This study is published here
The world is in health transition, and obesity is greatest threat to human health (Jose Hernandez–Morante et al., 2011). Obesity associates with various lifestyle-related diseases, such as diabetes, cardiovascular disease, hypertension and fatty liver disease (Jebb and Moore, 1999; Nakamura et al., 1994), causing a major health burden in terms of morbidity and mortality (Sturm, 2007). Although there has a few drugs in market to ameliorate or prevent obesity, costs and side effects limited their application (Chuah et al., 2013). It is well known that anti-obesity food ingredients could control and reduce body weight (Kim et al., 2008b).
Garcinia Cambogia extracts are found in northeastern India and Andaman Islands, and it has been extensively used for centuries throughout Southeast Asia as a food preservative, flavoring agent, and carminative (Jena et al., 2002).
Garcinia cambogia extracts is now popularly used as an ingredient of dietary supplements for weight loss (Saito et al., 2005), anti-obesity (Kim et al., 2008a; Kim et al., 2004), hypolipidaemic (Altiner et al., 2012) and anticancer activity (Mazzio and Soliman, 2009).
(-)-Hydroxycitric acid (HCA) is the major active ingredient present in the fruit rind of Garcinia cambogia (Jena et al., 2002; Marquez et al., 2012). The Garcinia cambogia extracts contains approximately 10~30% (-)-HCA, which can be isolated in the free form (Lewis and Neelakantan, 1965). Previous studies had identified (-)-HCA as a potent competitive inhibitor of adenosine triphosphate-citrate lyase (Watson et al., 1969), which is an extra-mitochondrial enzyme catalyzing the cleavage of citrate to oxaloacetate and acetyl-CoA (Watson and Lowenstein, 1970). (-)
Hydroxycitric acid reduced the availability of acetyl coenzyme A for the fatty acids and cholesterol synthesis (Watson et al., 1969). Many studies had reported that supplemental with (-)-HCA can promote weight reduction through suppressing de novo fatty acid synthesis, increasing lipid oxidation and reducing food intake (Chuah et al., 2013; Downs et al., 2005).
Recently, our laboratory also certified that Garcinia cambogia extracts could attenuate fat accumulation through regulating lipolysis gene expression by affecting adiponectin-AMPK signaling pathway in rat obesity model induced with high-fat diet (Liu et al., 2015).
(-)-Hydroxycitric acid is structurally similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002). Thus, (-)-HCA supplementation is expected to alter metabolic pathways. Amino acids are the fundamental building blocks of proteins (Jiang et al., 2014). In body, amino acids can be transformed to α-ketoglutarate by transamination reactions associated with multiple metabolic pathways such as glycolysis, gluconeogenesis, and the citric acid cycle (TCA) (Fauth et al., 1990).
Previous study showed that α-ketoglutarate is not only a key intermediate in the TCA cycle but also can replenish the cycle in anaplerotic reactions (Fink, 2008). Thus, amino acids are involved in protein synthesis, the energy production, gluconeogenesis, and lipogenesis (Wang et al., 2013). In addition, some amino acids are important for body development because they are the precursors of hormones.
It had been shown that phenylalanine and tyrosine are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Also, it had reports that insulin and growth hormone enhance amino acid uptake and protein synthesis, meanwhile the increase of amino acid contents stimulates glucagon secretion that promotes rapid conversion of amino acids to glucose (Rhoades and Rhoades, 2012).
Although dietary supplements of Garcinia cambogia extracts may be a practical way to reduce excessive fat accumulation in human or animal production, the precise physiological mechanism of HCA has not yet been fully clarified. Therefore, present study was conducted to investigate the effect of a long-term Garcinia cambogia extracts supplement on body weight gain, energy metabolism and the changes of amino acids content in serum, liver, and muscle of rats.
To our knowledge, this is the first report to investigate the impact of Garcinia cambogia extracts on the amino acid profile in different tissue. Our results not only provided information about how Garcinia cambogia extracts exerts its action but also certified the use of Garcinia cambogia extracts to control body weight.
MATERIALS AND METHODS
Garcinia cambogia extracts. Garcinia cambogia extracts was purchased from An Ynn Co. Ltd (Zhengzhou, China). The Garcinia cambogia extracts contain 56.0% ~ 58.0% (-)-Hydroxycitric acid including its free and lactone form, and it also contains 12.0%~14.0% cellulose, 5.5%~6.0% α-Dmelibiose, 2.5% ~ 3.0% β-D-lactin, 1.5% ~ 2.0% D-mannopyranose, 11%~12% oxophenic acid, 2.0% ~3.0% octadecyl alcohol, 3.5%~4.0% Coenzyme A, and 1.5% ~ 2.0% inorganic elements.
