Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.

The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle.

Effects of Delaying Post-hatch Feeding on the Plasma Metabolites of Broiler Chickens Revealed by Targeted and Untargeted Metabolomics.

Exogenous nutrients are essential for body and skeletal muscle growth in newly hatched chicks, and delaying post-hatch feeding negatively affects body growth, meat yield, and meat quality. The aim of this study was to investigate the effects of delayed post-hatch feeding on the metabolic profiles of broiler chickens using a combination of targeted and untargeted metabolomics. Newly hatched chicks had either immediate free access to feed (freely fed chicks) or no access to feed from 0 to 2 days of age (delayed-fed chicks); both groups were subsequently provided feed ad libitum until 13 days of age. Untargeted metabolomic analysis was performed using gas chromatography-mass spectrometry, whereas targeted metabolomic analysis of amino acids was performed using high-performance liquid chromatography with ortho-phthalaldehyde derivatization. Delayed feeding increased the plasma levels of sucrose, maltose, serotonin, lactitol, gentiobiose, xylitol, threonic acid, and asparagine, and decreased the plasma levels of creatinine, aspartic acid, and glutamic acid. In addition, the digestibility of the nitrogen-free extract (starch and sugar) and the cecal butyric acid concentration increased in chicks subjected to delayed feeding. In contrast, delayed feeding did not affect muscle protein degradation or digestibility in chicks. Taken together, our results indicate that delaying feeding until 48 h post-hatch alters multiple metabolic pathways, which are accompanied by changes in intestinal carbohydrate digestion and cecal butyric acid content in broiler chickens.

Changes in metabolite content in the kidneys and skeletal muscles of rats fed magnesium-restricted diets.

A metabolomic study was performed on the kidneys and skeletal muscles of rats fed diets containing varying contents of Mg for 4 weeks. The kidneys are divided into two parts, the aerobic cortex and the anaerobic medulla, that differ in metabolism. The relative contents of 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvic acid increased with Mg restriction in both renal regions. In contrast, pyruvic acid content decreased with Mg restriction in the diets, suggesting an inhibitory conversion of phosphoenolpyruvic acid to pyruvic acid. The lactic acid content increased in both regions of the kidneys of Mg-restricted rats, implying changes towards a more glycolytic metabolism, possibly resulting from the impairment of mitochondrial function. There are two types of muscle fibers: glycolytic fast and oxidative slow muscle fibers. The soleus muscle consists of slow muscle fibers, whereas the gastrocnemius muscle consists of a combination of fast and slow muscle fibers. Similar to the changes in the kidneys, the contents of 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid increased in the soleus and gastrocnemius muscles with dietary Mg restriction. Unlike in the kidney, pyruvic acid content increased in the soleus muscle in response to Mg restriction. Severe Mg restriction decreased contents of carnosine and its constituent ß-alanine and increased the levels of purine derivatives such as xanthine and uric acid in the gastrocnemius muscle. The present study suggests a region-dependent sensitivity to dietary restriction of Mg, which may lead to the onset of various metabolic disorders.

Quantification of Nτ -Methylhistidine and Nπ-Methylhistidine in Chicken Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The concentration of Nτ-methylhistidine in plasma provides an index of skeletal muscle protein breakdown. This study aimed to establish a quantitative method for measuring the concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in chicken plasma, using liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were 1.56-50.00 µmol/L for Nτ-methylhistidine and 0.78-25.00 µmol/L for Nπ-methylhistidine. The proposed method detected changes in the plasma levels of Nτ-methylhistidine and Nπ-methylhistidine in response to fasting and re-feeding. These results suggest that the method developed in this study can be used for the simultaneous measurement of Nτ-methylhistidine and Nπ-methylhistidine in chicken plasma.

L-amino acid oxidase-1 is involved in the gut-liver axis by regulating 5-aminolevulinic acid production in mice.

