International Journal of Obesity (2004) 28, S38–S44 & 2004 Nature Publishing Group All rights reserved 0307-0565/04 $30.00 www.nature.com/ijo PAPER Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores

M Rossmeisl1, P Flachs1, P Brauner1, J Sponarova1, O Matejkova1, T Prazak1, J Ruzickova1, K Bardova1, O Kuda1 and J Kopecky1*

1Department of Adipose Tissue Biology, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

As indicated by in vitro studies, both lipogenesis and lipolysis in adipocytes depend on the cellular ATP levels. Ectopic expression of mitochondrial uncoupling protein 1 (UCP1) in the white adipose tissue of the aP2-Ucp1 transgenic mice reduced obesity induced by genetic or dietary manipulations. Furthermore, respiratory uncoupling lowered the cellular energy charge in adipocytes, while the synthesis of fatty acids (FA) was inhibited and their oxidation increased. Importantly, the complex metabolic changes triggered by ectopic UCP1 were associated with the activation of AMP-activated protein kinase (AMPK), a metabolic master switch, in adipocytes. Effects of several typical treatments that reduce adiposity, such as administration of leptin, b-adrenoceptor agonists, bezafibrate, dietary n-3 polyunsaturated FA or fasting, can be compared with a phenotype of the aP2-Ucp1 mice. These situations generally lead to the upregulation of mitochondrial UCPs and suppression of the cellular energy charge and FA synthesis in adipocytes. On the other hand, FA oxidation is increased. Moreover, it has been shown that AMPK in adipocytes can be activated by adipocyte-derived hormones leptin and adiponectin, and also by insulin-sensitizes thiazolidinediones. Thus, it is evident that metabolism of adipose tissue itself is important for the control of body fat content and that the cellular energy charge and AMPK are involved in the control of lipid metabolism in adipocytes. The reciprocal link between synthesis and oxidation of FA in adipocytes represents a prospective target for the new treatment strategies aimed at reducing obesity. International Journal of Obesity (2004) 28, S38–S44. doi:10.1038/sj.ijo.0802855