Animals and diets. Five-week old male Sprague– Dawley (SD) rats weighing 200 ± 20 g were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (China). Rats were housed individually under constant temperature of 25 ° C and humidity of 50% ~ 60% and maintained on a 12:12 h light/dark cycle. All animal handling procedures were performed in strict accordance with guidelines established by Institutional Animal Care and Use Committee of Nanjing Agricultural University.
Before initiation of experiment, rats were acclimatized to the environmental conditions for 1 week. A total of 60 rats were randomly assigned to one of four groups: control group, low dose of (-)-HCA-treated group, medium dose of (-)-HCA-treated group, and high dose of (-)HCA-treated group.
Rats were supplemented with Garcinia cambogia extracts at 0, 25, 50, and 75 g/kg diet, and the contents of Garcinia cambogia extracts were equivalent to 0, 1000, 2000, and 3000 mg/kg diet of (-)HCA level. Rats were fed ad libitum with free access to water for 8 weeks, diet was removed for the last 12 h of the experimental term, and then the rats were anesthetized with ether and scarified by decapitation. Rats were weighed at the beginning and the end of experiment to determine average daily gain.
Daily feed consumption per day was recorded; the average daily feed intake and feed conversion ratio were then determined. At the end of the experiment, blood samples were allowed to clot at 4 °C and centrifuged at 1520 × g for 20min before harvesting the serum. Then, the serum, liver, and muscle samples were collected and kept at 70 °C until further analysis. Liver was weighted to determine the liver index, which indicated by the liver weight (mg)/body weight (g).
Measurement of serum glucose and glycogen content.
Serum glucose (catalog#: F006), hepatic glycogen (catalog#: A034), and muscle glycogen contents (catalog#: A034) were measured using commercial kits according to the manufacturers’ protocol (Nanjing Jiancheng Biotechnology Institution, Nanjing, China).
Measurement of protein content in liver and muscle.
Protein content in liver and muscle was measured using commercial kits (catalog#: P0012), which were purchased from the Beyotime Biotechnology Institution Shanghai, China.
Measurement of serum hormone content.
Serum triiodothyronine (T3, catalog#: A01PZC), thyroxine (T4, catalog#: A02PZC), insulin (catalog#: F01PJB), glucagon (catalog#: F03PJB) and Leptin (catalog#: C16DJB) contents were measured using Radioimmunoassay Kit according to the manufacturers’ protocol (Beijing North Institute of Biotechnology, Beijing, China). The intracoefficients of variation for all hormones detection kit were less than 10% and inter-coefficients of variation were less than 15%.
Analyses of amino acids profile by high pressure liquid chromatography
Instrumentation and reagents. High pressure liquid chromatography (HPLC) analyses were carried out on a benchtop Agilent1100 series LC chromatographic system (Agilent Technologies, Waldbronn, Germany) equipped with a vacuum degasser, autosampler, thermostated column compartment, quaternary pump and a diodearray detector. The chromatographic column (XTerra®MS C18, 5 μm, 4.6 × 250 mm) was purchased from Waters (Waters Co., Milford, MA, USA).
The standards of alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine(Gly), glutamine (Gln), leucine (Leu), valine(Val), tryptophan (Trp), phenylalanine (Phe), arginine (Arg), asparagine (Asn), threonine (Thr), tyrosine (Tyr), isoleucine (Ile), serine (Ser), methionine (Met), proline (Pro), cysteine (Cys), histidine (His), and lysine (Lys) were purchased from Sigma. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Tedia Company Inc. (Fairfield, OH, USA). Tetrahydrofuran (HPLC grade) and Ophthaldialdehyde (OPA) were purchased from Merck KGaA (Darmstadt, Germany).
High-purity water was prepared from a Milli-Q gradient water purification system (Millipore, MA, USA) and was used for all protocols in this study.