L-amino acid oxidase (LAAO) is a metabolic enzyme that converts L-amino acids into ketoacids, ammonia, and hydrogen peroxide (H2O2). The generated H2O2 has previously been shown to have antibacterial and gut microbiota-modulatory properties in LAO1 knock-out (KO) mice. Since most microbial metabolites reach the liver through the portal vein, we examined gut-liver interactions in LAO1 KO mice. We found lower total cholesterol levels, higher glutamic pyruvic transaminase (GPT) levels in the serum, and higher pro-inflammatory cytokine mRNA expression in the liver tissue. In wild-type (WT) mice, LAO1 was expressed in gut tissues (ileum and colon). Microbiome analysis revealed that the abundance of some bacteria was altered in LAO1 KO mice. However, short-chain fatty acid (SCFAs) levels in cecal feces and gut permeability did not change. Fecal microbiota transplantation (FMT) revealed that feces from LAO1 KO mice slightly stimulated pro-inflammatory cytokine expression in the liver. During metabolomic analysis, 5-aminolevulinic acid (5-ALA) was the only metabolite found to be significantly upregulated in the portal and abdominal veins of the LAO1 KO mice. Intraperitoneal administration of 5-ALA to WT mice significantly increased IL-6 mRNA expression in the liver. These observations suggest that gut LAO1 plays a role in regulating 5-ALA production and that a high level of 5-ALA stimulates the liver to increase pro-inflammatory cytokine expression by disrupting LAO1 in mice.

Stimulation of uncoupling protein 1 expression by ß-alanine in brown adipocytes.

Carnosine, which is abundant in meat, is a dipeptide composed of ß-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that ß-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of ß-alanine (12.5-100 µM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of ß-alanine. Treatment with ß-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with ß-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, ß-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that ß-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, ß-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

Metabolism of Imidazole Dipeptides, Taurine, Branched-Chain Amino Acids, and Polyamines of the Breast Muscle Are Affected by Post-Hatch Development in Chickens.

To explore metabolic characteristics during the post-hatch developmental period, metabolomic analyses of breast muscle and plasma were performed in chickens. The most significant growth-related changes in metabolite levels were observed between seven and 28 days of age. Some of these metabolites are essential nutrients or reported as growth-promoting metabolites. In the muscle, two imidazole dipeptides-carnosine and its methylated metabolite, anserine-increased with the development. These dipeptide levels may be, in part, regulated transcriptionally because in the muscle mRNA levels of carnosine synthase and carnosine methylation enzyme increased. In contrast, taurine levels in the muscle decreased. This would be substrate availability-dependent because some upstream metabolites decreased in the muscle or plasma. In branched-chain amino acid metabolism, valine, leucine, and isoleucine decreased in the muscle, while some of their downstream metabolites decreased in the plasma. The polyamines, putrescine and spermidine, decreased in the muscle. Furthermore, mRNA levels associated with insulin/insulin-like growth factor 1 signaling, which play important roles in muscle growth, increased in the muscle. These results indicate that some metabolic pathways would be important to clarify metabolic characteristics and/or growth of breast muscle during the post-hatch developmental period in chickens.

The use of behavioral tests of fearfulness in chicks to distinguish between the Japanese native chicken breeds, Tosa-Kukin and Yakido.

The aim of this study was to investigate the differences in fearfulness between two Japanese native chicken breeds, Tosa-Kukin (TOK) and Yakido (YKD). In a tonic immobility test, TOK breed chicks showed longer duration and lower induction number compared with YKD. The number of peeps in YKD in an isolation test was lower than that in TOK, whereas there were more bouts of peeping in YKD than in TOK. In a manual restraint test, YKD showed more active responses and initiated peeps and struggling earlier than TOK. The three behavioral tests all indicated that YKD are less fearful than TOK chickens. A latent structures discriminant (OPLS-DA) analysis was used to identify behavioral parameters that contributed to the differences between the breeds. The major parameters were duration and number of inductions in the tonic immobility test and number of struggle in the manual restraint test. These results suggest that three behavioral tests can be used together to evaluate fearfulness of Japanese native breeds of chickens.

Effects of Dietary Defatted Meat Species on Metabolomic Profiles of Murine Liver, Gastrocnemius Muscle, and Cecal Content.

