Ablation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and brown adipose tissues

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) improves insulin sensitivity during aging. Compared to wild-type (WT) mice, old Ghsr(-/-) mice have reduced fat and preserve a healthier lipid profile. Old Ghsr(-/-) mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS-R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS-R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age-associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS-R antagonist abolishes ghrelin's effects and increases UCP1 expression. Hence, GHS-R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging-associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.     

Ghrelin octanoylation by ghrelin O-acyltransferase: protein acylation impacting metabolic and neuroendocrine signalling

The acylated peptide hormone ghrelin impacts a wide range of physiological processes but is most well known for controlling hunger and metabolic regulation. Ghrelin requires a unique posttranslational modification, serine octanoylation, to bind and activate signalling through its cognate GHS-R1a receptor. Ghrelin acylation is catalysed by ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) enzyme family. The ghrelin/GOAT/GHS-R1a system is defined by multiple unique aspects within both protein biochemistry and endocrinology. Ghrelin serves as the only substrate for GOAT within the human proteome and, among the multiple hormones involved in energy homeostasis and metabolism such as insulin and leptin, acts as the only known hormone in circulation that directly stimulates appetite and hunger signalling. Advances in GOAT enzymology, structural modelling and inhibitor development have revolutionized our understanding of this enzyme and offered new tools for investigating ghrelin signalling at the molecular and organismal levels. In this review, we briefly summarize the current state of knowledge regarding ghrelin signalling and ghrelin/GOAT enzymology, discuss the GOAT structural model in the context of recently reported MBOAT enzyme superfamily member structures, and highlight the growing complement of GOAT inhibitors that offer options for both ghrelin signalling studies and therapeutic applications.     

Water deprivation induces appetite and alters metabolic strategy in Notomys alexis: unique mechanisms for water production in the desert

Like many desert animals, the spinifex hopping mouse, Notomys alexis, can maintain water balance without drinking water. The role of the kidney in producing a small volume of highly concentrated urine has been well-documented, but little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. In Notomys, we found that water deprivation (WD) induced a sustained high food intake that exceeded the pre-deprivation level, which was driven by parallel changes in plasma leptin and ghrelin and the expression of orexigenic and anorectic neuropeptide genes in the hypothalamus; these changed in a direction that would stimulate appetite. As the period of WD was prolonged, body fat disappeared but body mass increased gradually, which was attributed to hepatic glycogen storage. Switching metabolic strategy from lipids to carbohydrates would enhance metabolic water production per oxygen molecule, thus providing a mechanism to minimize respiratory water loss. The changes observed in appetite control and metabolic strategy in Notomys were absent or less prominent in laboratory mice. This study reveals novel mechanisms for appetite regulation and energy metabolism that could be essential for desert rodents to survive in xeric environments.     

Effects of fat, protein, and carbohydrate and protein load on appetite, plasma cholecystokinin, peptide YY, and ghrelin, and energy intake in lean and obese men

While protein is regarded as the most satiating macronutrient, many studies have employed test meals that had very high and unsustainable protein contents. Furthermore, the comparative responses between lean and obese subjects and the relationships between energy intake suppression and gut hormone release remain unclear. We evaluated the acute effects of meals with modest variations in 1) fat, protein, and carbohydrate content and 2) protein load on gastrointestinal hormones, appetite, and subsequent energy intake in lean and obese subjects. Sixteen lean and sixteen obese men were studied on four occasions. Following a standardized breakfast, they received for lunch: 1) high-fat (HF), 2) high-protein (HP), 3) high-carbohydrate/low-protein (HC/LP), or 4) adequate-protein (AP) isocaloric test meals. Hunger, fullness, and gut hormones were measured throughout, and at t = 180 min energy intake at a buffet meal was quantified. In lean subjects, hunger was less and fullness greater following HF, HP, and AP compared with HC/LP meals, and energy intake was less following HF and HP compared with HC meals (P < 0.05). In the obese subjects, hunger was less following HP compared with HF, HC/LP, and AP meals, and energy intake was less following HP and AP compared with HF and HC meals (P < 0.05). There were no major differences in hormone responses to the meals among subject groups, but the CCK and ghrelin responses to HP and AP were sustained in both groups. In conclusion, HP meals suppress energy intake in lean and obese subjects, an effect potentially mediated by CCK and ghrelin, while obese individuals appear to be less sensitive to the satiating effects of fat.     