High pressure liquid chromatography was performed according the method described by Shen et al (Shen et al., 2010). Briefly, a ternary system was used as mobile phase: solvent A is methanol, solvent B is acetonitrile, and solvent C is 10 mmol · L 1 Na2HPO4–NaH2PO4 (pH = 7.2, containing 0.3% tetrahydrofuran). A gradient elution program is mobile phase A, B, and C is 9%, 6%, and 85% for 10min, and then change to 12%, 8%, and 80% for 25 min, finally 15%, 15%, and 70% of mobile phase A, B, and C was used for another 25min. Flow rate: 1.0 mL · min 1. Fluorescence: excitation wave length = 340 nm and emission wavelength of 450 nm. Oven temperature: 40 °C. Injection volume: 20 μL.
Samples prepared and analyzed. Approximately 100 mg of liver and muscle was homogenized on ice with 1 mL of saline, and then centrifuged at 3000 g for 15 min before harvesting the supernatant. One hundred microliter of serum or tissue supernatant samples were mixed with 200 μL acetonitrile for 30 min at room temperature and then centrifuged at 12000 g for 30 min to harvest the supernatant for HPLC analysis. High pressure liquid chromatography analysis was performed after automatic pre-column derivatization with O-phthaldialdehyde (OPA) according the method described by Zeng et al.
(Zeng et al., 2013). Briefly, 20μL samples were mixed with 40 μL OPA-solution for 2 min at room temperature, and then 20 μL of the mixture was loaded into column. For identification purposes, the amino acid standards were used by spiking the samples, as well as by comparing the relative retention time. For quantification purposes, calibration curves using external standard methodology were performed. For recovery calculations, peak areas obtained from each sample was compared with the peak areas of standard used for spiking (Di Pierro et al., 2000; Uhe et al., 1991).
Data were analyzed using the Statistical Package for Social Science (SPSS Inc., Chicago, IL, USA) and expressed as mean values ± SE. Treatment differences were subjected to a Duncan’s multiple comparison tests, and the differences were considered significant at P < 0.05 RESULTS
Effect of (-)-HCA on body weight and feed intake in rats
Compared with the control group, the body weight gain was significantly decreased in 2000 mg/kg (-)-HCA treatment group (P < 0.05) (Fig. 1A). No significantly differences were observed on the feed intake (p > 0.05)
No statistical differences were observed on the serum glucose content in (-)-HCA treatment groups at different doses compared with the control group (p>0.05) (Fig. 2A). The hepatic glycogen (Fig. 2B) and muscle glycogen (Fig. 2C) contents were significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05). These results indicated that (-)-HCA supplement could promote the glycogen synthesis in rats. Effect of (-)-HCA on protein content in rats As shown in Fig. 3, the protein contents in liver (Fig. 3A) and muscle (Fig. 3B) were significantly increased in (-)-HCA treatment groups at three doses compared with the control group (p<0.05). These results indicated that (-)-HCA supplement could enhance protein synthesis in rats. Effect of (-)-HCA on serum metabolic hormone content in rats
A significantly increase of serum T4 content was observed in 1000 mg/kg and 2000 mg/kg (-)-HCA treatment groups when compared with the control group (p < 0.05) (Fig. 4A). The T3 content was significantly higher in 1000mg/kg (-)-HCA treatment group than that in control group (p < 0.05) (Fig. 4B). No noticeable changes were observed on glucagon content (p>0.05) (Fig. 4D), while the insulin content was significantly increased in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups than that in control group (p < 0.05) (Fig. 4C). In addition, serum Leptin content was significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05) (Fig. 4E). These results indicated that (-)-HCA supplement might regulate the glucose metabolism by regulating serum metabolic hormones content in rats. Effect of (-)-HCA on amino acid profile in rats
A chromatogram of synthetic mixture of amino acid standards was shown in Fig. 5. Each peak represents one of specific amino acid, and 15 amino acids were separated (T<50min) under the experimental conditions used. As shown in Table 1, in 7.8125 ~ 500 μmol/L concentration range, amino acid standard concentration was linear related to the peak area and the correlation coefficients is 0.9983 ~ 0.9999. The Intra-day RSD and Inter-day RSD is between 0.47% ~ 2.37% and 1.68% ~ 5.50%, respectively, which are within 6%. In addition, the recovery rate of 15 amino acid standards was between 93.88% ~ 105.20%. These parameters results indicated that this sensitive procedure could be used for the quantitative analysis of amino acid in tissues. Amino acid content in serum