Keywords: adipose tissue; AMPK; transgenic mice; uncoupling protein

Introduction lism within individual tissues. Due to the complex nature of Obesity, an excessive accumulation of adipose tissue, leads to this process, any treatment strategy for obesity is difficult. various metabolic complications including cardiovascular Many pieces of evidence suggest that body fat content is disease, hypertension, dyslipidemia, and type II diabetes, controlled, at least partially, by the metabolism of adipose together designated as metabolic syndrome. The prevalence tissue itself. First, many candidate genes for obesity have of obesity has increased in industrialized countries and the important roles in adipocytes.5 Second, mice that are prone economic costs of obesity range from 2 to 7% of their total or resistant to obesity were created by transgenic modifica- health-care budget.1 In order to preserve energy stored as tion of adipose tissue (for a review, see Kopecky et al6). In triacylglycerols (TG) in adipose tissue, a number of regula- these transgenic models, metabolic changes in white adipose tory mechanisms have developed in order to help the body tissue are mostly responsible for the altered accretion of body maintain stable levels of energy stores.2–4 Such mechanisms fat and associated metabolic disorders. The characteristic involve the central control of behavior and thermogenesis feature associated with obesity and the pivotal factor in the mediated by neuroendocrine system, control of metabolite pathogenesis of type II diabetes is insulin resistance.7,8 fluxes among various organs, and, finally, energy metabo- Reduced ability of peripheral tissues, particularly skeletal muscle and liver, to respond to insulin stimulation can be detected many years before the clinical onset of hyperglyce- mia. In obese subjects, insulin resistance most likely results *Correspondence: Dr J Kopecky, Institute of Physiology, Academy of from an increased accumulation of lipids in peripheral Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic. tissues due to enhanced release of fatty acids (FA) from 9 E-mail: [email protected] hypertrophic fat cells. In fact, hypertrophic adipocytes Energy charge and AMP-activated protein kinase M Rossmeisl et al S39 themselves become resistant to insulin, which results in a UCP1 was capable of decreasing mitochondrial membrane lower clearance of plasma TG and higher FA release from potential in adipocytes24 and elevating oxygen consumption adipose tissue. It was observed that insulin resistance also two-fold.21 Importantly, mitochondrial biogenesis in uni- develops as the consequence of a lack of white adipose locular adipocytes of transgenic mice was induced, probably tissue.10,11 Under these circumstances, levels of circulating due to upregulation of the transcription factor nuclear FA are elevated due to insufficient ‘buffering’ capacity of respiratory factor (NRF-1).23 Since the total content of adipocytes for FA. Consequently, excess lipids are deposited transgenic UCP1 in white fat of adult mice did not exceed in other organs and insulin resistance develops.12–14 In 2% of UCP1 levels found in the interscapular brown fat of addition to FA, also various adipocyte-secreted proteins control mice,19 it is clear that only minute amounts of (‘adipokines’) like leptin, adiponectin, tumor necrosis factor ectopic UCP1 in unilocular adipocytes of white fat were a, interleukin-6, and resistin modulate sensitivity of other sufficient to uncouple oxidative phosphorylation24 and tissues to insulin and may be involved in the induction of reduce accumulation of body fat. systemic insulin resistance.15,16 Thus, adipose tissue meta- In agreement with a low oxidative capacity of white fat, bolism together with adipokines are involved in the control the increase in tissue oxygen consumption brought about by of systemic insulin sensitivity. transgenic UCP1 (see above) resulted in only a marginal Only a few laboratories have focused their research efforts stimulation of resting metabolic rate in transgenic mice.22 on the link between energy and lipid metabolism in white This suggests that, in addition to increased energy expendi- adipose tissue. In vitro studies demonstrate that a decrease in ture, there must be another way how transgenic UCP1 mitochondrial ATP production resulted in the inhibition of reduces adiposity. Indeed, a strong diminution of FA both FA synthesis17 and lipolytic action of catecholamines,18 synthesis was found in greatly reduced subcutaneous fat and in the enhancement of glycolysis due to respiratory depots of transgenic mice, while changes in gonadal fat were uncoupling in adipocytes.17 Without complementary in vivo less obvious.26 This probably reflects the magnitude of UCP1 studies, the significance of these findings would be limited. expression19,23 as well as changes in the ATP/ADP ratio. The We contributed to this topic through our studies on the latter was observed only in the subcutaneous but not mechanism, by which ectopic expression of mitochondrial gonadal fat depot of transgenic mice.25 Moreover, decreased uncoupling protein 1 (UCP1) in white fat of the aP2-Ucp1 FA synthesis was accompanied by a downregulation of transgenic mice protects against obesity. These studies acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS),26 clarified several aspects involved in the modulation of lipid and peroxisome proliferator-activated receptor g (PPARg)27 in metabolism in adipose tissue by cellular energy charge, as the white fat. Transgenic UCP1 lowered FA synthesis by well as the role of AMP-activated protein kinase (AMPK) in means of respiratory uncoupling, as confirmed by experi- this process. ments in vitro.17,26 Lipolysis and the activity of adipose tissue lipoprotein lipase (LPL) were also affected by the ectopic expression of UCP1 in white fat. For instance, the maximum lipolytic effect of noradrenaline was suppressed by 50% in Reduced adiposity due to respiratory uncoupling subcutaneous but not gonadal fat of transgenic animals. In in white fat of the aP2-Ucp1 transgenic mice parallel, UCP1 transgene caused a downregulation of Transgenic aP2-Ucp1 mice, in which the UCP1 gene is driven hormone-sensitive lipase (HSL), lowered its activity, and by the fat-specific aP2 promoter to achieve enhanced altered the expression of G-proteins in adipocytes.25 The LPL expression in both brown and white fat, represent an activity in gonadal fat was higher in the transgenic excellent model to study the effects of respiratory uncou- compared to control mice, especially when the animals were pling on fat accumulation in vivo.19 These animals are fed a high-fat diet.21 In agreement, we found that plasma TG partially resistant to obesity induced by age, genetic back- levels were significantly lower in transgenic mice.20 These ground,19 and high-fat diet.20,21 Their resistance to obesity data suggest that respiratory uncoupling in adipose tissue reflects lower accumulation of TG in all fat depots except for stimulates LPL-mediated clearance of TG. Phenotype of the gonadal fat, which becomes relatively large.19,20 Impor- aP2-Ucp1 transgenic mice indicates that the main physiolo- tantly, obesity resistance results exclusively from a trans- gical function of UCPs and respiratory uncoupling in white genic modification of white fat,6 since brown fat in these adipose tissue could be modulation of lipogenesis, oxidation mice is greatly atrophied.22 of substrates, lipolysis, and hormonal control of lipid Ectopic expression of transgenic UCP1 in white fat and its metabolism. consequences have been studied in great detail. All uni- locular adipocytes contained transgenic UCP1; however, the level of transgene expression differed in various fat depots with gonadal fat showing relatively low expression.19,23 This AMP-activated protein kinase may partially explain why the effect of transgene on lipid The broad range of metabolic effects triggered by ectopic accumulation was less pronounced in gonadal fat (see UCP1 in the white adipose tissue of the aP2-Ucp1 transgenic above). Nevertheless, even in this fat depot transgenic mice (see above) is stunning and makes one search for a