Meal timing and composition influence ghrelin levels, appetite scores and weight loss maintenance in overweight and obese adults

BackgroundAlthough dietary restriction often results in initial weight loss, the majority of obese dieters fail to maintain their reduced weight. Diet-induced weight loss results in compensatory increase of hunger, craving and decreased ghrelin suppression that encourage weight regain. A high protein and carbohydrate breakfast may overcome these compensatory changes and prevent obesity relapse.MethodsIn this study 193 obese (BMI 32.2 ± 1.0 kg/m²), sedentary non diabetic adult men and women (47 ± 7 years) were randomized to a low carbohydrate breakfast (LCb) or an isocaloric diet with high carbohydrate and protein breakfast (HCPb). Anthropometric measures were assessed every 4 weeks. Fasting glucose, insulin, ghrelin, lipids, craving scores and breakfast meal challenge assessing hunger, satiety, insulin and ghrelin responses, were performed at baseline, after a Diet Intervention Period (Week 16) and after a Follow-up Period (Week 32).ResultsAt Week 16, groups exhibited similar weight loss: 15.1 ± 1.9 kg in LCb group vs. 13.5 ± 2.3 kg in HCPb group, p = 0.11. From Week 16 to Week 32, LCb group regained 11.6 ± 2.6 kg, while the HCPb group lost additional 6.9 ± 1.7 kg. Ghrelin levels were reduced after breakfast by 45.2% and 29.5% following the HCPb and LCb, respectively. Satiety was significantly improved and hunger and craving scores significantly reduced in the HCPb group vs. the LCb group.ConclusionA high carbohydrate and protein breakfast may prevent weight regain by reducing diet-induced compensatory changes in hunger, cravings and ghrelin suppression. To achieve long-term weight loss, meal timing and macronutrient composition must counteract these compensatory mechanisms which encourage weight regain after weight loss.     

Central and peripheral effects of ghrelin on energy balance, food intake and lipid metabolism in teleost fish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue receptor. Ghrelin and its receptor system are present not only in peripheral tissues such as stomach and intestine, but also in the central nervous system of mammals. Interestingly, administration of ghrelin induces an orexigenic effect and also modifies locomotor activity, suggesting its involvement in feeding control and the regulation of energy balance, in addition to the regulation of growth hormone release. Information about ghrelin in non-mammals, such as teleost fish, has also been increasing, and important data have been obtained. An understanding of the evolutionary background of the energy regulation system and the central and peripheral roles of ghrelin in teleost fish could provide indications as to their roles in mammals, particularly humans. In this review, we overview the central and peripheral effects of ghrelin on energy balance, locomotor activity, and lipid metabolism in teleost fish.     

Bacterial protein meal in diets for growing pigs: effects on protein and energy metabolism

This experiment investigated the effects of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism of pigs from weaning to a live weight of 80 kg. Four litters with four castrated male pigs in each litter were used. The litters were divided into two blocks according to age. One pig from each litter was fed one of the four experimental diets. Soya-bean meal was replaced with BPM on the basis of digestible protein, and the BPM contents in the four diets were 0% (BP0), 5% (BP5), 10% (BP10) and 15% (BP15), corresponding to 0%, 17%, 35% and 52% of the digestible nitrogen (N), respectively. Four balance periods were performed, at the start of which the pigs weighed 9.5 kg, 20.7 kg, 45.3 kg and 77.2 kg, respectively. Once during each balance period, 22-h respiration experiments were performed using indirect calorimetry. Daily weight gain, feed intake and feed conversion rate were the same for all diets. The apparent digestibility of N was lower on diet BP10 than on BP0 (P = 0.002), whereas the apparent digestibility of energy was similar on all diets. The retention of nitrogen did not differ between diets and was 1.50, 1.53, 1.33 and 1.46 g N per kg^0.75 per day on BP0, BP5, BP10 and BP15, respectively. Neither metabolisable energy intake nor heat production were affected by inclusion level of BPM. Retention of energy was 620 (BP0), 696 (BP5), 613 (BP10) and 664 kJ/kg^0.75 per day (BP15), the differences among diets being non-significant. The N-free respiratory quotient was similar on all diets. It was concluded that the overall protein and energy metabolism in growing pigs were not affected when up to 50% of dietary N was derived from BPM.     