As shown in Fig. 6, most of the amino acid contents were higher in (-)-HCA treatment groups than that in the control group. Among glucogenic amino acid, the threonine content in 2000 mg/kg (-)-HCA treatment group (p < 0.05) and the threonine, arginine, alanine, valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.05) were significantly increased than that in the control group (Fig. 6A). Among aromatic amino acid, the tyrosine and phenylalanine contents were significantly increased in 3000 mg/kg (-)-HCA treatment group (p < 0.05) than that in the control group (Fig. 6B). Among branched amino acid, the valine and leucine contents were significantly increased in 3000 mg/kg (-)HCA treatment group (p < 0.05) than that in the control group (Fig. 6C). These results indicated that (-)-HCA supplement could increase the glucogenic amino acid, aromatic amino acid, and branched amino acid in serum of rats. Amino acid content in liver
Similarity, most of the amino acid contents were also higher in (-)-HCA treatment groups than that in the control group (Fig. 7). Among glucogenic amino acid, the asparagine and threonine contents in 1000mg/kg (-)-HCA treatment group (p<0.05) and the aspartic acid, glutamic acid, threonine, and valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased than that in the control group (Fig. 7A). Among aromatic amino acid, the tryptophan and phenylalanine contents in 1000 mg/kg (p < 0.05) and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly increased (Fig. 7B). Among branched amino acid, the isoleucine content in (-)-HCA treatment group at various doses (p < 0.05) and the valine and leucine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased (Fig. 7C). These results indicated that (-)-HCA supplement could increase amino acid contents, especially aromatic amino acid and branched amino acid, in the liver of rats. Amino acid content in muscle
Contrary to serum and liver, most of the amino acid contents were lower in (-)-HCA treatment groups (Fig. 8). Among glucogenic amino acid, the glutamic acid content in (-)-HCA treatment group at all dose (p < 0.05) and the valine and glutamine contents in 1000 mg/kg (-)-HCA treatment group (p < 0.05) and aspartic acid content in 2000mg/kg (-)-HCA treatment group (p < 0.05) were significantly decreased (Fig. 8A). Among aromatic amino acid, 1000 mg/kg (-)-HCA treatment significantly decreased tyrosine and phenylalanine contents in muscle (p < 0.05) when compared with the control group (Fig. 8B). Among branched amino acid, the valine content in 1000mg/kg (-)-HCA treatment group (p < 0.05) and the leucine content in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly decreased (Fig. 8C). These results indicated that (-)-HCA supplement could decrease amino acid contents, especially aromatic amino acid and branched amino acid, in the muscle of rats. DISCUSSION
Present results showed that diet supplement with (-)-HCA reduced the body weight gain in male rats, and 2000mg/kg (-)-HCA treatment significantly decreased the body weight gain. This observation was consistent with the study of Leonhardt et al. (2001), who reported that (-)-HCA could promote the body weight loss in male rats. Many studies demonstrate that (-)-HCA reduced the body weight gain in rats (Kim et al., 2008b), human (Marquez et al., 2012) and broilers (Liu et al., 2015), and suggesting feed intake inhibition maybe a major mechanism of how Garcinia cambogia extracts exerts its function in controlling body weight (Leonhardt et al., 2001).
Nevertheless, no differences was observed on feed intake in rats supplemented with (-)-HCA, and this results indicated that the inhibition effect of (-)-HCA on body weight gain is not mainly via regulating feed intake in male rats.
The results presented here were consistent with the previous study, which demonstrated that 2 weeks (-)-HCA supplement has no significant effects on appetite in human (Kovacs et al., 2001). In addition, it is reported that Garcinia cambogia leaf supplementation had no effect in male Ross 308 broiler chickens on feed intake in finisher stage (Sebola et al., 2011).
We presumed that the differences in the preparation of (-)-HCA and the different animal or rat strains used in those study could contribute to such discrepancy. As a weight loss agent, it had presume that an increased fatty acid oxidation and decreased fat accumulation in animal with (-)-HCA-treated contributed to decrease its weight (Sullivan et al., 1972).
In addition, our recently study demonstrated that Garcinia Cambogia extracts could attenuated fat accumulation and body weight gain through activating the Adiponectin-AMPK signaling pathway in rat obesity model induced by high-fat diet (Liu et al., 2015). Although we did not investigated the effect of (-)-HCA on fat accumulation in this study, taken our results and other reported, we think that suppression fat accumulation may be a major mechanism of weight loss by (-)HCA-induced.
It is well known that body weight gain depends on the balance between energy intake and energy expenditure. Previous study suggests that Garcinia cambogia extracts could enhance energy expenditure in rats (Vasselli et al., 1998), and the suppressive effect of Garcinia cambogia extracts on body weight gain might also depend on increased thermogenesis, except for the reduction in feed intake (Leonhardt et al., 2001).
