Effect of growth hormone on glycogenesis in cerebral cortical slices

The uptake of glucose by cerebral cortical slices of rats was found to be enhanced by insulin by Rafaelsen (1961) and Genes and Charnaya (1966). This was confirmed by Prasannan and Subrah-manyam (1965) and more recently by Nelson , Schultz , Pasoneau and Wry (1968). Eisenberg and Seltzer (1962) and Gotistein , Held , Sebenng and Walpurger (1965) obtained evidence for a direct effect of insulin on the entry of glucose into brain and on its metabolism in this tissue. A marked resynthesis of glycogen was demonstrated with glucose as substrate by Lebaron (1955) and Mcilwain and Tresize (1956) in cerebral cortical slices of the guinea pig. Prasannan and Subrahmanyam (1965) obtained evidence for a similar resynthesis of glycogen in cerebral cortical slices of the rat. Addition of 0.2 unit of insulin per 3.5 ml of incubating medium gave rise to an increase of 60 per cent in the resynthesis of glycogen in these slices. The incorporation of ¹⁴C from labelled glucose into glycogen and CO₂ by cerebral cortical slices of normal and alloxan diabetic rats and the stimulation of the incorporation into glycogen by insulin in vitro was reported by Visweswaran , Prasannan and Subrahmanyam (1969). An insulin-like action of growth hormone on the carbohydrate metabolism was reported by Ketterer , Randle and Young (1967) and Manchester and Young (1961). It was believed to be due to the formation of a polypeptide breakdown product of growth hormone which has biological insulin-like properties. Park , Brown , Cornbluth , Daughaday and Krahl . (1952) reported an increased uptake of glucose by isolated rat diaphragm due to the action of growth hormone which is similar to that of insulin. Hence, it was considered appropriate to study the incorporation of ¹⁴C from labelled glucose into glycogen and CO₂ by cerebral slices of growth hormone treated rats and the effect of growth hormone treatment on the activities of the enzymes concerned with glycogenesis in rat cerebral cortex.     

Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-<Superscript>13</Superscript>C]Glucose and [1,2-<Superscript>13</Superscript>C]Acetate as Substrates

Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15–90 min with unlabeled glucose in combination with [U-¹³C]glucose or [1,2-¹³C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for ¹³C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography–mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured ¹³C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of ¹³C-labeling observed with [U-¹³C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2-¹³C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using ¹³C-labeling (%) data obtained from mass spectrometry. Based on this approach we suggest that cellular metabolic compartmentation in hippocampus and cerebral cortex is very similar.     

Towards a valid preparation for the in vitro study of energy metabolism in fish muscles

Slices of lateral red muscle and isolated intact Musculus protractor hyoidei of goldfish were examined for their suitability as model systems for the in vitro study of muscular energy metabolism. Slicing of red muscle causes a strong breakdown of ATP and creatine phosphate and a lowering of the adenylate energy charge. Furthermore, slices do not recover when incubated in an oxygenated balanced salt solution, but show a continuous depletion of direct energy reserves. In contrast with red muscle slices, the Musculus protractor hyoidei can be isolated in an intact state, as shown by constancy of creatine phosphate and adenylate levels and stability of the adenylate energy charge during incubation. Therefore, isolated M. protractor hyoidei seems to be promising as a model system for the in vitro study of muscular energy metabolism.     

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage

Summary.  Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Effect of phosphatidylethanolamine and its constituent base on the metabolism of linoleic acid in rat liver

Dietary phosphatidylethanolamine (PE) contributes the circulatory and hepatic free-ethanolamine in rats (Ikeda et al. (1987) Biochim. Biophys. Acta 921, 245). A role for circulatory ethanolamine has not been defined; however, our recent studies have shown that exogenous ethanolamine influences cholesterol and linoleic acid metabolism in rats (Imaizumi et al. (1983) J. Nutr. 113, 2403). In order to understand the role of dietary PE the effects of PE and its base on the hepatic metabolism of linoleic acid were investigated in vivo and in primary cultured hepatocytes in rats. Dietary PE increased the plasmic level of ethanolamine from 37 to 52 microM and decreased the ratio of arachidonate to linoleate in hepatic phospholipids. Activity of hepatic delta 6-desaturase decreased in rats given PE and the desaturation of [14C]linoleate in the cultured hepatocytes decreased by the addition of ethanolamine. Secretion [14C]linoleate labeled very-low-density lipoprotein from the cultured hepatocytes decreased by the addition of ethanolamine. Dietary PE caused an increased formation of CO2 from [14C]acetate by liver slices, and ethanolamine added to the hepatocytes caused an increased oxidation of [14C]linoleate and a suppression of fatty acid synthesis from [3H]serine. These results suggest that ethanolamine derived from the dietary PE plays a regulatory role in the linoleate metabolism in the liver.     