Rise of plasma ghrelin with weight loss is not sustained during weight maintenance

Objective: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects. Research Methods and Procedures: This study was a randomized clinical trial, with a 12‐month follow‐up period. Obese Mexican‐American women matched for age and BMI were randomized to a 12‐month WL program (n = 25) or no intervention (controls, n = 23). Interventions included diet, exercise, and orlistat. Body weight and fasting ghrelin, leptin, insulin, and glucose concentrations were measured at baseline and 6 and 12 months. Results: The WL group lost 8.5% of body weight after 6 months and maintained the new weight for the next 6 months. Ghrelin concentrations increased significantly at 6 months but returned to baseline at 12 months. Baseline ghrelin concentrations were directly related to the degree of WL achieved after 12 months. Controls experienced no change in BMI or ghrelin levels. There were no associations between plasma ghrelin and leptin or insulin concentrations. Discussion: Consistent with previous results, ghrelin rises in response to WL, perhaps as a counterregulatory mechanism. However, the present results indicate that ghrelin concentrations return to baseline with sustained weight maintenance, suggesting that its effects are unlikely to regulate long‐term energy balance. Baseline ghrelin concentrations are related to the degree of WL that can be achieved by active weight reduction.     

Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [125I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.     

Effect of bacterial protein meal on protein and energy metabolism in growing chickens

Abstract

This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2% (D2), 4% (D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3 – 7, 10 – 14, 17 – 21, 23 – 27, and 30 – 34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with groups of 6 chickens (period 1), 5 chickens (period 2), and one chicken (periods 3 – 5). After each balance period, one chicken in each cage was killed and the carcass weight was recorded. Chemical analyses were performed on the carcasses from periods 1, 3, and 5. Weight gain, feed intake, and feed conversion rate were found to be similar for all diets. Chickens on D0 retained 1.59 g N · kg^?0.75 · d^?1, significantly more than chickens on D2, D4, and D6, which retained 1.44 g, 1.52 g, and 1.50 g N · kg^?0.75 · d^?1, respectively. This was probably caused by the higher nitrogen content of D0. Neither the HE (p = 0.92) nor the retention of energy (p = 0.88) were affected by diet. Carcass composition was similar between diets, in line with the values for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.     

The hypoalbuminaemia of ovine fascioliasis: the influence of protein intake on the albumin metabolism of infected and of pair-fed control sheep.

Studies using ¹²⁵I-albumin and ⁵¹Cr-labelled plasma proteins showed that the hypoalbuminaemia which developed in sheep during the migratory stage of Fasciola hepatica infections was brought about by a combination of reduced albumin synthesis and plasma volume expansion. It was suggested that these changes were a reflection of the attendant liver damage and possibly of preferential utilisation of amino acids for immunoglobulin production. During the biliary stage of the disease, when the animals developed even more marked hypoalbuminaemia, increased albumin degradation arising from excessive plasma leakage into the gut were the outstanding features. The severity of these changes was closely linked to the state of the albumin pools which in turn was related to such factors as the plane of nutrition, appetite and fluke burden of the host. More albumin was catabolised by sheep with low fluke burdens, and in animals with the same level of infection, greater rates of catabolism were associated with a high protein intake. Sheep which catabolised most albumin became the least hypoalbuminaemic and survived longest. These animals also synthesised most albumin. It was shown that by impairing albumin synthesis, inappetence was an important additional factor in the hypoalbuminaemia of heavy infections, particularly if superimposed on a low protein diet. Nevertheless, irrespective of the size of their adult fluke burden, chronically-infected sheep were able to synthesise more albumin than pair-fed controls.     

Ghrelin, des-acyl ghrelin and obestatin on the gastrointestinal motility

Ghrelin, des-acyl ghrelin and obestatin are derived from a common prohormone, preproghrelin by posttranslational processing, originating from endocrine cells in the stomach. Ghrelin exerts stimulatory effects on the motility of antrum and duodenum in both fed and fasted state of animals. On the other hand, des-acyl ghrelin exerts inhibitory effects on the motility of antrum but not on the motility of duodenum in the fasted state of animals. Obestatin exerts inhibitory effects on the motility of antrum and duodenum in the fed state but not in the fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of ghrelin, CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, whereas CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin.     

