Emerging aspects of pharmacotherapy for obesity and metabolic syndrome

Pharmacological Research 50 (2004) 453–469

Emerging aspects of pharmacotherapy for obesity and metabolic syndrome Enzo Nisoli a,b,∗ , Michele O. Carruba a,b a

Department of Preclinical Sciences, Center for Study and Research on Obesity, L. Sacco Hospital, University of Milan, LITA Vialba, via G.B. Grassi 74, 20157 Milan, Italy b Istituto Auxologico Italiano, Milan, Italy Accepted 9 February 2004

Abstract Obesity is a multifactorial, chronic disorder that has reached epidemic proportions in most industrialized countries and is threatening to become a global epidemic. Obese patients are at higher risk from coronary artery disease, hypertension, hyperlipidemia, diabetes mellitus, cancers, cerebrovascular accidents, osteoarthritis, restrictive pulmonary disease, and sleep apnoea. In particular, visceral fat accumulation is usually accompanied by insulin resistance or type 2 diabetes mellitus, hypertension, hypertriglyceridemia, high uremic acid levels, low high density lipoprotein (HDL) cholesterol to define a variously named syndrome or metabolic syndrome. Metabolic syndrome is now considered a major cardiovascular risk factor in a large percentage of population in worldwide. Both obesity and metabolic syndrome are particularly challenging clinical conditions to treat because of their complex pathophysiological basis. Indeed, body weight represents the integration of many biological and environmental components and relationships among fat and glucose tolerance or blood pressure are not completely understood. Efforts to develop innovative anti-obesity drugs, with benefits for metabolic syndrome, have been recently intensified. In general two distinct strategies can be adopted: first, to reduce energy intake; second, to increase energy expenditure. Here we review some among the most promising avenues in these two fields of drug therapy of obesity and, consequently, of metabolic syndrome. © 2004 Elsevier Ltd. All rights reserved. Keywords: Obesity; Anti-obesity drugs; Metabolic fitness; Obesity-related disorders; Metabolic syndrome; Leptin; Thermogenic drugs; Mitochondria

1. Introduction Obesity is a multifactorial, chronic disorder that has become a global epidemic (globesity) [1]. It is not just a concern for adults as the number of overweight/obese children and adolescents has doubled in the past 2–3 decades in the US [2]. Overweight children and adolescents are more likely to become overweight or obese adults [2,3]. Obese patients are at higher risk from coronary artery disease, hypertension, hyperlipidemia, diabetes mellitus, cancers, cerebrovascular accidents, osteoarthritis, restrictive pulmonary disease, and sleep apnoea [4,5]. The risk of morbidity and mortality increases with an increase in body weight beyond a body mass index (BMI) (weight in kg/height in m2 ) of 27 and with an increase in waist circumference (as an index of visceral localization of fat). Obesity reduces life expectancy [1]. In the Nurses’ Health Study, in which over 115,000 women 30–55 ∗ Corresponding author. Tel.: +39-02-50319682; fax: +39-02-50319683. E-mail address: [email protected] (E. Nisoli).

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years of age and without known cardiovascular disease were followed for 16 years, the risk of death was 60–70% higher among subjects with a BMI between 29 and 32 kg/m2 than among subjects with a BMI of 25–27 kg/m2 . These figures translate into 1260 excess lives lost per million women per year as a consequence of an average weight difference of only 13 kg (28 lbs) [6]. In the US, approximately 300,000 deaths a year are currently associated with overweight and obesity [3]. People with the metabolic syndrome are at increased risk for developing diabetes mellitus [7] and cardiovascular disease [8] as well as increased mortality from cardiovascular disease and all causes [9]. The recently released Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III; ATP III) draws attention to the importance of the metabolic syndrome and provides a working definition of this syndrome for the first time [10]. In addition, the report has drawn specific attention to the importance of this syndrome as a new target of risk-reduction therapy.

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A recent report suggested that the prevalence of the metabolic syndrome increases with age, affecting more than 40% of those older than 60 years [11]. Based on ageadjusted estimates, about a quarter of the population has the metabolic syndrome, representing about 47 million Americans [11]. As detailed in the ATP III report, participants having three or more of the following criteria were defined as having the metabolic syndrome: abdominal obesity (waist circumference >102 cm in men and >88 cm in women); hypertriglyceridemia (150 mg/dl or 1.69 mmol/l); low high density lipoprotein (HDL) cholesterol (<40 mg/dl (1.04 mmol/l) in men and <50 mg/dl (1.29 mmol/l) in women); high blood pressure (130/85 mmHg); high fasting glucose (110 mg/dl or 6.1 mmol/l). Williamson et al. [12] reported that the association between intentional weight loss and longevity in middle-aged overweight women appears to depend on their health status: in women with obesity-related health conditions (n = 15,069), intentional weight loss of any amount was associated with a 20% reduction in all-cause mortality, primarily due to a 40–50% reduction in mortality from obesity-related cancers; in women with no preexisting illness (n = 28,388), intentional weight loss of greater than or equal to 9.1 kg that occurred within the previous year was associated with about a 25% reduction in all-cause, cardiovascular, and cancer mortality; however, loss of <9.1 kg or loss that occurred over an interval of >1 year was generally associated with small to modest increases in mortality. More recently, to examine the relationships among intention to lose weight, weight loss, and all-cause mortality, a prospective cohort study using a probability sample of the US population was performed [13]. Participants were 6391 overweight and obese persons (BMI ≥ 25 kg/m2 ) who were at least 35 years of age. Intention to lose weight and weight change during the past year were assessed by self-report in 1989. Vital status was followed for 9 years. Hazard rate ratios (HRRs) were adjusted for age, sex, ethnicity, education, smoking, health status, health care utilization, and initial BMI. Compared with persons not trying to lose weight and reporting no weight change, those reporting intentional weight loss had a 24% lower mortality rate (HRR: 0.76 (95% CI, 0.60–0.97)) and those with unintentional weight loss had a 31% higher mortality rate (HRR: 1.31 (CI, 1.01–1.70)). However, mortality rates were lower in persons who reported trying to lose weight than those in not trying to lose weight, independent of actual weight change. Compared with persons not trying to lose weight and reporting no weight change, persons trying to lose weight had the following HRRs: no weight change, 0.80 (CI, 0.65–0.99); gained weight, 0.94 (CI, 0.65–1.37); lost weight, 0.76 (CI, 0.60–0.97). Thus, attempted weight loss was associated with lower all-cause mortality, independent of weight change. Self-reported intentional weight loss was associated with lower mortality rates, and weight loss was associated with higher mortality rates only if it is unintentional (see also Section 2).

