa null mice.10 PPARs A wide range of lipid factors including ceramides, gangliosides, sphingolipids, prostaglandins, eicosanoids and sterols are activators or ligands of PPARs.11 PPARg has been shown to play a critical role in adipocyte differentiation, especially since one isoform, PPARg 2, is almost exclusively expressed in adipose tissue.10 The homozygous deletion of PPARg is not compatible with life because the protein is an essential factor in cardiac and placental development, in addition to adipocyte differentiation.11 However, heterozygous PPARgC/- mice are resistant to obesity and the development of fatty liver under a high-fat diet.60 Moreover, these mice are protected from insulin resistance induced by hypercaloric feeding. Adipocytes from PPARgC/K mice have impaired lipid accumulation with decreased expression of C/EBPa, indicating that the cross-talk of these transcription factors is bi-directional.60 Moreover, data from further in-vitro studies demonstrate that re-introduction of PPARg into PPARg null cells restores adipocyte differentiation, whereas restored C/EBPa expression has no such effect on adipogenesis.89 These data strongly suggest that PPARg is the direct modulator of adipogenesis, while the primary role of C/EBPa is maintainance of PPARg levels.10 Transgenic mice lacking PPARa show a disruption of fatty acid oxidation in the liver with increased WAT deposition.90 The inactivation of the ubiquitously expressed PPARb, also named PPARd, leads to reduced gonadal fat pads. PPARb null mice are a little smaller than wild-type controls and develop otherwise normally.91 However, adipose-tisssue-selective reduction in PPARb does not cause alterations in adipose tissue, indicating that decreased fat mass in PPARb null mice is the consequence of its expression in tissues other than fat.10 Retinoid X receptors (RXRs) heterodimerize with PPARs to bind DNA and activate transcription. WAT expresses high levels of RXRa; however, the role of this molecule can not be investigated in vivo because RXRa null mice die in utero.92 Mice with an adipocyte disruption of RXRa display a similar phenotype of that of PPARgC/K mice,60 suggesting that heterodimers of PPARg/RXRa are essential for the development of adipocyte hypertrophy. Adipose-specific knockout of RXRa

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leads to an impaired increase in plasma free fatty acid levels during fasting.93 This model also revealed that RXRa is an important factor in fat mobilization.10 SREBPs SREBP-1c, which is also known as adipocyte determination and differentiation factor 1, is a transcription factor expressed in BAT, WAT, liver and kidney.10 SREBP-1c has been shown to regulate adipogenesis and to stimulate gene expression of genes involved in lipogenesis. More than 80% of SREBP-1c knockout mice die in utero at embryonic day 11.94 The few survivors appear to be normal with no effect on fat mass, gene expression in adipose tissue or adipogenesis.94 Unexpectedly, the overexpression of a constitutively active form of SREBP-1c specifically in adipose tissue causes lipodystrophy (Table 1).24 The expression of a dominant positive form of another member of the SREBP family, SREPB2, does not change lipogenic enzyme gene expression, but increases mRNA expression of LDL receptors and enzymes of cholesterol synthesis, suggesting a specific role of SREBP2 as an activator of cholesterol biosynthesis in adipose tissue.95 Forkhead box C2 Forkhead box C2 (FOXC2) is exclusively expressed in differentiated adipocytes of adult mice and humans and counteracts obesity, hypertriglyceridaemia and dietinduced insulin resistance.10,11 Disruption of FOXC2 causes embryonic or perinatal death with severe vascular and skeletal defects.96 Transgenic mice with selective overexpression in adipose tissue are lean because of reduced intra-abdominal fat depots (Table 3).48 Moreover, white adipocytes of these mice appear like brown fat cells with reduced size, multilocular droplets, increased mitochondria, and profound changes in WAT gene expression.48 These changes in WAT may increase thermogenic capacity and contribute to the lean phenotype as well as to the resistance of FOXC2-Tg mice against diet-induced obesity. FOXC2 has been suggested as a master regulator of adipocyte differentiation because transgenic overexpression of FOXC2 also causes increased expression of C/EBPa, PPARg and SREBP-1c.

CONCLUSIONS Transgenic animal models have influenced and challenged our understanding of adipose tissue biology remarkably. In the past years, many genetically modified animal models have revealed further insights into the role of adipose tissue in energy homeostasis, in endocrine function and in relation to other physiological systems. Continued generation and characterization of transgenic animal models will add pieces to the puzzle for a better understanding of the pathophysiology of adipose tissue, including obesity, lipodystrophy and insulin resistance. Transgenic animal models are valuable tools for the functional characterization of single genes in vivo, and for the search of unknown genes or unrecognized functions of genes. Nevertheless, transgenic animal models have limitations and the study of single genes independently through the generation of genetically modified animals only provides a fragmented view on complex physiological processes. The major limitations in studying

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transgenic animal models are that a phenotype can be masked by compensatory mechanisms for the loss or overexpression of individual proteins, and that it may be difficult to distinguish phenotypes arising from developmental defects from those resulting from gene targeting. Taken together, transgenic animal models with single genetic defects can yield important information on adipose tissue biology and pathophysiological states, but may not always match the human disease. Thus, it is important that new relevant animal models employing more sophisticated strategies for gene inactivation or overexpression are developed to extend our understanding of the mechanisms underlying obesity and its metabolic consequences. Finally, a better understanding of the function of adipose tissue biology will likely permit more rational approaches in the prevention and treatment of excess and deficiency of adipose tissue.

Practice points † more than 500 transgenic animals have contributed to a better understanding of adipose tissue biology † with the Cre/LoxP system, conditional transgenesis and knockout became available † adipose-tissue-specific knockouts are generated using Cre recombinase expressed under the control of the promoter for adipocyte lipid binding protein aP2 † complete absence of adipose tissue leads to neonatal lethality † genetic ablation of adipose tissue in lipodystrophy models causes insulin resistance, hepatic steatosis and diabetes † depletion of BAT causes decreased energy expenditure, hyperphagia, diabetes and hypertriglyceridaemia † disruption of GLUT4 in adipose tissue has no effect on fat mass, but causes impaired insulin action in muscle and liver † FIRKO mice have reduced fat mass and are protected from age- and hyperphagia-induced obesity and its associated metabolic alterations † knockout of lipoprotein lipase leads to neonatal death due to marked hypoglycaemia † mice deficient in HSL have normal fat content, despite adipocyte hypertrophy † deletion of all b-adrenergic receptors demonstrates that b-adrenergic signalling is required for diet-induced thermogenesis † leptin deficiency (ob/ob mice) causes extreme obesity, hyperphagia, hyperinsulinaemia, dyslipidaemia and other metabolic alterations † adiponectin/Acrp30 has insulin-sensitizing effects in vivo † TNFa may be involved in obesity-induced insulin resistance † the RAS contributes to adipose tissue development † C/EBPs and PPARs are essential for adipogenesis † knockout of C/EBPa, b and d, PPARg, RXRa, SREBP-1c and FOXC2 are lethal, suggesting that these proteins are essential factors in early development † FOXC2 seems to be a master regulator of adipocyte differentiation

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ACKNOWLEDGEMENTS This work was supported by a grant from the Deutsche Forschungsgemeinschaft, BL 580/3-1 and the Bundesministerium fu¨r Bildung und Forschung, Interdisciplinary Centre for Clinical Research at the University of Leipzig (01KS9504/1, project N03).

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