International Journal of Obesity Energy charge and AMP-activated protein kinase M Rossmeisl et al S40 common governing mechanism. In the white fat, similar to Role of UCPs in white adipose tissue other tissues, lipid and carbohydrate metabolism is modu- Similar to other cell types, mitochondria represent the main lated by AMPK, which serves as a metabolic master switch. In source of ATP in white adipocytes. Efficiency of ATP response to a fall in the cellular ATP/AMP ratio, AMPK synthesis during oxidative phosphorylation depends on a becomes activated through phosphorylation of its a-subunit proton leak through the inner mitochondrial membrane. by an upstream kinase. Activated AMPK then phosphorylates Experiments of Brand et al34 described basal proton leak in and inactivates a number of enzymes involved in biosyn- mitochondria as a general phenomenon. Several candidate thetic pathways, thus preventing further ATP utilization (for genes encoding for proteins that could enable the regulated a review, see Winder and Hardie28). In the skeletal muscle, proton leak are expressed in adipocytes, namely the genes for AMPK also upregulates NRF-1, which induces mitochondrial various UCPs. In the brown fat, UCP1, UCP2, UCP3, and biogenesis.29 In the liver, the inhibitory effect of AMPK on UCP5 genes are expressed, while in the white fat only UCP2 lipogenesis is indirect, being mediated by suppression of the and UCP5 genes are normally active.35,36 Similar to UCP1, transcription factor sterol regulatory element binding pro- UCP2 and UCP3 might also enhance proton leak, induce tein (SREBP-1),30 which upregulates genes engaged in respiratory uncoupling, and decrease ATP synthesis.36,37 It lipogenesis.31 In adipocytes, AMPK is known to inhibit both may be hypothesized that respiratory uncoupling in the lipolysis and lipogenesis by regulating directly the enzymes white fat would increase energy expenditure and thermo- engaged in lipid metabolism,32 as well as by downregulating genesis. However, the effect on energy balance may be PPARg expression.33 All the effects of transgenic UCP1 on the relatively small, since white fat contributes to the resting biochemical properties of white fat in the aP2-Ucp1 mice metabolic rate in humans by only about 5%.38 On the other seem to be in agreement with the activation of AMPK (see hand, a decrease in ATP synthesis due to respiratory above). Indeed, it was observed that UCP1-induced decrease uncoupling in white fat with a consequent rise in ADP and in the ATP/ADP ratio in the subcutaneous white fat of AMP levels may result in allosteric regulations of key transgenic mice25 was also associated with a reduced ATP/ enzymes of carbohydrate and lipid metabolism. AMP ratio, increased activity of AMPK, and enhanced Based on its function during adaptive thermogenesis in oxidation of FA.27 Less dramatic changes were also observed adipocytes of the brown fat, UCP1 is the only homologue in the gonadal fat of these mice.27 Thus, activation of AMPK with a proven uncoupling activity.39 Brown fat is present in by decreased intracellular energy charge in the white fat of all mammalian neonates, but in large nonhibernating aP2-Ucp1 mice explains the complexity of changes in adipose species its content is greatly reduced. However, some brown tissue metabolism observed in this obesity-resistant trans- adipocytes interspersed in white fat depots could be detected genic model (Figure 1). even in humans,40 especially in perirenal depot,41 and adult humans retain some capacity to induce brown adipocytes, as shown in patients with pheochromocytoma.42 In abdominal