2. Management of obesity and metabolic syndrome Obesity is a particularly challenging clinical condition to treat because of its complex pathophysiological basis. Indeed, body weight represents the integration of many biological and environmental components [14,15]. Rather than focusing primarily on body weight, many experts are focusing on the so-called “metabolic fitness”, that tracks the metabolic health of obese individuals. Metabolic fitness is defined as the absence of biochemical risk factors associated with obesity (elevated fasting concentrations of cholesterol, triglycerides, glucose, or insulin; impaired glucose tolerance; or elevated blood pressure). Thus, weight loss should be viewed as a modality to improve health [16]. Modest weight reduction, in the range of 5–10% of initial body weight, has been shown to improve obesity-related morbidity and mortality [1]. In women 40–60 years of age who had never smoked, moderate but intentional weight loss reduced all-cause mortality by 20% and diabetes-associated mortality by 30–40% [12]. Modest weight reduction has also been associated with clinically significant improvements in hypertension [17], lipid abnormalities [18] and glycaemic control [19,20]. Recently, the Finnish Diabetes Program [21] and the Diabetes Prevention Program [22] both reported that, in overweight patients losing approximately 5% of their body weight and increasing their physical activity the risk of developing type 2 diabetes was reduced by 58%. Caloric restriction, physical exercise and behavioral modification constitute the standard model for obesity treatment. Successful weight management implies not only initial weight loss over a short period of time, but also maintenance of reduced weight over a period of years. In most cases, dietary changes, exercise and behavioral modification, either alone or in combination, are generally met with poor long-term outcomes [23]. Pharmacological therapy is an adjunct to the treatment of obesity. Anti-obesity drugs must be used only in the context of a comprehensive management programme that includes the standard model. If physiological intervention does not induce weight loss after 6 months, the use of anti-obesity drugs may be considered for weight management in high-risk patients [24–26]. Thus anti-obesity drugs may have a role in weight reduction in patients whose condition is refractory to nonpharmacological measures and for the maintenance of weight loss. To date, agents for the management of obesity have been limited and unsatisfactory. Amphetamine have profound euphoric actions and carry the potential for abuse [27]. Phentermine has stimulant and sympathomimetic effects through catecholaminergic pathways [27]. Phenylpropanolamine-containing appetite suppressants have been associated with increased risk of hemorrhagic stroke in women [28], which resulted in their withdrawal by the U.S. Food and Drug Administration (FDA). Fenfluramine and dexfenfluramine, which are also centrally acting appetite suppressants, act predominantly by releasing serotonin. These agents were also withdrawn from the market

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because of their association with pulmonary hypertension and heart valve damage [28,29]. Sibutramine and orlistat, two newer therapies with relatively favorable efficacy and safety profiles, have been approved for weight management in conjunction with lifestyle modifications [161]. These mentioned drugs remain “symptomatic” and they are not “curative” drugs. Thus, exploding research has been finding many new potential drug targets. This review will focus on central and peripheral molecular processes involved in the maintenance of energy balance.

3. Central and peripheral processes that control energy balance Both central and peripheral mechanisms are involved in energy balance. In particular, fat depots are able to storage energy as fatty acids in response to fuel availability, and this information can be transfer to brain centers by means of different molecules. In particular, the production of the hormone leptin by adipose tissue is regulated by energy balance. When energy (i.e. fat) stores are replete, leptin production is high. Conversely, leptin production is inhibited when energy stores are depleted during, for example, prolonged fasting [30,31]. Circulating leptin levels thus reflect the status of body energy reserves, and energy balance throughout the body is regulated via leptin-mediated control of processes that are involved in energy intake and utilization. Thus, the fall in leptin levels when energy stores are inadequate (e.g. during starvation) enhances appetite and decreases energy utilization. By contrast, when energy stores are adequate, high leptin levels decrease the drive to eat and enable utilization of energy by the systems described below. There are multiple leptin receptor (LR) isoforms, which result from alternative mRNA splicing of the transcript of the lepr gene and/or from proteolytic processing of the subsequent protein products [32,33]. The lepr gene contains 17 common exons and several alternatively spliced exons. In mice, the five distinct LR isoforms that have been identified are designated LRa–LRe. In all species, LR isoforms can be divided into three classes: secreted, short and long. The secreted forms are either alternative splice products (e.g. murine LRe, which contains only the first 14 exons of lepr) or proteolytic cleavage products of membrane-bound forms of LR. These secreted forms contain only extracellular domains that bind circulating leptin, perhaps regulating the concentration of free leptin [34]. Short forms (LRa, LRc and LRd in mice) and the long form (LRb in mice) contain exons 1–17 of lepr and therefore have identical extracellular and transmembrane domains as well as the same first 29 intracellular amino acids, but then diverge in sequence because of alternative splicing of 3 exons. LRb is crucial for leptin action. Indeed, the db/db mice described originally lack only LRb because of a mutation that causes mis-splicing of LRb mRNA, but have a phenotype

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that is indistinguishable from that of db3J /db3J mice (which are deficient in all LR isoforms) and of leptin-deficient ob/ob animals [30–33]. The function of short-form LRs is less clear, although proposed roles include the transport of leptin across the blood–brain barrier [35,36]. Many of the effects of leptin are attributed to effects in the CNS, particularly in the basomedial hypothalamus, the site of highest LRb mRNA expression [37–39]. LRb is present with the highest levels in neurons of the nuclei of the basomedial hypothalamus, including the arcuate (ARC), dorsomedial hypothalamic (DMH) and ventromedial hypothalamic (VMH) nuclei [38,39]. Chemical and physical ablation of these nuclei results in increased feeding and neuroendocrine abnormalities that are similar to the phenotypes of db/db and ob/ob mice. This indicates that these hypothalamic nuclei, which make up the so-called ‘satiety center’, are crucial sites of leptin action [37,40]. Within these basomedial hypothalamic nuclei, LRb mRNA is expressed most highly in the ARC, where it is found in at least two distinct populations of neurons (Fig. 1). One population synthesizes neuropeptide Y (NPY) and agouti-related peptide (AgRP) and the other synthesizes pro-opiomelanocortin (POMC) [37,40]. POMC is processed to produce the powerful anorectic (appetite-suppressing) peptide ␣-melanocyte-stimulating hormone (␣-MSH) in LRb/POMC neurons. LRb stimulates the synthesis of POMC and activates LRb/POMC neurons [40,41]. AgRP inhibits ␣-MSH signaling and NPY is an orexigenic (appetite-stimulating) hormone that also suppresses the central LRb-mediated growth and reproductive axes [42–45]. Leptin acts via LRb to inhibit NPY/AgRP neurons and suppress expression of these neuropeptides. Thus, LRb signaling stimulates the production of anorectic neuropeptides and suppresses levels of orexigenic peptides. Conversely, a decrease or deficiency in leptin activity (e.g. during starvation and in ob/ob and db/db mice) stimulates appetite by suppressing synthesis of anorectic neuropeptides (e.g. POMC) and increasing expression of orexigenic peptides (e.g. NPY and AgRP) [37,40].

