Pharmacological substances C75
C75, an inhibitor of the enzyme FAS, was initially developed for the treatment of certain cancers (Kuhajda et al. 2000) because many common human cancers express high levels of FAS. Subsequent tests revealed that systemic and intracerebroventricular (i.c.v.) administration of C75 in mice reduced food intake and body weight (Loftus et al. 2000), making FAS also an interesting target in the therapy of obesity. C75 blocks the conversion of malonyl-CoA into fatty acids and, hence, increases tissue levels of malonyl-CoA
(Loftus et al. 2000). A C75-induced increase in hypothalamic malonyl-CoA and a decrease in AMP-activated kinase activity and subsequent changes in the expression of hypothalamic orexigenic (NPY, AgRP) and anorectic (POMC, CART) neuropeptides are presumably involved in the feeding-inhibitory effect of C75 (Kumar et al. 2002; Ronnett et al. 2005). Yet, not all findings support the assumption that hypothalamic malonyl-CoA levels are involved in the feeding-inhibitory effect of i.c.v. C75 (Wortman et al. 2003). In addition, some results (Clegg et al. 2002; Takahashi et al. 2004; Rohrbach et al. 2005) indicate that intraperitoneally (i.p.) injected C75 has unspecific aversive effects in rodents.
Although C75 clearly increases malonyl-CoA, which should inhibit CPT 1 and, hence, mitochondrial fatty acid oxidation (McGarry and Foster 1980) (Fig. 1.1), the published results on the effect of C75 on CPT 1 activity and fatty acid oxidation are controversial. Bentebibel et al. (2006) demonstrated that the CoA derivative of C75 is a potent inhibitor of CPT 1 and fatty acid oxidation, whereas Thupari et al. (2002) showed that i.p. injected C75 increased CPT 1 and fatty acid oxidation in adipose tissue and liver despite a high tissue level of malonyl-CoA. C75 stimulated CPT 1 and increased intracellular ATP levels also in primary cortical neuronal cultures, similar to its effects in peripheral tissues (Kim et al. 2004; Landree et al. 2004). C75 also increased whole-body and in particular skeletal-muscle fatty acid oxidation when injected i.c.v. in mice. Phentolamine, an a-adrenergic blocking agent, prevented the C75-induced increases in whole-body fatty acid oxidation, implicating the sympathetic nervous system in this effect (Cha et al. 2005). One consequence of an increase in whole-body fatty acid oxidation is that energy expenditure is also increased, and this effect in response to C75 was more pronounced in mice with diet-induced obesity compared with lean mice (Tu et al. 2005). Finally, FAS seems to generate signals that may be essential for the differentiation of preadipocyte, because inhibition of FAS by C75 prevented preadipocyte differentiation (Schmid et al. 2005).
All in all, C75 [for review see also Ronnett et al. (2005)] is presumably not a drug that can be used for the treatment of human obesity, but it is an interesting substance that allows researchers to study the roles of FAS and CPT 1 in the control of food intake, body weight and adipose tissue mass (Kuhajda et al. 2005).
Cerulenin and 5-(tetradecyloxy)-2-furoic acid
Cerulenin is another inhibitor of FAS that reduced food intake when injected i.p. in mice, albeit not as potently as C75 (Loftus et al. 2000). Another difference between cerulenin and C75 is that cerulenin does not activate CPT 1 (Jin et al. 2004). In vitro cerulenin decreased fatty acid oxidation by increasing cytosolic malonyl-CoA levels (Thupari et al. 2001). When cerulenin was injected i.c.v., however, it increased energy expenditure and CPT 1 activity in soleus muscle, possibly via sympathetic nervous system activation (Jin et al. 2004). 5-(Tetradecyloxy)-2-furoic acid (TOFA) is an inhibitor of the enzyme acetyl-CoA carboxylase; it increases ATP levels in neuronal cells in vitro (Landree et al. 2004), but does not affect food intake in vivo. Pretreatment with TOFA actually reversed the anorectic effect of C75 (Loftus et al. 2000), suggesting that the absolute energy status of hypothalamic neurons is not crucial for the control of food intake. As is the case for C75, cerulenin and TOFA can presumably not directly be used for the treatment of obesity. Yet again, all these substances are interesting tools with which to study the role of DNL in the control of food intake and body weight.
Hydroxycitric acid (HCA) is a compound found in fruit rinds of Garcina cambogia, Garcina indica and Garcina atroviridis. These plants are cultivated on the Indian subcontinent and in western Sri Lanka (Jena et al. 2002). HCA has been shown to potently inhibit the extramitochondrial enzyme CL (Fig. 1.1), which catalyses the cleavage of citrate to acetyl-CoA and oxalacetate, another key step in DNL (Sullivan et al. 1972). Sullivan et al. (1974b) demonstrated that oral administration of HCA dose-dependently reduced in vivo lipogenesis in liver, adipose tissue and small intestine. Furthermore, HCA caused a significant reduction in food consumption and body weight in rodent studies when animals had access to a high-glucose diet that contained only 1% fat (Sullivan et al. 1974a). Recently, we demonstrated that HCA also reduced food intake and body weight in adult rats after substantial body weight loss, when HCA was given with a high-glucose (Leonhardt et al. 2001, 2004c; Leonhardt and Langhans 2002) or a high-fructose diet (Brandt et al. 2006) that contained 120 g/kg of fat.