In both humans and animals, meat not only constitutes one of the sources of protein, but also includes various water-soluble bioactive substances such as imidazole peptides (carnosine and anserine) and taurine. Previous studies demonstrated that dietary meat species could differently affect physiological functions; however, the mechanisms of this remain unclear. To explore the physiological effects of dietary defatted meat species, especially on metabolism, we investigated their influence on the metabolomic profiles of the liver, gastrocnemius muscle, and cecal content in mice. Casein (control) or a defatted meat species (beef leg, pork leg, chicken leg, or chicken breast) was supplied as the major protein source in the diet for four weeks, and metabolism-related molecules were measured by gas chromatography-mass spectrometry. We found that various metabolite levels in tissues and cecal content differed according to the types of dietary protein consumed. Specifically, differences in carnosine, 1,5-anhydro-glucitol, inositol, butyric acid, and propionic acid were clearly observed. Among them, the highest carnosine intake by dietary pork leg was clearly related to the highest carnosine level in the liver. In addition, taurine intake was suggested to be linked to some metabolic pathways including taurine and hypotaurine metabolism in cecal content. These results provide additional knowledge of the effects of different dietary meat species on human and animal health.

Transcriptional Activation of Chac1 and Other Atf4-Target Genes Induced by Extracellular l-Serine Depletion is negated with Glycine Consumption in Hepa1-6 Hepatocarcinoma Cells.

Mouse embryonic fibroblasts lacking D-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step of de novo synthesis of l-serine, are particularly sensitive to depletion of extracellular L-serine. In these cells, depletion of l-serine leads to a rapid reduction of intracellular L-serine, cell growth arrest, and altered expression of a wide variety of genes. However, it remains unclear whether reduced availability of extracellular l-serine elicits such responses in other cell types expressing Phgdh. Here, we show in the mouse hepatoma cell line Hepa1-6 that extracellular l-serine depletion transiently induced transcriptional activation of Atf4-target genes, including cation transport regulator-like protein 1 (Chac1). Expression levels of these genes returned to normal 24 h after l-serine depletion, and were suppressed by the addition of l-serine or glycine in the medium. Extracellular l-serine depletion caused a reduction of extracellular and intracellular glycine levels but maintained intracellular l-serine levels in the cells. Further, Phgdh and serine hydroxymethyltransferase 2 (Shmt2) were upregulated after l-serine depletion. These results led us to conclude that the Atf4-mediated gene expression program is activated by extracellular l-serine depletion in Hepa1-6 cells expressing Phgdh, but is antagonized by the subsequent upregulation of l-serine synthesis, mainly from autonomous glycine consumption.

Analysis of infant microbiota composition and the relationship with breast milk components in the Asian elephant (Elephas maximus) at the zoo.

The prevention of diseases through health control is essential at zoos. Here, we investigated the gut microbiota formation during infancy in an Asian elephant and compared the composition between infant and mother. Besides, we analyzed the components of breast milk and examined the correlation with the infant gut microbiota. Analysis revealed the gut microbiota of the infant contained high amount of Lactobacillales and its diversity was relatively low compared to that of the mother. We found several milk components, showed a positive correlation with the change of Lactobacillales. The present study revealed the mechanism of gut microbiota formation during infancy in an Asian elephant and provides important insights into the health control of Asian elephants in zoos.

Effects of Cyclic High Ambient Temperature and Dietary Supplementation of Orotic Acid, a Pyrimidine Precursor, on Plasma and Muscle Metabolites in Broiler Chickens.

The aim of this study was to evaluate the effects of high ambient temperature (HT) and orotic acid supplementation on the plasma and muscle metabolomic profiles in broiler chickens. Thirty-two 14-day-old broiler chickens were divided into four treatment groups that were fed diets with or without 0.7% orotic acid under thermoneutral (25 ± 1 °C) or cyclic HT (35 ± 1 °C for 8 h/day) conditions for 2 weeks. The chickens exposed to HT had higher plasma malondialdehyde concentrations, suggesting an increase in lipid peroxidation, which is alleviated by orotic acid supplementation. The HT environment also affected the serine, glutamine, and tyrosine plasma concentrations, while orotic acid supplementation affected the aspartic acid, glutamic acid, and tyrosine plasma concentrations. Untargeted gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS)-based metabolomics analysis identified that the HT affected the plasma levels of metabolites involved in purine metabolism, ammonia recycling, pyrimidine metabolism, homocysteine degradation, glutamate metabolism, urea cycle, ß-alanine metabolism, glycine and serine metabolism, and aspartate metabolism, while orotic acid supplementation affected metabolites involved in pyrimidine metabolism, ß-alanine metabolism, the malate-aspartate shuttle, and aspartate metabolism. Our results suggest that cyclic HT affects various metabolic processes in broiler chickens, and that orotic acid supplementation ameliorates HT-induced increases in lipid peroxidation.