4. Interventions to reduce energy intake 4.1. Leptin and leptin receptor The complete congenital absence of the adipocyte-derived hormone leptin leads to a syndrome of intense hyperphagia and morbid obesity in humans [46,47] and rodents [48], which can be reversed by the administration of recombinant leptin [49,50]. So, for children with the rare congenital leptin deficiency, recombinant leptin represents a pronounced and, indeed, life-saving therapy. Unfortunately, severe leptin deficiency is not often found in human obesity, and leptin supplementation in obese individuals seems to have only modest effects on fat mass [51]. Given that they are fat, but

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Fig. 1. Diagrams of rat brain, showing major hypothalamic regions implicated in adiposity signaling and regulation of food intake. The small figure at the top is a longitudinal view of a rat brain, with olfactory bulb at the anterior end on the left and the caudal hindbrain on the right. Cross-sections of the brain at two levels (indicated by vertical lines) are shown at middle (left and right). First-order neurons responding to adiposity signals are located in the arcuate nucleus (ARC) and project anteriorly to the PVN as well as the PFH. Other regions implicated in regulating food intake include the ventromedial nucleus (VMN) and dorsomedial nucleus (DMN). Abbreviations of brain structures: AM, amygdala; CC, corpus callosum; CCX, cerebral cortex; HI, hippocampus; OC, optic chiasm; SE, septum; TH, thalamus; 3V, third ventricle. NPY/AGRP and POMC/CART neurons in the ARC (bottom, right), adjacent to the third ventricle, are first-order neurons in the hypothalamic response to the circulating adiposity signals insulin and leptin. NPY and POMC are expressed in discrete populations of arcuate nucleus neurons. NPY release in the PVN and LHA/PFA regions stimulates eating, whereas release of ␣-MSH (derived from POMC) in the PVN has an anorexic effect. Locations of candidate second-order neurons (bottom, left) involved in the hypothalamic response to insulin and leptin adiposity signaling. Second-order neurons include those that express TRH and CRH in the PVN (which cause anorexia), and neurons that express orexins and MCH in the PFA and LHA (which increase feeding).

generally have high plasma levels of leptin, obese subjects can be considered to be ‘leptin resistant’ [50]. There is some evidence for impaired transport of leptin into the CNS in states of leptin resistance [52]. But resistance is also likely to be occurring at the level of neurons in the CNS that express the leptin receptor, and their downstream targets. The recent observation that subjects who are heterozygous for leptin mutations have modest reductions in plasma leptin and show increased fat mass [53], provides some hope that there might be a subgroup of subjects with relative hypoleptinaemia who might respond to leptin supplementation.

Leptin passes through the blood–brain barrier and enters the brain. The problems of peripheral delivery and CNS penetration for agonists of the leptin receptor could, of course, be solved by using a small-molecule agonist for the leptin receptor. An alternative approach has been to search for ligands that mimic the actions of leptin and might be able to either overcome or bypass leptin resistance. One such molecule is ciliary neurotrophic factor (CNTF) [54] and its analogue axokine. These molecules seem to activate signal-transduction mechanisms within the hypothalamus similar to those that

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are activated by leptin. In contrast to leptin, these agents cause weight loss in animals with mutated leptin receptors, and also seem to be effective in diet-induced obesity [55]. 4.2. Small molecules insulin mimetics Insulin has been suggested to function as one of the adiposity signals to the brain for modulation of energy balance. Administration of insulin into the brain reduces food intake and body weight [15], and mice with a genetic deletion of neuronal insulin receptors are hyperphagic and obese [56]. However, insulin is also an anabolic factor; when administered systemically, pharmacological levels of insulin are associated with body weight gain in patients [57]. Air et al. [58] investigated the efficacy and feasibility of small molecule insulin mimetic compounds (Cpd1 and Cpd2) [59,60] to regulate key parameters of energy homeostasis. Central intracerebroventricular (i.c.v.) administration of an insulin mimetic resulted in a dose-dependent reduction of food intake and body weight in rats, and altered the expression of hypothalamic genes known to regulate food intake and body weight. In particular, POMC expression, normally suppressed by food deprivation, increased 183% in fasted, Cpd1-treated animals compared to those treated with vehicle [58]. Neuropeptide Y (NPY) expression, normally elevated in fasted animals, was suppressed by Cpd1 to 38% of fasted, vehicle-treated animals [58]. This effect is larger than that seen following insulin administration [61]. These data indicate that Cpd1 reduces food intake and body weight, at least in part, by upregulating the catabolic melanocortin system and downregulating the anabolic NPY system in the hypothalamus. Oral administration of a mimetic in a mouse model of high-fat diet-induced obesity reduced body weight gain, adiposity and insulin resistance. Thus, insulin mimetics have a unique advantage over insulin in the control of body weight and hold potential as a novel anti-obesity treatment. 4.3. Pro-opiomelanocortin and melanocortin receptors As previously mentioned a large body of both genetic and pharmacological research clearly implicates hypothalamic neurons that express pro-opiomelanocortin (POMC) as key elements in the control of appetite and energy balance [62]. A role for melanocortin signaling in the control of energy homeostasis first emerged after the cloning of the MC3- and MC4-receptor genes and the demonstration that they are expressed primarily in the brain [63]. This discovery was followed by evidence that a synthetic agonist of these receptors suppresses food intake, whereas a synthetic antagonist has the opposite effect [42]. The report that mice lacking the MC4 receptor (owing to gene targeting) are hyperphagic and very obese [64] indicates that tonic signaling by MC4 receptors limits food intake and body fat mass. Mice heterozygous for the deleted MC4 allele also become obese, although less so than homozygous knockouts [64]. Lack of a full complement of central MC4 receptors, there-

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fore, predisposes to hyperphagia and pathological weight gain. This finding has since been extended to humans with MC4-receptor mutations [65–67]. The subsequent cloning of the Agrp gene [68] identified a peptide, AGRP, with homology to agouti that is an antagonist of MC3 and MC4 receptors [69]. The demonstration that hypothalamic AGRP expression, like that of NPY and POMC, is localized to ARC, and that it is upregulated by fasting and by leptin deficiency, indicates that antagonism of CNS melanocortin receptors is important in body-weight regulation (see [40]). Consistent with its role as an anabolic signaling molecule, AGRP causes hyperphagia when administered i.c.v. [70] or expressed transgenically [40], and the increase of food intake following a single i.c.v. injection of AGRP is sustained for up to a week [70]. Thus, the central role of the MC4 receptor in controlling body weight makes it a very attractive target for anti-obesity drug development. Interesting at this point it is the observation that i.c.v. infusion of ␣-MSH at a dose below that needed to reduce food intake, both insulin-stimulated glucose disposal and insulin-induced suppression of hepatic glucose production were increased [71]. Increased neuronal melanocortin receptor signaling is therefore sufficient to increase insulin sensitivity in vivo, although it remains to be determined whether this effect occurs independently of melanocortin-induced decreases of body adiposity. 4.4. Neuropeptide Y and its receptors NPY is one of the most abundant peptides of the hypothalamus and, when administered centrally, has the powerful and sustained effect of increasing food intake [72]. Conversely, reduction in endogenous NPY by means of antisense-oligonucleotide [73] or immunoneutralization techniques [74] leads to a decrease in food intake. NPYergic neurons are present in the ARC, where they co-express Agrp and are negatively regulated by leptin. Paradoxically for the receptors of an orexigenic factor, mice that lack Y1 , Y2 and Y5 receptors all develop mild late-onset obesity [75–77]. Npy1r- and Npy2r-deficient animals [72–77] show a normal response to the orexigenic effects of NPY, whereas the Npy5r knockout mouse [78] has a greatly reduced feeding response to NPY administration. Despite the discrepancy between pharmacological and genetic studies, Y1 - and Y5 -receptor antagonists remain attractive candidates for drug development, and several such compounds are now in development. 4.5. Other neuropeptides/receptors There are an increasing number of neuropeptides for which a central role in the control of food intake and energy balance has been proposed (Table 1). In all cases, there is good evidence that the particular peptide is synthesized in and/or acts on the hypothalamus. Furthermore, all of these peptides have been shown to reduce food intake after acute