The mechanism involved in the feeding-inhibitory effect of HCA is poorly understood. HCA reduces the availability of cytosolic acetyl-CoA level (Michno et al. 2004) and thereby prevents the formation of malonyl-CoA (Fig. 1.1). HCA should therefore stimulate fatty acid oxidation. Changes in peripheral, in particular, hepatic fatty acid oxidation are supposed to be involved in the control of food intake [for review see Leonhardt and Langhans (2004)]. In cell culture experiments HCA reduced cytosolic malonyl-CoA levels (Saha et al. 1997) and increased fatty acid oxidation (Chen et al. 1994). However, whether this also occurs in vivo is unclear. In one of our studies (Leonhardt et al. 2004a) HCA reduced the respiratory quotient (RQ); this has also been shown in other animal (Ishihara et al. 2000) and human (Lim et al. 2002; Tomita et al. 2003) studies. A reduction in RQ could be related to an increase in fatty acid oxidation and/or a reduction in DNL. DNL is an energy-consuming process, and the conversion of carbohydrate to fat costs about 0.25 MJ per MJ of ingested carbohydrates (Acheson and Flatt 2002). As a result, inhibition of DNL should decrease energy expenditure. Indeed, HCA reduced energy expenditure in addition to the RQ (Leonhardt et al. 2004a), suggesting that the reduced RQ was mainly related to the suppression of DNL. HCA also reduced DNL, RQ and energy expenditure in humans (Kovacs and Westerterp-Plantenga, 2006). Another argument against the assumption that an increase in hepatic fatty acid oxidation causes the feeding-inhibitory effect of HCA is that this effect was shown to be independent of vagal afferents (Leonhardt et al. 2004b), whereas several findings strongly suggest that the feeding-stimulatory effect caused by an inhibition of peripheral fatty acid oxidation is signaled to the brain by vagal afferents [for review see Leonhardt and Langhans (2004)].
Wielinga et al. (2005) recently demonstrated that HCA delayed glucose absorption, but it is unclear whether this effect is involved in the feeding-inhibitory effect of HCA. Finally, HCA inhibited serotonin reuptake, thereby increasing serotonin availability in isolated rat brain cortical slices (Ohia et al. 2002) and HCA reduced the synthesis and release of acetylcho-line in experiments on slices of rat caudate nuclei (Ricny and Tucek 1982). However, so far it is unknown whether HCA can cross the blood-brain barrier, which would be a precondition for a direct effect of HCA on brain areas involved in food intake control.
Therefore, whereas a reduction in food intake and body weight by HCA was shown in many rodent studies, the efficacy of HCA in humans appears to be inconsistent and variable: effects of HCA on food intake, body weight, visceral fat accumulation or fatty acid oxidation have been reported in some (Lim et al. 2002, 2003; Westerterp-Plantenga and Kovacs 2002; Hayamizu et al. 2003; Tomita et al. 2003; Preuss et al. 2004), but not all (Kriketos et al. 1999; Mattes and Bormann 2000; van Loon et al. 2000; Kovacs et al. 2001a,b), studies. Different experimental designs or differences in the HCA preparations employed might explain the discrepant findings. For example, the bioavailability of various HCA preparations differs (Lim et al. 2005).
Finally, so far it is unclear whether long-term HCA treatment may have adverse effects. Most animal studies indicate that HCA is a safe, natural supplement that does not cause any changes in major organs or in hematol-ogy, clinical chemistry and histopathology (Ohia et al. 2002; Shara et al. 2004; Soni et al. 2004; Oikawa et al. 2005). However, in one study a high dose of HCA caused potent testicular atrophy and toxicity (Saito et al. 2005), and we recently observed that long-term application of HCA increased liver lipid content and plasma cholesterol levels in rats (Brandt et al. 2006). One explanation for the unexpected effects of HCA on lipid metabolism might be that HCA stimulates the enzyme ACC (Hackenschmidt et al. 1972). Thus, HCA acts as an inhibitor of DNL only if cytoplasmatic acetyl-CoA is produced by the citrate cleavage enzyme reaction, whereas HCA will not affect (Zambell et al. 2003) or even activate fatty acid synthesis whenever an alternative source of cytoplasmatic acetyl-CoA, e.g. acetate, is available (Hackenschmidt et al. 1972). It is still unclear whether HCA also enhances lipid synthesis and hepatic lipid accumulation in humans under certain cir cumstances. Consequently, long-term treatment with HCA may only be recommended with caution.