Thyroid hormones, including triiodothyronine (T3) and thyroxine (T4), are recognized as the key metabolic hormones in body. Thyroid hormones essentially modulate all metabolic pathways through alterations in oxygen consumption and carbohydrate metabolism (Smith et al., 2002).
Serum thyroid hormones contents are associated with energy expenditure and other effects, such as lipid metabolism and protein synthesis (Hornick et al., 2000).
Present study showed that serum T4 content in 1000 mg/kg and 2000mg/kg (-)-HCA treatment groups and serum T3 content in 1000mg/kg (-)-HCA treatment group were significantly increased in rats. Leptin, the ‘satiety hormone’, is an important hormone that helps to regulate energy balance (Pan et al., 2014).
In present study, it was demonstrated that 1000 mg/kg (-)-HCA treatment significantly increased serum Leptin content in rats.
One of the major functions of Leptin is control energy balance by binding to receptors in hypothalamus, which results in the increase energy expenditure (Lerario et al., 2001; Watson et al., 2000). Thus, the above data suggested that one possible mechanism of (-)-HCA supplement reduced body weight gain via enhancing energy expenditure in rats, which might be associated with the increase of thyroid hormones and Leptin levels.
It had been reported that aromatic amino acids, including phenylalanine, tryptophan, and tyrosine, have important roles in body development because they are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Our results showed that (-)-HCA treatment significantly increased serum tyrosine and phenylalanine contents in rats, and the tryptophan and phenylalanine contents also significantly increased in liver. Tyrosine and phenylalanine are premise material for the synthesis of thyroid hormones, and up to 80% of the T4 is then converted to T3 by organs such as the liver, kidney, and spleen (Koehrle and Brabant, 2010).
The changes of aromatic amino acids content were consistent with the significant increase of serum T4 and T3 contents in rats after (-)HCA treatment. In addition, tryptophan is premise material for the synthesis of serotonin (Boopathi and Ramasamy, 2014); thus, an increase of serum tryptophan levels could affect food intake behavior (Lopez et al., 2015). Although the serotonin content was not detected in this study, the significant increase of tryptophan contents indirectly indicated that (-)-HCA treatment might increase the serotonin release and availability.
This results was also consistent with previous reports that shows the increase of serotonin content after (-)-HCA treatment might be the main reason for appetite suppression (Ohia et al., 2002; Preuss et al., 2004; Roy et al., 2004).
Taken together, these results further confirmed that (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormones levels.
(-)-Hydroxycitric acid inhibits ATP-citrate lyase and increases the cellular pool of citrate, which in turn inhibits glycolysis and thus redirects the carbon sources for glycogen production within the liver or muscle (Cheng et al., 2012; Shara et al., 2003). No changes were observed on the glucose content, while 1000 mg/kg (-)HCA treatment significantly increased the hepatic glycogen and muscle glycogen contents in rats. Our results is similar to previous study results that show glycogen levels in skeletal muscle are increased after (-)-HCA supplementation in animal models (Ishihara et al., 2000) or in human (Cheng et al., 2012). Our results showed that 2000 mg/kg and 3000 mg/kg (-)-HCA treatment significantly increased insulin content in rats. Insulin can promote the storage of glucose and inhibit lipolysis and gluconeogenesis (Bernard et al., 2013; Bernard et al., 2011).
It has been reported that treated with Garcinia cambogia extracts for 4weeks significantly increased plasma insulin content (Hayamizu et al., 2003). In addition, previous study certifies that (-)-HCA can inhibit phosphofructokinase, a key enzyme controlling glycolysis (McCune et al., 1989). Once glucose was absorbed, it is rapidly phosphorylated to glucose-6-phosphate and then converted into glycogen through the glycogen synthesis pathway or lactate through the glycolytic pathway. Although we did not measure phosphofructokinase activity in this study, the inhibitory action of (-)-HCA on phosphofructokinase that results in the inhibition of glycolysis is consistent with the higher glycogen content in liver and muscle reported in here.
Therefore, the mechanism that (-)-HCA could increase the glycogen content in liver and muscle might be due to its inhibitory effect on the glycolytic pathway, and this action might be related to its ability to increase insulin content in rats.