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Table 1 Peptides and neurotransmitters affecting feeding behavior Peptide/neurotransmitter

Acute effects on feeding

Chronic effects on feeding

Leptin ␣-MSH CART GLP1 CRF Urocortin Neuromedin U CCK IL-6 CNTF PYY3–36 Oxyntomodulin AGRP NPY MCH Galanin Ghrelin Endocannabinoids Orexins

− − − − − − − − − − − − + + + + + + +

− − − − − ND ND ND ND − ND ND + + + ND + ND +/−

−: Inhibition of food intake; +: stimulation of food intake; ND: experiment has not been done.

administration. However, in contrast to the situation with leptin and POMC, in few of the cases described below are there either compelling genetic experiments or convincing results from long-term administration of the peptides on fat mass. (i) CART and its receptor (yet to be identified). Fasted animals show a decrease in expression of CART (cocaine- and amphetamine-regulated transcript) messenger RNA in the ARC [79]. However, the CART knockout mouse does not become obese on a normal diet [80]. In addition, CART has effects on the control of movement, which could complicate food-intake studies [81]. Initial evidence suggested an anorexigenic role for this peptide and decreased hypothalamic CART expression was found in animal models with an obese phenotype [79]. However, recent data challenged previous observations on the appetite-inhibiting effect of CART, showing that intrahypothalamic injection of the peptide might even increase food intake [82]. (ii) Corticotrophin-releasing factor (CRF) and urocortin, and their receptors. Although the effects of CRF and urocortin on appetite seem compelling [83], it is notable that deletion of neither the CRF1 nor the CRF2 receptor [84,85] has any pronounced effect on body weight. (iii) GLP-1. The glucagon-like peptide 1 (GLP1) is a product of post-translational modification of proglucagon in the pancreas, intestinal cells and the CNS [86]. Intracerebroventricular GLP1 suppresses appetite and, more impressively, i.c.v. administration of a GLP1 antagonist increases food intake [87]. However, animals that are deficient in the GLP1 receptor show no systematic alteration in mass of body fat [88].

(iv) IL-6. Interleukin-6 (IL-6) is expressed both in adipose tissue and centrally in the hypothalamus. IL-6-deficient mice develop mature-onset obesity that is partially reversed by central IL-6 administration [89]. (v) BDNF. Brain-derived neurotrophic factor (BDNF) was studied initially for its role in the development of sensory neurons. Homozygous Bdnf-deficient mice die soon after birth, but heterozygous knockout mice show a tendency towards obesity. However, these mice also have abnormalities in locomotor activity, which would have important implications for any attempt to develop BDNF mimetics [90]. (vi) Other peptides. Finally, other peptide–receptor systems that might be worthwhile targets for anorexic drugs include the prolactin-releasing peptide and its receptor GRP10 (G-protein-coupled receptor 10) [91], as well as neuromedin U and the Neuromedin U receptor 2 (FM4), which is selectively expressed in brain [92], and oxyntomodulin, which is released from the gut postprandially in proportion to energy intake [93]. 4.6. Melanin-concentrating hormone and its receptors The melanin-concentrating hormone (MCH) is an orexigenic neuropeptide that is found in the lateral hypothalamus [94]. The Mch transcript is upregulated in the hypothalamus of leptin-deficient (ob/ob) mice, and fasting further increases this expression in both normal and ob/ob mice [95]. Intracerebroventricular injections of MCH into normal rats lead to a marked increase in food intake [95]. Borosky et al. [96] have recently reported that SNAP-7941, a selective, high-affinity MCH1 receptor (MCH1-R) antagonist, inhibited food intake stimulated by central administration of MCH, reduced consumption of palatable food, and, after chronic administration to rats with diet-induced obesity, resulted in a marked, sustained decrease in body weight. Interestingly, besides these effects on food intake, SNAP-7941 produced effects similar to clinically used antidepressants and anxiolytics in three animal models of depression/anxiety. Given these observations, an MCH1-R antagonist may be useful not only in the management of obesity but also as a treatment for depression and/or anxiety. 4.7. Ghrelin and its receptor Great excitement has been engendered by the discovery that the stomach can secrete a peptide, termed ghrelin [97], which, on administration, whether peripherally or centrally, can increase food intake in rodents [98]. Consistent with a role in initiating feeding, ghrelin levels are increased by fasting and decreased after a meal [99]. The feeding response to ghrelin seems to be dependent on NPY and AGRP, as central administration of neutralizing antibodies or antagonists to these agents abolishes the acute effect of ghrelin on feeding [98]. Agonists that interfere with the action of ghrelin on its receptor and inverse agonists

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that modulate ghrelin receptor activity might be candidates for anti-obesity molecules [162], although impairment of growth-hormone secretion, with resultant adverse effects on metabolism, is a potential risk.

tor down-regulation may limit the efficacy of prolonged PYY3–36 administration.