Green and black teas are popular drinks consumed all over the world. Epidemiological studies suggest that green tea in particular has preventive effects on chronic inflammatory diseases, cardiovascular diseases and cancer (Sueoka et al. 2001). Green tea or green tea extracts contain large amounts of polyphenolic components such as epicatechin, epicatechin gallate, epigallo-catechin and epigallocatechin gallate (EGCG) (Dulloo et al. 1999). EGCG is the most abundant of these substances, constituting more than 50% of the total amount of polyphenolic components in green tea, and is believed to be the most pharmacologically active tea catechin (Dulloo et al. 1999). Dulloo et al. (1999) demonstrated in humans that treatment with green tea extracts resulted in a significant increase in 24-h energy expenditure and a stimulation of fat oxidation. In rats, orally and i.p. administered EGCG reduced food intake and body weight (Kao et al. 2000). Recently Wolfram et al. (2005) confirmed that EGCG prevents body weight gain in mice, although in their study EGCG had no effect on food intake. Further, FAS and ACC 1 mRNA levels were decreased in adipose tissue of EGCG-supplemented mice (Wolfram et al. 2005) suggesting that EGCG might inhibit DNL (Fig. 1.1). Indeed Wang and Tian (2001) demonstrated that EGCG is an inhibitor of FAS and that the inhibition is related to P-ketoacyl reductase activity of FAS. In vitro, EGCG inhibits FAS as effectively as C75 (Wang et al. 2003), but the inhibition kinetics of the two substances differ considerably: the inhibition of FAS by EGCG is mainly a reversible fast-binding inhibition, whereas C75 causes an irreversible, slow-binding inactivation (Wang and Tian 2001). In a recent study (Zhang et al. 2006), the ability of green tea extracts to inhibit FAS was even more potent than that of EGCG, suggesting that other components of green tea can also inhibit FAS. The same group also identified catechin gallate in green tea extracts as a very potent inhibitor of FAS (Zhang et al. 2006). Finally, it is not only green tea extracts that can inhibit FAS; keemun black tea extracts also contain potent FAS inhibitors (Du et al. 2005), and these components are possibly theaflavins. Unfortunately, only 10-23% of the inhibitory activity of black tea is extracted by the general method of boiling with water (Du et al. 2005).
All in all, green and black tea extracts seem to have anti-obesity effects, i.e. they decrease in body weight and adipose tissue mass, in particular by increasing energy expenditure and by reducing DNL through inhibition of FAS.
Polyunsaturated fatty acids (PUFAs), in particular those of the n-3 family, seem to act as fuel partitioners in that they direct glucose away from glycolysis towards glycogen storage and shift fatty acids away from TAG synthesis and storage in adipose tissue towards fatty acid oxidation (Clarke 2000, 2001). These effects appear to be mainly related to the fact that PUFAs activate PPARs (Fig 1.1), induce genes encoding proteins involved in fatty acid oxidation (Clarke 2000) and inhibit genes involved in DNL such as FAS, presumably by suppressing the abundance of the sterol regulatory element-binding protein (Sekiya et al. 2003; Jump et al. 2005). Recently, Dentin et al. (2005) demonstrated that some of the n-3 PUFAs suppressive effects on glycolysis and lipogenesis are also mediated through the inhibition of carbohydrate responsive element-binding protein. Most of the n-3 PUFAs-induced changes in fatty acid metabolism have been shown for the liver. As the liver plays a central role in whole-body lipid meta-bolism, effects on whole-body lipid metabolism can be expected (Jump et al. 2005).
Another feature of n-3 PUFAs is that they also increase brown adipose tissue uncoupling protein 1 mRNA level in rats (Takahashi and Ide 2000) and induce a marked stimulation of brown fat thermogenesis (Oudart et al. 1997). In both studies, n-3 PUFAs had no major effect on food intake, but epididymal white fat mass was reduced, suggesting that n-3 PUFA indeed increased energy expenditure (Oudart et al. 1997; Takahashi and Ide 2000). Whether PUFAs also increase thermogenesis in humans is unknown.
n-3 PUFAs may be interesting as a nutrient supplement in the therapy of obesity, but it has to be mentioned that n-3 PUFAs had adverse effects on glycemic control in obese individuals (Mori et al. 2000; Woodman et al. 2002). The mechanism of this negative effect is unknown, but an increase in hepatic gluconeogenesis caused by an increase in hepatic fatty acid oxidation might contribute (Woodman et al. 2002). Nevertheless, in another study with overweight patients, n-3 PUFAs (as fish oil) combined with a weight-loss regimen were more effective at improving glucose-insulin metabolism than either weight loss or fish oil supplementation alone (Mori et al. 1999). Therefore, further studies should examine whether n-3 PUFAs have adverse effects on insulin sensitivity in obese people under certain circumstances.
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