Respiratory uncoupling fat, UCP1 mRNA levels are inversely related to the level of (UCP1) obesity,43 similarly to UCP2.44 Research efforts in many laboratories have focused on the biology of UCP1 expression in white adipose tissue as the possible means of reducing ATP/AMP adiposity. For instance, treatments with b3-adrenergic ago- nists or exposure of animals to a cold stress upregulated the Phosphorylation AMPK HSL Lipolysis UCP1 expression, induced brown adipocytes in white fat, 45,46 47 FAS and reduced adiposity in mice and rats. In rats, brown Lipogenesis adipocyte-like cells induced in white fat seem to have two SREBP-1 ACC Malonyl-CoA expression Expression origins; that is, most come from convertible unilocular white adipocytes and express UCP3, while the rest (B8%) comes 48 PPAR
from precursor cells and expresses UCP1. The induction of expression FA oxidation UCP1 in white fat depots, at least in mice, appears to be a 46 NRF-1 highly variable genetic trait and first analyses of this Mitochondrial biogenesis expression quantitative trait are underway.49,50 Importantly, human Figure 1 The key role of AMPK in metabolic effects of respiratory adipocytes in culture can be stimulated to express UCP1 gene uncoupling in the white fat of aP2-Ucp1 transgenic mice. Ectopically following the exposure to adrenergic agonists51 or thiazoli- expressed UCP1 leads to a fall in the cellular ATP/AMP ratio and stimulation dinediones (TZD).52 Adenovirus-mediated transfer of human of AMPK. In turn, AMPK phosphorylates and inactivates a number of enzymes involved in lipid metabolism, including HSL, ACC, and FAS. It also down- PPARg coactivator 1a (PGC-1a) into human subcutaneous regulates the expression of lipogenic genes, possibly through the effect on white adipocytes induced the expression of UCP1, respira- SREBP-1 and PPARg expression. This results in the inhibition of lipolysis and tory chain proteins, FA oxidation enzymes, and stimulated lipogenesis, while FA oxidation is increased probably due to low malonyl-CoA palmitate oxidation rate.53 The signaling pathways that levels. AMPK also upregulates transcription factor NRF-1, which stimulates mitochondrial biogenesis. Full arrows indicate the changes detected in the would orchestrate the induction of UCP1 and conversion of aP2-Ucp1 mice. See text for details. white adipocytes to fat-oxidizing brown fat-like cells in vivo

International Journal of Obesity Energy charge and AMP-activated protein kinase M Rossmeisl et al S41 are largely unknown, but a number of transcription factors glucose uptake into adipocytes by inducing AMPK.81 In and coactivators including PGC-1a54,55 and PPARd56 are rodents, TZD stimulate AMPK in adipose tissue,82 while certainly involved. Interestingly, a negative correlation parallel inductions of glycerol kinase83 and phosphoenol- between heat production in adipocytes and body fat content pyruvate carboxykinase84 presumably lead to a futile cycling has also been found.38 All these results suggest that white of FA within the cells. However, such mechanism may not adipocytes are capable of acquiring the features of brown operate in human patients treated with TZD.85 Recent adipocytes and could be directed toward a transcriptional studies even indicate the involvement of AMPK in the program linked to energy dissipation and in situ fat effects of physical exercise in adipose tissue.86 oxidation.