4.8. PYY3–36

The appetite-stimulating effect of marijuana in humans has been well known for centuries [104]. Several reports have demonstrated that administration of cannabinoids stimulates food intake in animal models [105,106]. Both peripheral and central administration of anandamide, one of the major endocannabinoids, increase food intake in rodents [107–109]. On the basis of the observation that cannabinoid receptor 1 (CB1) and endocannabinoids are present in the brain regions controlling food intake [110], the endogenous cannabinoid system has been proposed as a putative modulator of feeding behavior [111,112]. This concept has been further substantiated by the use of specific CB1 antagonists, which provided evidence for the role of CB1 in mediating the orexigenic effect of exogenous or endogenous cannabinoids [113]. Importantly, the levels of hypothalamic endocannabinoids were shown to be decreased after leptin administration, and the blockade of CB1 was demonstrated to inhibit starvation-induced hyperphagia in mice [114]. However, the phenotype of neurons in the hypothalamic appetite center that are directly affected by cannabinoids had not been elucidated. Cota et al. [115] have recently provided compelling evidence that endocannabinoids act at various regions of the hypothalamus that regulate energy homeostasis, including the lateral hypothalamus, ARC, and paraventricular nucleus, in conjunction with direct effects in fat cells on lipogenesis to promote a positive energy balance. If the receptor, CB1, is blocked (in this case by knockout of the gene), the overall effect will be to decrease appetite and lipogenesis in white fat: CB−/− animals are leaner because their caloric intake is lower when they are young. However, in older animals, increased peripheral energy expenditure appears to be the predominant defense against adiposity in these animals compared with their wild-type littermates [115]. Cota et al. have shown that CB1 is expressed in key hypothalamic peptidergic systems of appetite regulation, including those producing CRH in the paraventricular nucleus, CART in the ARC, and MCH and orexin in the lateral hypothalamus–perifornical region. Therefore, it is likely that by modulating food intake and mechanisms regulating energy expenditure, endocannabinoids or their antagonists will affect the signaling flow between these hypothalamic circuits and other neuronal networks with which these peptidergic systems interact. This central mechanism may dominate cannabinoid effects at younger ages but appears to diminish with age [115]. Whether it is chronological aging per se or other developmental processes, for example, puberty, that affects the function of relevant hypothalamic circuitry and thereby enables this impairment of cannabinoid signaling still needs to be clarified. In addition, it must be determined whether such a shift in endocannabinoid signaling during the aging process also occurs in hu-

The NPY Y2 receptor (Y2R), a putative inhibitory presynaptic receptor, is highly expressed on NPY neurons. As we seen, NPY is the most potent orexigenic hypothalamic petide, and NPY neurons are in the ARC, which is accessible to peripheral hormones. Peptide YY3–36 (PYY3–36 ), a Y2R agonist, is released from the gastrointestinal tract postprandially in proportion to the calorie content of a meal [100]. Batterham et al. [101] have recently shown that peripheral injection of PYY3–36 in rats inhibits food intake and reduces weight gain. PYY3–36 also inhibits food intake in mice but not in Y2r-null mice, which suggests that the anorectic effect requires the Y2R. Peripheral administration of PYY3–36 increases c-Fos immunoreactivity in the ARC and decreases hypothalamic NPY messenger RNA. Intra-arcuate injection of PYY3–36 inhibits food intake. PYY3–36 also inhibits electrical activity of NPY nerve terminals, thus activating adjacent POMC neurons [102]. In humans, infusion of normal postprandial concentrations of PYY3–36 significantly decreases appetite and reduces food intake by 33% over 24 h [101]. These results suggest that a gut–hypothalamic pathway that involves postprandial PYY3–36 acting at the arcuate Y2R has a role in regulating feeding. Thus, the PYY3–36 system may provide a therapeutic target for the treatment of obesity. Given, however, that the majority of obese subjects are resistant to the effects of leptin, the effects of PYY3–36 infusion on appetite and food intake were compared in obese and lean subjects. Batterham et al. [103] have found more recently that a single infusion of PYY3–36 , as compared with an infusion of saline, reduced appetite and food consumption by approximately 30% at an all-you-want-to-eat buffet lunch provided 2 h after the infusion. In the obese subjects, the endogenous postprandial PYY3–36 response was diminished as compared with that in the lean subjects, even though the obese subjects consumed a greater number of calories. The PYY3–36 infusion reduced hunger in both the obese and lean groups and had no effect on subjects’ reports of the palatability of the meals or their feelings of nausea. The study by Batterham et al. [103] also shows that infusion of PYY3–36 decreases fasting concentrations of the orexigenic peptide ghrelin. However, the extent to which suppression of ghrelin secretion contributes to a PYY3–36 -mediated reduction in food intake is unclear. Although single intraperitoneal injections of PYY3–36 decrease food intake for up to 7 days in rats, the results of a single infusion in humans cannot be extrapolated to predict long-term outcomes. The use of PYY3–36 may prevent counterregulatory mechanisms from overriding the stimulation of anorexigenic pathways. However, the development of antibodies or tachyphylaxis through recep-

4.9. Endocannabinoids and CB1 cannabinoid receptor

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mans. Nevertheless, the observation that CB1 is present in adipocytes and that CB1 activation affects lipogenesis provides an alternative and/or complementary mechanism for the unidirectional effect of cannabinoids on energy balance. 4.10. FAS inhibitors An important step toward development of drugs to treat obesity was the serendipitous discovery that fatty acid synthase (FAS) inhibitors, originally developed to treat cancer, including cerulenin, produce profound weight loss and reduction in food intake [116]. Despite these promising results, mechanisms mediating the weight-reducing effects of FAS inhibitors remained to be elucidated. A particularly intriguing finding was that FAS inhibitors are more potent in obese than in lean mice. This observation suggested that FAS inhibitors may enhance the effects of factors such as insulin, glucose and fatty acids, which are elevated in obese individuals and act through hypothalamic mechanisms to reduce body weight. A common nutrition-sensing pathway through which these factors act might involve the stimulation of acetyl CoA carboxylase through a PI(3) kinase-dependent pathway [117], which appears to mediate some effects of leptin [118] and insulin [119]. In turn, increased acetyl CoA

carboxylase activity stimulated malonyl CoA production. Hypothalamic responses to glucose appeared to be mediated through a mechanism that would also lead to enhanced production of malonyl CoA [120]. The accumulation of hypothalamic fatty acids, which produce profound metabolic effects [121], was also enhanced by malonyl CoA because this inhibits the enzyme carnitine palmitoyltransferase-1 (CPT1), which is required for entry of long-chain fatty acids (LCFAs) into mitochondria, where they are oxidized. Although a single injection of C75 could influence NPY and POMC mRNA [122], the chronic effects of FAS inhibitors appeared to be independent of at least these known hypothalamic regulators of body weight, suggesting that as yet undiscovered hypothalamic neuropeptides or systems mediated these chronic effects. Recently, Obici et al. [123] have proposed that the mechanism of action of FAS inhibitors does not simply involve increasing concentrations of malonyl-CoA that subsequently inhibit the flux of LCFAs into the mitochondria. Instead, the critical component may be an intracellular increase in LCFAs themselves that then induces a still ill-defined sensing mechanism in hypothalamic neurons (Fig. 2). This is in agreement with their previous observations that hypothalamic infusions of fatty acids decrease food intake and glucose production. The authors

Fig. 2. Neurons in the hypothalamus (in particular, the arcuate nucleus) are primary targets of a number of key hormones and metabolic cues. (Enlargement) Proposed model for function of CPT1 in hypothalamic regulation of food intake. Anorectic drugs such as fatty acid synthase (FAS) inhibitors increase malonyl-CoA, which is derived from the carboxylation of acetyl-CoA by the enzyme acetyl-CoA carboxylase (ACC). Large amounts of malonyl-CoA inhibit CPT1-dependent oxidation of LCFA-CoA molecules. ICV administration of exogenous fatty acids (LCFAs) directly increases cellular LCFA-CoA. The resulting increase in intracellular LCFA-CoA concentration leads to inhibition of feeding behavior.