杏仁中央核损毁对缺钠大鼠钠欲行为启动和表达的影响

目的:探讨杏仁中央核(CeA)损毁对缺钠大鼠钠欲行为启动和表达的影响。方法:将18只成年雄性SD大鼠随机分为3组(n=6):双侧Ce A损毁组、假损毁组和不损毁组,手术恢复后给予大鼠14 d低钠饲料摄食以建立缺钠大鼠模型,运用单笼双瓶选择测试方法观察缺钠大鼠在24 h内5个不同时间段对0.3 mol/L NaCl和自由饮水的摄入情况。应用免疫荧光化学染色方法观察杏仁中央核损毁与否对缺钠或正常大鼠孤束核内醛固酮敏感神经元活动的影响。结果:低钠饮食14 d后,大鼠对0.3 mol/L NaCl 24 h内饮用量和偏爱率比低钠饮食前明显增加(P<0.01);杏仁中央核损毁后缺钠大鼠对0.3 mol/L Na Cl溶液的摄入量和偏爱率显著下降(P<0.01)。杏仁中央核损毁对低钠饮食诱发的大鼠孤束核内醛固酮敏感神经元活动增加没有影响。结论:低钠饮食诱导大鼠钠欲行为表达增加;杏仁中央核损毁压抑缺钠大鼠钠欲行为的表达,而对缺钠大鼠的钠欲行为的启动没有影响。     

Genetic parameters for feed intake,litter weight,body condition and rebreeding success in primiparous Norwegian Landrace sows

The aim of this study was to estimate genetic parameters for feed intake recorded as farmers’ perception of young sows’ appetite for the first 3 weeks of lactation (APP) and feed intake recorded for one day in the 3rd week of lactation (FEED), litter weight (LW) at 3 weeks, sow body condition at weaning (BC) and the following five reproduction traits: weaning-to-service interval of 1 to 7 days (WSI7), weaning-to-service interval of 1 to 50 days (WSI50), delayed service or not (DELAYED), pregnant on first service or not (PREGNANT) and litter size in 2nd parity (NBT2). The analyses included data on 4606 Norwegian Landrace 1st-parity sows and their litters. The Gibbs sampling method was used. The traits DELAYED and PREGNANT were analysed as threshold traits and APP, FEED, LW, BC, WSI7, WSI50 and NBT2 were analysed as linear traits. The heritability estimates for APP and FEED were low (<0.1), whereas the estimates for DELAYED and PREGNANT were rather high (0.4 and 0.3). The heritability estimate for BC was 0.2. The genetic correlations confirmed the complexity of breeding for sow performance; selection for heavy 1st litters may lead to lower body condition at weaning, which in turn leads to lower reproductive performance and smaller litters in 2nd parity. Selection for higher sow feed intake would improve body condition, but the simple way of measuring feed intake tested in this study (APP and FEED) cannot be recommended because of the low heritability obtained for these traits.     

Effects of cold exposure on cyclic AMP concentration in plasma,liver, and brown and white adipose tissues in cold-acclimated rats

Effects of acute cold exposure on plasma energy substrates and tissue 3,5-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to –5°C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to –15°C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.Abbreviations cAMP cyclic 3,5-adenosine monophosphate - NST nonshivering thermogenesis - FFA free fatty acids - IBAT brown adipose tissue - WAT white adipose tissue     

Roles of protein kinase Cα isozyme in the regulation of oxidative stress and neuropeptide Y gene expression in phenylpropanolamine-mediated appetite suppression