Conclusions Metabolism of adipose tissue as well as its secretory function Physiological relevance of energy metabolism and are deeply involved in the pathophysiology of metabolic AMPK in white adipocytes syndrome. It appears that the cellular energy charge of It is necessary to assess whether changes in energy metabo- adipocytes and AMPK contributes to the regulation of lism occur in adipocytes during treatments that affect adipose tissue metabolism. AMPK thus represents a switch adiposity, insulin resistance, and other components of the that might participate in the conversion of lipid-storing metabolic syndrome. In this respect, the effects of several white adipocytes to lipid-oxidizing cells, while suppressing in 57–61 typical treatments such as administration of leptin, situ lipogenesis. The reciprocal link between synthesis and 62,63 48,64–68 bezafibrate, adrenoreceptor agonists, dietary n-3 oxidation of FA in adipocytes may be a promising target for 69–71 66,72–74 polyunsaturated FA or fasting can be compared treatment strategies, including dietary manipulations and 20,21,26 with a phenotype of the aP2-Ucp1 transgenic mice. In pharmacological approaches, aimed at reducing adiposity all of these situations, fat accumulation is reduced and (Figure 2). Future studies should elucidate how adipocytes disturbances related to the metabolic syndrome are im- respond to the above-mentioned treatments with respect to proved. Importantly, in most cases the expression of various changes in their cellular energy charge and malonyl-CoA UCPs is upregulated, FA oxidation increased, in situ lipogen- levels, and the mechanisms of their metabolic adaptations. esis suppressed, and LPL-mediated clearance of TG in the New drugs developed to increase AMPK activity in the white fat is augmented. Opposite changes in the activity of muscle and liver may also reduce body fat stores by FA oxidation and lipogenesis probably reflect the elimina- activating AMPK in adipose tissue. tion of an inhibitory effect of malonyl-CoA on the transport of FA into mitochondrial matrix mediated by carnitine palmitoyl transferase-1 (CPT-1).75 In the white fat, due to a Acknowledgements low activity of CPT-1, FA oxidation is relatively slow and FAs This work was supported by the Grant Agency of the Czech are directed towards esterification,76 unless the oxidation is Republic (303/02/1220 and 303/03/P127), Grant Agency of activated by leptin77 and probably also by other treatments the Academy of Sciences of the Czech Republic (see above). Alterations in the energy charge of adipocytes, as (KJB5011303), and research project AVOZ 5011922. revealed by changes in the cellular ATP content and/or ATP/ ADP and ATP/AMP ratios, were detected not only in the aP2- Ucp1 mice,25,27 but also during fasting66 and after adminis- 64,66 Respiratory uncoupling Futile metabolic cycles ? tration of b-adrenergic agonists. ? Experiments in the aP2-Ucp1 transgenic mice strongly ? support a role of adipocyte AMPK in situations that lead to a ATP/AMP reduction of adiposity. Adenovirus-induced hyperleptinemia Lipogenesis in rats activates AMPK and induces mitochondrial biogenesis and transcription factors PGC-1a and FOXC2.61 Under these ? ? conditions, body fat is depleted, expression of UCP1 and ? AMPK ACC Malonyl-CoA UCP2 in white adipose tissue is upregulated, FA oxidation is increased, and expression of lipogenic genes is profoundly ? ? FA oxidation suppressed. In fact, the induction of UCPs by leptin in ? adipose tissue, leading to a drop in the intracellular energy charge, would partly explain the activation of AMPK. Correspondingly, in the skeletal muscle, AMPK mediates Adiposity the effects of leptin on lipid metabolism78,79 and antidiabetic drugs such as TZD stimulate AMPK by decreasing the ATP/ Figure 2 Schematic description of regulatory pathways reducing adiposity 80 by converting the adipocytes into lipid-oxidizing cells. Malonyl-CoA is AMP ratio. Furthermore, adipocyte-secreted hormone probably crucial for the reciprocal link between lipogenesis and FA oxidation. adiponectin with insulin-sensitizing properties increases See text for details.

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