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go on to show that increases in LFCAs not only decrease food intake, but also reduce glucose production by the liver. These results and other recent findings suggest that manipulation or dysregulation of hypothalamic pathways can induce hallmarks of type 2 diabetes: hyperinsulinemia, decreased glucose uptake and increased hepatic glucose production. How might LCFAs regulate cellular events? That remains to be determined, but recent studies have indicated that fatty acids act as membrane ligands of G-protein-coupled receptors [124]. Whatever the molecular mechanism, LCFAs may now be viewed as CNS signaling molecules, and FAS and CPT1 as potential pharmaceutical targets for the treatment of obesity and diabetes. Indeed, the potent orexigenic effects of cannabinoids and the potent anorectic effects of CB1 receptor antagonism may also be partly mediated through modulation of hypothalamic CPT1 activity and of LCFA-CoA. In fact, endocannabinoids stimulate CPT1 activity and fatty acid oxidation in cultured astrocytes independently of malonyl-CoA and through interaction with CB1 receptors [114]. Thus, central inhibition of fatty acid oxidation may represent an innovative approach to the prevention and treatment of obesity and type 2 diabetes mellitus. 4.11. Oleylethanolamide Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signaling. However, OEA does not activate cannabinoid receptors and its biological functions remained unknown for long time. It was demonstrated that, in rats, food deprivation markedly reduced OEA biosynthesis in the small intestine [125]. Moreover, administration of OEA caused a potent and persistent decrease in food intake and gain in body mass. This anorexic effect was behaviorally selective and was associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA did not affect food intake when injected into the brain ventricles, and its anorexic actions were prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results suggested that OEA is a lipid mediator involved in the peripheral regulation of feeding. Indeed, in a more recent work the same authors have showed that OEA binds with high affinity to the peroxisomeproliferator-activated receptor-␣ (PPAR-␣), a nuclear receptor that regulates several aspects of lipid metabolism [126]. Administration of OEA produces satiety and reduces body weight gain in wild-type mice, but not in mice deficient in PPAR-␣. In the small intestine of wild-type but not PPAR-␣-null mice, OEA regulates the expression of several PPAR-␣ target genes: it initiates the transcription of proteins involved in lipid metabolism and represses inducible nitric oxide synthase (iNOS), an enzyme that may contribute to

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feeding stimulation [127]. These results, which show that OEA induces satiety by activating PPAR-␣, identify an unexpected role for this nuclear receptor in regulating behavior, and raise possibilities for the treatment of eating disorders.

5. Interventions to increase energy expenditure Two types of adipose tissue are present in mammals: white adipose tissue (WAT) stores energy as triglycerides, and brown adipose tissue (BAT) burns energy for heat production (adaptive thermogenesis). In situations of energy deficit, such as fasting, lipolysis in WAT controls the supply of energy to the body through the release of fatty acids into the plasma. BAT is specialized in adaptive thermogenesis, the part of energy expenditure induced by cold exposure or diet. Fatty acid oxidation and heat production by brown adipose cells are due to intense metabolic activity because of the presence of a large number of mitochondria, and the expression of uncoupling protein 1 (UCP1) [128]. UCP1 allows the dissipation of the proton electrochemical gradient generated by the mitochondrial respiratory chain. Uncoupling between oxygen consumption and ATP synthesis promotes energy dissipation as heat. In neonatal mammals, hibernators and rodents, cold-induced thermogenesis in BAT contributes to the maintenance of body temperature. Fuel is provided as fatty acids from BAT and WAT lipolysis. Thus, two potential drug strategy interventions for the control of fat mass and obesity could be to increase BAT thermogenesis or to induce conversion from white to brown adipocytes. BAT is present throughout the lifespan in rodents but disappears soon after birth in large mammals. In humans, there are no BAT depots in adults and UCP1 mRNA is expressed at low levels in WAT [129]. BAT is not thought to contribute significantly to thermogenesis [130]. However, UCP1 is expressed in hibernomas and in perirenal WAT of adult patients with phaeochromocytoma and primary aldosteronism, revealing that UCP1 expression can be induced in rare tumors and endocrinological disorders [131]. Appearance of brown adipocytes is therefore possible in certain conditions in adults. The thermogenetic relevance of ␤3 -adrenergic receptors, which are selectively expressed in brown adipocytes, has been widely shown in laboratory animals and ␤3 -adrenergic receptor agonists have been proposed as anti-obesity drugs [132]. Recently, Bachman et al. [133] provide direct evidence that ␤-adrenergic receptors directly mediate the stimulatory activity of sympathetic nervous system (SNS) on diet-induced thermogenesis. These authors reveal that mice lacking all three ␤-adrenergic receptors (␤1 AR, ␤2 AR, and ␤3 AR) cannot increase thermogenesis and become massively obese during overfeeding. In contrast to these “␤-less” mice, wild-type mice are able to resist obesity during overfeeding by activating diet-induced thermogenesis. The ␤-less mice are also intolerant to cold exposure, suggesting that the SNS and ␤-adrenergic receptor signaling

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pathways overlap in their control of heat production in response to both diet and cold. Interestingly, the BAT of ␤-less mice is unresponsive to physiological (cold exposure) and pharmacological (nonselective ␤-agonist) stimulation. The earlier discovery that noradrenaline activates thermogenesis in BAT primarily through ␤3 -adrenergic receptor signaling prompted the realization that the SNS-␤3 -adrenergic receptor-UCP-1 axis regulates thermogenesis in response to both diet and cold. Previous attempts to induce obesity in mice by selectively deleting genes encoding UCP-1, ␤3 -adrenergic receptor or other proteins of the SNS have ended in failure. In particular, the production of transgenic mice lacking ␤3 -adrenergic receptor or UCP-1 failed to induce obesity, although varying degrees of cold sensitivity and small increases in body fat (less than two-fold) were observed [134]. These discrepancies may reflect the influence of genetic background on phenotypic outcomes of transgenic manipulations, and the existence of compensatory mechanisms that enable the transgenic mice to stay lean. The importance of PPAR-␥ and its cofactors in the differentiation of brown and white fat cells has been substanti-

ated by several studies (Fig. 3). PPAR-␥ is associated with white fat cell differentiation and hypertrophy. Somewhat unexpectedly, a critical enhancer in rodent and human UCP1 genes contains a PPAR-␥-responsive element that mediates the stimulation induced by thiazolidinediones, a novel class of anti-diabetic drugs [131]. Because PPAR-␥ is expressed in both WAT and BAT, the basis for the specific activation of UCP1 gene transcription in BAT remained elusive until the identification of the PPAR-␥ coactivator 1␣ (PGC-1␣) [135]. PGC-1␣ is expressed at higher levels in BAT than in WAT and its expression is increased in response to cold exposure and ␤3 -adrenergic stimulation. PGC-1␣ coactivates the PPAR–RXR heterodimer to stimulate the UCP1 promoter. The cofactor also plays a crucial role in mitochondrial biogenesis and stimulates the expression of electron-transport chain genes through induction of nuclear respiratory factors 1 and 2 and cooperation with nuclear respiratory factor 1 [136]. Recently, two different experimental approaches have given new insights for understanding whether it is possible to induce a metabolic shift in white fat cells from lipid