Our results showed that (-)-HCA treatment significantly increased the protein contents in liver and muscle, which indicated that administration of (-)-HCA could promote protein synthesis in rats. This was consistent with the feed conversion ratio, which was significantly increased in rats supplemented with (-)HCA. Amino acids are not only the fundamental building blocks for protein synthesis (Jiang et al., 2014); they are also used for energy dissipation or other metabolic purposes (Conceicao et al., 2003). (-)-HCA has a structure similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002).
Thus, the indirect inhibition of cytosolic pool of citrate by (-)-HCA and subsequent reduction in acetyl coenzyme A and oxaloacetate alter the citric acid cycle (TCA) that is expected to alter metabolic pathways. Importantly, oxaloacetate is not only an important intermediate of the TCA cycle but also the first designated substrate of the gluconeogenic pathways of all other cycle intermediates, glycerol, or amino acids (Homem de Bittencourt et al., 1993).
Thus, we presume that (-)-HCA supplement may alter metabolic directions of amino acids, which in turn promoted protein synthesis in rats. In the present study, most of amino acid contents in serum and liver were increased, while its content in muscle were decreased in rats supplemented with (-)-HCA. Amino acids are used in a variety of cellular metabolism pathways, such as provision of energy (Conceicao et al., 2003), protein, and nucleotide precursors (Jiang et al., 2014), signaling molecules (Wu et al., 2000) and protection against oxidative stress (Nasresfahani et al., 1992). As energy requirements of body are met, amino acids will be mainly used for protein synthesis rather than for provision energy.
Our results showed that no changes were observed on serum glucose content, while hepatic glycogen and muscle glycogen contents were significantly increased in rats supplemented with (-)-HCA, which indicated that there was sufficient energy to meet the requirement of body. Under this condition, the increased of amino acid contents in serum and liver might be used to promote protein synthesis. In addition, alanine in muscle can be used to transport the ammonia to liver, and then the liver delivers glucose to muscle through serum, which is called as alanine-glucose cycle, and it can provide the adequate glucose for muscle (Rijkers, 2015).
Present study showed that (-)-HCA treatment obviously increase alanine contents in serum and liver of rats, this indirectly indicated that sufficient glucose in muscle provided an essential prerequisite for the protein synthesis. As mentioned earlier, insulin can enhance amino acid uptake and protein synthesis (Bernard et al., 2013; Bernard et al., 2011). Our result showed that serum insulin content was significantly increased in rats after (-)HCA treatment, which was consistent with the significant increase of the protein content in liver and muscle of rats. Therefore, we conjecture that (-)-HCA treatment could promote protein synthesis via regulating metabolic directions of amino acids in rats.
The amino acids predominantly involved in energy metabolic processes are branched amino acids, which include leucine, isoleucine, and valine (Assenza et al., 2004). Under normal conditions, the branched amino acids are selectively excluded from hepatic uptake and are metabolized predominantly in the skeletal muscle (Adibi, 1980; Tsuchiya et al., 2005; Urata et al., 2007).
Our results showed that (-)-HCA treatment significantly increased the contents of valine and leucine in serum and liver, and decreased their contents in muscle of rats. Previous study shows that excessive oxidation of branched amino acids may inhibit citric acid cycle via depleting the glutamate and ketoglutarate pool (Trottier et al., 2002). The changes of serum glucose and glycogen contents in this study indicated that branched amino acids might be mainly used for protein synthesis in muscle rather than for the source of energy. Muscle is one of the main target organs of insulin action, and insulin can promote the protein synthesis (Rijkers, 2015).
Escobar et al. demonstrated that insulin can stimulate the influx of branched amino acids into the skeletal muscle (Escobar et al., 2006). In addition, previous study suggests that branched amino acids play important role in skeletal muscles, specifically in the regulation of protein synthesis metabolism (Desikan et al., 2010). Wang et al. reports that amino acids can enhance protein synthesis (Wang and Proud, 2008). Considering the increase of serum insulin level and muscle protein content, we suspected that decrease of branched amino acids content after (-)-HCA treatment might be due to its enhance on protein synthesis.
In conclusion, in spite that (-)-HCA could promote protein synthesis, it can also promote weight loss by suppressing de novo of fatty acid synthesis and promoting energy expenditure. This study demonstrated that supplement with (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormone levels. Meanwhile, (-)-HCA treatment could promote protein synthesis in male rats by altering the metabolic directions of amino acids. The elucidation of the precise mechanism involved in this action of (-)-HCA needs further investigation.