Nutritional Characteristics and Functions of D-Amino Acids in the Chicken.

D-Amino acids occur in modest amounts in bacterial proteins and the bacterial cell wall, as well as in peptide antibiotics. Therefore, D-amino acids present in terrestrial vertebrates were believed to be derived from bacteria present in the gastrointestinal tract or fermented food. However, both exogenous and endogenous origins of D-amino acids have been confirmed. Terrestrial vertebrates possess an enzyme for converting certain L-isomers to D-isomers. D-Amino acids have nutritional aspects and functions, some are similar to, and others are different from those of L-isomers. Here, we describe the nutritional characteristics and functions of D-amino acids and also discuss the future perspectives of D-amino acid nutrition in the chicken.

Beef extract supplementation promotes myoblast proliferation and myotube growth in C2C12 cells.

PURPOSE: We previously determined that the intake of beef extract for 4 weeks increases skeletal muscle mass in rats. Thus, this study aimed to clarify whether beef extract has a hypertrophic effect on muscle cells and to determine the signaling pathway underlying beef extract-induced myotube hypertrophy. METHODS: We assessed the effects of beef extract supplement on mouse C2C12 skeletal muscle cell proliferation and differentiation and myotube growth. In addition, the phosphorylation of Akt, ERK1/2, and mTOR following beef extract supplementation was examined by western blotting. Furthermore, the bioactive constituents of beef extract were examined using amino acid analysis and dialysis. RESULTS: In the proliferative stage, beef extract significantly increased myoblast proliferation. In the differentiation stage, beef extract supplementation did not promote myoblast differentiation. In mature myotubes, beef extract supplementation increased myotube diameter and promoted protein synthesis. Although Akt and ERK1/2 levels were not affected, beef extract supplementation increased mTOR phosphorylation, which indicated that the mTOR pathway mediates beef extract-induced myotube hypertrophy. The hypertrophic activity was observed in fractions of > 7000 Da. CONCLUSIONS: Beef extract promoted C2C12 myoblast proliferation and C2C12 myotube hypertrophy. Myotube hypertrophy was potentially induced by mTOR activation and active components in beef extract were estimated to be > 7000 Da.

Feeding the Outer Bran Fraction of Rice Alters Hepatic Carbohydrate Metabolism in Rats.

Dietary intake of fiber-rich food has been reported to contribute to multiple health benefits. The aim of the current study is to investigate the effects of a diet containing the outer bran fraction of rice (OBFR), which is rich in insoluble fiber, on the intestinal environment and metabolite profiles of rats. Fourteen 8-week-old male Sprague-Dawley rats were divided into a control group and an OBFR group. For a period of 21 days, the control group was fed a control diet, while the OBFR group was fed a diet containing 5% OBFR. Metabolomics analysis revealed drastic changes in the cecal metabolites of the rats fed the OBFR diet. Furthermore, in the plasma and liver tissue, the concentrations of metabolites involved in pyruvate metabolism, the pentose phosphate pathway, gluconeogenesis, or valine, leucine, isoleucine degradation were changed. Concordantly, the OBFR diet increased the expression of genes encoding enzymes involved in these metabolic pathways in the livers of the rats. Collectively, these results suggest that the OBFR diet altered the concentrations of metabolites in the cecal contents, plasma, and liver, and the hepatic gene expressions of rats, and that this may have mainly contributed to carbohydrate metabolism in the liver.

Elevated quinolinic acid levels in cerebrospinal fluid in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a rare neurodegenerative disorder caused by a persistent infection with aberrant measles virus. Indoleamine-2, 3-dioxygenase (IDO) initiates the increased production of kynurenine pathway (KP) metabolites quinolinic acid (QUIN), which has an excitotoxic effect for neurons. We measured serum IDO activity and cerebrospinal fluid (CSF) levels of QUIN. The CSF QUIN levels were significantly higher in SSPE patients than in controls, and increased according as neurological disability in a patient studied. Elevation of CSF QUIN and progression of SSPE indicate a pathological role of KP metabolism in the inflammatory neurodestruction.

Metabolomics Approach Reveals the Effects of Breed and Feed on the Composition of Chicken Eggs.