Fig. 3. Molecular processes involved in fat cell differentiation. (Enlargement) Coordination of transcription of mitochondrial and nuclear genes encoding subunits of OXPHOS by different extracellular stimuli, such as hormone. In the nucleus, the hormone–receptor complex can interact with the hormone response elements (HREs) of OXPHOS genes, to directly activate them, and also with the HREs of transcription factor genes (NRF, PGC-1␣), to induce transcription factors which exert a positive effect on the OXPHOS genes. The effect of extracellular stimuli, such as hormones, on mitochondrial OXPHOS can be direct, by interaction of the hormone–receptor complex with mitochondrial HREs, or indirect, via induction of nuclear-encoded mitochondrial transcription factors. NR, nuclear receptor.

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storage towards fatty acid utilization. Firstly, human subcutaneous white adipocytes have been transduced with an adenovirus expressing human PGC-1␣ [137,138]. Expression of the coactivator induced the expression of UCP1, respiratory chain proteins, fatty acid oxidation enzymes and other brown adipocyte markers. The coordinated regulation of gene expression suggested a potential increase in the capacity of fatty acid oxidation in white adipocytes. On the other hand, nitric oxide (NO) production, which is induced by noradrenergic stiimulation of BAT through ␤3 -adrenergic receptors, has been found to increase PGC-1␣ gene expression and, consequently, mitochondrial biogenesis in brown fat cells [139]. The relevance in vivo of these findings was strengthened by studying the BAT functions in wild-type and eNOS−/− mice before and after cold exposure. At both temperatures, histological analysis indicated that eNOS−/− BAT was functionally inactive, and mitochondrial biogenesis was impaired. When the authors looked at the control of biogenesis in the brain, liver and heart of the knockout mice, they found that deletion of eNOS was enough to reduce the number of mitochondria even in tissues that have a basal level of neuronal, and possibly inducible, NOS expression [139]. In eNOS−/− mice, oxygen consumption rates—an indicator of metabolic rate—were decreased, indicating that BAT-dependent thermogenesis might be impaired. In genetic models of obesity, defective energy expenditure is involved in increased food intake and body-weight gain; 8-week-old eNOS−/− mice showed similar food consumption but weighed more than wild-type mice. So, the increased body weight of eNOS−/− mice could be accounted for by higher feed efficiency (i.e. weight gain/food intake) caused by defective energy expenditure. These results suggest that pharmacological manipulation of both NO system and PGC-1␣ transcriptional machinaries could be of relevance for drug treatment of obesity. Two other recent transgenic experiments open up other potential avenues for modulating the brown-fat phenotype. Transgenic mice that overexpress the winged helix–forkhead transcription factor Foxc2 (forkhead box c2) specifically in adipose tissue have enlarged BAT depots, whereas their WAT pads are reduced in size and, on histological examination, have increased numbers of multilocular (as opposed to unilocular) adipocytes [140]. The expression of several genes that are associated with mitochondrial biogenesis and respiration, including UCP1 and PGC-1␣, was also upregulated in WAT. Of further interest, these mice were more insulin sensitive than their non-transgenic littermates, indicating that pharmacological manipulation could lead to a further antidiabetic action. Also subject to genetic manipulation was the eukaryotictranslation-initiation-factor-4E binding protein 1 (EIF4EBP1), which is highly expressed in WAT and reversibly represses cap-dependent translation by preventing the formation of the EIF4E complex [141]. Homozygous EIF4EBP1-deficient mice (Eif4ebp1−/− ) manifested smaller WAT pads than wild-type littermates. In addition,

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the WAT depots had six-fold higher UCP1 expression and contained increased numbers of multilocular adipocytes that are more characteristic of a brown adipocyte phenotype. Moreover, although these mice showed an unaltered level of PGC-1␣ mRNA expression, they did manifest a two-fold higher PGC-1␣ protein level.

6. Thermogenic drugs 6.1. β3 -Adrenergic receptor agonists First-generation ␤3 -adrenergic receptor agonists were selected on the basis of their thermogenic and anti-obesity activities in rats and mice. In obese rodents, they produce good weight loss (or prevent weight gain) because they increase metabolic rate. There may be an initial reduction in food intake [142], but no effect on total intake is detectable over a few days or longer [143]. All the weight lost is fat, and when given with food, there even may be an increase in body protein [144]. In addition to their anti-obesity activity, ␤3 -adrenergic receptor agonists elicit marked improvements in insulin sensitivity in obese and insulin-resistant rodents. Such effects are seen at doses below those that elicit significant anti-obesity activity. This suggests that ␤3 -adrenergic receptor agonists (and perhaps other thermogenic drugs) have even greater potential as anti-diabetic than as anti-obesity agents [145,146]. An improvement in the blood lipid profile elicited by the ␤3 -adrenergic receptor agonist GR 265261 at a dose that does not have an anti-obesity effect has been described in monkeys [147]. The thermogenic, anti-obesity and anti-diabetic effects of ␤3 -adrenergic receptor agonists in rodents are achieved without significant cardiovascular effects, lowering of blood potassium or tremor, because they lack potency or efficacy at rodent ␤1 - and ␤2 -adrenoceptors. Unfortunately, clinical experience with the first-generation compounds has been less encouraging. Although some compounds have been shown to be thermogenic, to produce weight loss with conservation of protein, or to improve insulin action, efficacy generally has been associated with ␤1 - or ␤2 -adrenoceptor-mediated side effects. Only the American Home Products (Madison, NJ, USA) compound CL-316243, which has little or no efficacy at rodent ␤1 - and ␤2 -adrenoceptors, has shown any beneficial activity without eliciting side effects. Unfortunately, CL-316243 had only 10% oral bioavailability in humans, and it was given at a dose of 1500 mg. Prodrugs with improved oral bioavailability have been described recently [148]. Despite the failure of the first-generation compounds in humans, their effects on metabolic rate and insulin action suggest that the ␤3 -adrenoceptor is a valid target. It might be argued that the beneficial effects of BRL-26830, BRL-35135, and ZD-2079 were due to stimulation of ␤2 - or even ␤1 -adrenoceptors. Indeed, 60% of the thermogenic effects of BRL-35135 was blocked with the ␤1 /␤2 -adrenoceptor antagonist nadolol. Nevertheless,