Hypothalamic neuropeptide Y (NPY) is an appetite stimulant in the brain. Although regulation of NPY expression has been reported to contribute to the appetite-suppressing effect of phenylpropanolamine (PPA), it is still unknown if protein kinase C (PKC) is involved in this effect. Rats were daily treated with PPA for 4 days. Changes in food intake, hypothalamic NPY, PKC, and proopiomelanocortin (POMC) mRNA levels were assessed and compared. Results showed that the NPY gene was down-regulated following PPA treatment, which was parallel with the decrease of feeding. Moreover, several isotypes of PKC mRNA level (α, βI, βII, γ, δ, η, λ, ε, and ζ) were changed. Among these, α, δ, and λ PKC were up-regulated along with POMC gene expression which coincided with down-regulation of the NPY gene. To further determine if PKCα was involved, infusions of antisense oligonucleotide into the cerebroventricle were performed at 1 h before daily PPA treatment in free-moving rats. Results showed that PKCα knock-down could modify both anorexia induced by PPA and the NPY mRNA levels. Moreover, PKCα knock-down could also modify superoxide dismutase (SOD) gene expression. It is suggested that PKCα participates in the regulation of PPA-mediated appetite suppression via the modulation of NPY and SOD gene expression.     

Effect of BCAA intake during endurance exercises on fatigue substances,muscle damage substances,and energy metabolism substances

The increase rate of utilization of branched-chain amino acids (BCAA) by muscle is reduced to its plasma concentration during prolonged exercise leading to glycogen. BCAA supplementation would reduce the serum activities of intramuscular enzymes associated with muscle damage. To examine the effects of BCAA administration on fatigue substances (serotonin, ammonia and lactate), muscle damage substances (CK and LDH) and energy metabolism substances (FFA and glucose) after endurance exercise. Subjects (n = 26, college-aged males) were randomly divided into an experimental (n = 13, EXP) and a placebo (n = 13, CON) group. Subjects both EXP and CON performed a bout of cycle training (70% VO₂max intensity) to exhaustion. Subject in the EXP were administrated BCAA (78ml/kg·w) prior to the bout of cycle exercise. Fatigue substances, muscle damage substances and energy metabolism substances were measured before ingesting BCAAs and placebos, 10 min before exercise, 30 min into exercise, immediately after exercise, and 30 min after exercise. Data were analyzed by two-way repeated measure ANCOVA, correlation and statistical significance was set at p < 0.05. The following results were obtained from this study; 1. In the change of fatigue substances : Serotonin in the EXP tended to decreased at the 10 min before exercise, 30 min into exercise, post exercise, and recovery 30 min. Serotonin in the CON was significantly greater than the EXP at the10 min before exercise and recovery 30. Ammonia in the EXP was increased at the 10 min before exercise, 30 min into exercise, and post exercise, but significantly decreased at the recovery 30min (p < 0.05). Ammonia in the CON was significantly lower than the EXP at the 10 min before exercise, 30 min into exercise, and post exercise (p < 0.05). Lactate in the EXP was significantly increased at the 30 min into exercise and significantly decreased at the post exercise and recovery 30 min. Lactate in the CON was significantly lower than the EXP at the post exercise (p < 0.05). 2. In the change of muscle damage substances : CK in the EXP was decreased at the 10 min before exercise and increased at the 30 min into exercise and then decreased at the post exercise and recovery 30 min. CK in the CON was greater than the EXP. LDH in the EXP was decreased at the 10 min before exercise and increased at the 30 min into exercise and then decreased at the post exercise and recovery 30 min. LDH in the CON was higher than the EXP. 3. In the change of energy metabolism substances :Glucose in the EXP tended to decrease at the 10 min before exercise, 30 min into exercise, post exercise and recovery 30 min. Glucose in the CON was significantly greater than the EXP at the recovery 30 min (p < .05). FFA in both EXP and CON was increased at the post exercise and recovery 30 min. % increase for FFA in the EXP was greater than the CON at the post exercise and recovery 30 min. 4. The relationship of the fatigue substances, muscle damage substances and energy metabolism substances after endurance exercise indicated strongly a positive relationship between LDH and ammonia and a negative relationship between LDH and FFA in the EXP. Also, there were a strong negative relationship between glucose and FFA and a positive relationship between glucose and serotonin in the EXP. There was a strong positive relationship between CK and LDH and a strong negative relationship between FFA and glucose in the CON. These results indicate that supplementary BCAA decreased serum concentrations of the intramuscular enzymes as CK and LDH following exhaustive exercise. This observation suggests that BCAA supplementation may reduce the muscle damage associated with endurance exercise.