Chicken eggs provide essential nutrients to consumers around the world. Although both genetic and environmental factors influence the quality of eggs, it is unclear how these factors affect the egg traits including egg metabolites. In this study, we investigated breed and feed effects on 10 egg traits, using two breeds (Rhode Island Red and Australorp) and two feed conditions (mixed feed and fermented feed). We also used gas chromatography-mass spectrometry (GC-MS/MS) to analyze 138 yolk and 132 albumen metabolites. Significant breed effects were found on yolk weight, eggshell weight, eggshell colors, and one albumen metabolite (ribitol). Three yolk metabolites (erythritol, threitol, and urea) and 12 albumen metabolites (erythritol, threitol, ribitol, linoleic acid, isoleucine, dihydrouracil, 4-hydroxyphenyllactic acid, alanine, glycine, N-butyrylglycine, pyruvic acid, and valine) were significantly altered by feed, and a significant interaction between breed and feed was discovered in one albumen metabolite (N-butyrylglycine). Yolk and albumin had higher levels of sugar alcohols when hens were fed a fermented diet, which indicates that sugar alcohol content can be transferred from diet into eggs. Linoleic acid was also enriched in albumen under fermented feed conditions. This study shows that yolk and albumen metabolites will be affected by breed and feed, which is the first step towards manipulating genetic and environmental factors to create "designer eggs."

Chronic retinoic acid treatment induces differentiation and changes in the metabolite levels of brown (pre)adipocytes.

Dietary vitamin A status affects energy metabolism. The present study explored the effect of all-trans retinoic acid (ATRA) on the expression levels of molecules and metabolites of brown adipocytes. Chronic ATRA treatment was initiated during the early stage (days 0-8) or late stage (days 8-12) of adipogenesis. Treatment with ATRA during the early and late stage of adipogenesis resulted in an increase in the expression level of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12, respectively, whereas expression of Pgc-1α, another gene expressed during brown adipogenesis, was unaffected by ATRA. Non-targeted metabolomic analyses indicated that the pathways related to the glucose metabolism were affected by ATRA, irrespective of the differentiation stage. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. ATRA decreased the cellular level of aconitic acid, fumaric acid, and malic acid on day 12 but not on day 8. Furthermore, ATRA increased the expression level of Hxk2 and downregulated the expressions of G6pdh and Pfkl/Pfkp on day 8 but not on day 12. Together, the results indicate that the chronic treatment with ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism. SIGNIFICANCE OF THE STUDY: Significance of the study treatment with all-trans retinoic acid (ATRA) during the early and late stage of adipogenesis increased the expression of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. The present results indicate that ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism.

Correlation between skeletal muscle fiber type and free amino acid levels in Japanese Black steers.

Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26-month-old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow-twitch fibers.

Metabolic changes in adipose tissues in response to ß3 -adrenergic receptor activation in mice.

Brown and beige adipocytes dissipate energy as heat. Thus, the activation of brown adipocytes and the emergence of beige adipocytes in white adipose tissue (WAT) are suggested to be useful for preventing and treating obesity. Although ß3 -adrenergic receptor activation is known to stimulate lipolysis and activation of brown and beige adipocytes, fat depot-dependent changes in metabolite concentrations are not fully elucidated. The current study examined the effect of treatment with CL-316,243, a ß3 -adrenergic receptor agonist, on the relative abundance of metabolites in interscapular brown adipose tissue (iBAT), inguinal WAT (ingWAT), and epididymal WAT (epiWAT). Intraperitoneal injection of CL-316,243 (1 mg/kg) for 3 consecutive days increased the relative abundance of several glycolysis-related metabolites in all examined fat depots. The cellular concentrations of metabolites involved in the citric acid cycle and of free amino acids were also increased in epiWAT by CL-316,243. CL-316,243 increased the expression levels of several enzymes and transporters related to glucose metabolism and amino acid catabolism in ingWAT and iBAT but not in epiWAT. CL-316,243 also induced the emergence of more beige adipocytes in ingWAT than in epiWAT. Furthermore, adipocytes surrounded by macrophages were detected in the epiWAT of mice given CL-316,243. The current study reveals the fat depot-dependent modulation of cellular metabolites in CL-316,243-treated mice, presumably resulting from differential regulation of cell metabolism in different cell populations.