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the remaining activity does appear to be mediated by the ␤3 -adrenoceptor [149]. Thus, although they may never be as remarkably effective in humans as in rodents, the main problem with the first-generation compounds appears to be that they are not as selective for human as for rodent ␤3 -adrenergic receptors. The reason for this became transparent when the human ␤3 -adrenergic receptor was cloned, and it was found that the first-generation compounds had low efficacies and potencies at the human receptor compared with their efficacies and potencies in rodent tissues that express ␤3 -adrenergic receptor [150]. Since potencies and efficacies at ␤1 - and ␤2 -adrenergic receptor appear similar between humans and rodents, selectivities for ␤3 -adrenergic receptor are low in humans. A suggestion to ameliorate the experimental methodologies, which limit the screening approaches to thermogenetic molecules active in humans as anti-obesity drugs, seems to be implied in a recent paper by Palma et al. [151]. While injecting heterogenic mRNAs, isolated from brains and other tissues, into Xenopus oocytes to express functional neurotransmitter receptors in the oocyte membrane [152], which was used extensively in the last 20 years to study the molecular structure and function of many receptors and voltage-operated channels [153], a method was developed to incorporate in the oocyte membrane foreign receptors that had already been assembled in their native cells [151]. Essentially, this method consists in injecting the oocytes with cell membranes prepared from foreign tissues. This approach has been used to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal 7 nicotinic acetylcholine receptors (␣7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal ␣4␤2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of ␤3 -adrenergic receptors expressed in the membrane of cultured cells. Finally, new concepts about drug–receptor interaction may add relevant changes in screening procedures to obtain therapeutically effective molecules. It is important to underline that the intimate relationship between affinity and efficacy in drug–receptor interaction is becoming appreciated as new technologies allow the detection of different receptor behaviors. Indeed, efficacy for G-protein-coupled receptors (GPCRs) should not be confined to G-protein

activation, but should also be expanded to the complete range of behaviors of GPCRs, such as receptor internalization, desensitization, oligomerization, phosphorylation and association with other membrane proteins [154]. The idea that affinity is linked to receptor conformational changes compels different views of how GPCRs could be screened for biologically active molecules. This idea also suggests that optimal drug screening processes should use maximal numbers of methods to detect receptor conformational changes. For example, if only one G-protein response is monitored for a given receptor screen (i.e. cAMP production or lipolytic activity by putative ␤3 -adrenergic receptor agonists), then a ligand–receptor interaction that produces a conformational change in the receptor, with a consequent “effect”, but does not produce a specific change in the monitored receptor–G-protein interaction would not be detected. Thus ideally, an assay in which protein conformational changes can be detected directly would optimal for screening for potential therapeutically useful molecules [154]. 6.2. PPAR-δ agonists A recent paper shows that a ligand-activated transcription factor known as peroxisome-proliferator-activated receptor-␦ (PPAR-␦) stimulates fat burning, which indicates that drugs that activate PPAR-␦ might have potential for treating obesity [155]. Selective expression of an activated form of PPAR-␦ in adipose tissue resulted in mice with a lean phenotype, although their food intake on a standard diet was normal. And when these mice were fed a high-fat diet, weight gain and lipid accumulation were markedly reduced compared with control mice [155]. Further experiments with db/db mice, which are predisposed to obesity, showed that expression of activated PPAR-␦ reverses the obesity phenotype [155]. Moreover, age- and weight-matched db/db mice were treated with vehicle or a PPAR-␦-specific agonist, GW501516 [156], for 7 days. The histology of BAT in vehicle-treated db/db mice resembles WAT, each cell filled with a single massive lipid droplet, pushing the nucleus to the cell periphery. Remarkably, in GW501516 treated db/db mice, brown fat adipocytes show increased mitochondria-rich eosinophilic staining, disappearance of large lipid droplets, and replacement with multiple small droplets [155]. Thus, even a relatively short-term agonist treatment leads to a rapid restoration of brown fat appearance with reduced lipid content and enhanced metabolic activity. In addition, the expression of genes involved in fatty acid metabolism was monitored, and it was found that PPAR-␦ activation specifically induces the expression of genes required for fatty acid oxidation and energy dissipation. The ability to induce adaptive thermogenesis and protection against both dietary and genetic obesity suggests that PPAR-␦ agonists may be promising drug candidates for anti-obesity therapies.

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6.3. KB-141 KB-141 is a thyroid hormone receptor (TR) agonist that binds to the human (h)TR␤ with a 14-fold higher affinity than to hTR␣ [157]. Recently, KB-141 was used to examine effects of selective activation of TR␤ in control and TR␣−/− mice, cholesterol-fed rats, and cynomolgus monkeys. The latter more closely resemble humans in terms of lipoprotein metabolism and regulation of body weight [158]. 3,5,3 -Triiodi-l-thyronine (T3 ), the major active form of thyroid hormone, increased the metabolic rate in both wild type and TR␣−/− mice, but the increase for the wild type animals was greater than with the TR␣−/− mice [159]. These data imply that both TR␣ and -␤ regulate the metabolic rate. As compared with T3 , KB-141 reduced plasma cholesterol levels selectively versus increasing heart rate in all three models. Relative to T3 , KB-141 also increased whole body oxygen consumption in both mice and rats more than heart rate. In monkeys, KB-141 decreased body weight after 1 week of treatment by up to 7% and Lp(a) by up to 56% without tachycardia. These studies suggest that selective stimulation of the TR␤ might be exploited as a therapeutically effective means to lower weight, plasma cholesterol, and Lp(a) without eliciting deleterious cardiac effects.

7. Conclusions The understanding that obesity is a chronic multifactorial disease, which is poorly treated with the available therapeutic approaches (diet, exercise, behavior), has stimulated a renewed interest in the use of drugs. Over the past 30 years relatively few drugs have been developed, or approved, for the treatment of obesity. Worldwide government regulations and treatment guidelines have been somewhat instrumental in impeding the development of these drugs. However, other drugs that control chronic diseases, such as hypertension or diabetes mellitus, are not expected to increasingly reduce high blood pressure or high blood glucose levels, but rather to maintain a target level [160]. The current view regarding weight loss is that obese patients should not be expected to reach their “ideal” bodyweight, but instead should be encouraged to initially resist further weight gain and subsequently to lose moderate amounts of weight (5–10%) and to maintain this weight loss. At present, few options exist for the pharmacological treatment of obesity. Dexfenfluramine was the only other centrally acting anti-obesity agent recently approved for use longer than 3 months. However, it, along with fenfluramine, has been voluntarily withdrawn from the market worldwide and is undergoing further evaluation because of potential cardiovascular safety concerns. Sibutramine, like dexfenfluramine, is a centrally acting agent which affects satiety, although the mechanism of action of each agent is different. Both agents appear devoid of abuse potential. New drug development is occuring throughout the pharmaceutical industry and several therapeutic agents are

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around the corner and still others are on the horizon. The advent of fully sequenced genomes and new genomics and proteomics technologies, which permit analyses of gene transcription profiles and protein interactions in complex cell circuitries, will lead to new target selection for drugs able to treat obesity and will enable obese individuals to be classified according to their likely response to an anti-obesity drug.

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