Phospholipid synthesis in aging potato tuber tissue

The effect of activation (“aging”) of potato tuber slices on their phospholipid metabolism was investigated. Aged slices were incubated with ¹⁴C labeled choline, ethanolamine, methionine, serine, and acetate. In all cases, the incorporation of radioactivity into the lipid fraction increased with the length of time the slices were aged. This incorporation was shown to be true synthesis and not exchange between precursors and existing phospholipids.

The increased incorporation of labeled choline into lipids was mainly due to an increase in its uptake by the tissue, the presence of actidione during aging prevented this increased uptake. The increase in the incorporation of labeled acetate into lipids resulted from the development of a fatty acid synthetase during aging. In the case of ethanolamine, both its uptake into the tissue and its incorporation into the lipid fraction increased.

The phospholipids formed from these precursors were identified by paper and thin-layer chromatography. The major compound formed from choline was lecithin, while phosphatidylethanolamine and a small amount of lecithin were formed from ethanolamine.

     

Comparison of Two Superfusion Systems for Study of Neurotransmitter Release from Rat Cerebral Cortex Slices

Depolarization-induced release of [3H]gamma-aminobutyric acid (GABA) and [3H]noradrenaline (NA) from rat cerebral cortex slices was studied in two superfusion systems: one with stationary and the other one with continuously shaken slice compartments. Calcium-dependent depolarization-induced release of GABA and NA could be demonstrated only with shaken slices. GABA, but not NA, could also be released by high K+ media and veratridine from stationary slices. Synaptic transmitter releasing mechanisms are apparently damaged in stationary slices, possibly due to impaired energy metabolism.     

Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out

Transduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (K(ATP)) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing K(ATP) channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to K(ATP) channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network.     

The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.     

EFFECTS OF OUABAIN ON ACID-SOLUBLE PHOSPHATES AND ELECTROLYTES OF ISOLATED CEREBRAL TISSUES IN PRESENCE OR ABSENCE OF CALCIUM

—(1) Cerebral slices were incubated in Ca²⁺-free media or in media which contained 2.8 mm -Ca²⁺. Omission of Ca²⁺ brought about a drop in creatine phosphate content of 28 per cent, as well as a drop of 3–10 per cent in non-inulin K⁺ content. There was little change in content of 10-min phosphate or of non-inulin Na⁺. (2) Ouabain in concentrations of to M increased the loss of K⁺ from the slice and caused a rise in Na⁺ content. The changes were most marked in Ca²⁺-free media. Creatine phosphate levels were depressed by ouabain both in the presence or absence of Ca²⁺. In the absence of Ca²⁺, the lowering of phosphocreatine did not occur until significant shifts in K⁺ had taken place. In contrast, slices incubated in Ca²⁺-containing media lost creatine phosphate and K⁺ at about the same rate. (3) When ouabain and labelled phosphate were added simultaneously, there was little difference in the rate of incorporation of label into creatine phosphate in media which differed in Ca²⁺ concentration. However, incorporation of ^azP-labelled phosphate into creatine phosphate was decreased by 30–40 per cent in media which lacked Ca²⁺ when ouabain was added 15 min prior to the labelled phosphate. This change was not observed when the media contained Ca²⁺. (4) Ouabain did not affect oxidative phosphorylation or respiratory control when added directly to bovine brain mitochondrial preparations. (5) The results suggest that the previously observed depression of respiration brought about by ouabain in Ca²⁺-deficient media is not a good indicator of the proportion of the cell's metabolism used for active cation transport. Under these conditions, the inhibition of cation transport is accompanied by a drop in slice content of high-energy phosphate which may represent a secondary effect of ouabain, or of cytoplasmic alterations brought about by ouabain, on energy-producing processes.     

Comparison of In Vitro Ischemia-Induced Disturbances in Energy Metabolism and Protein Synthesis in the Hippocampus of Rats and Gerbils

Abstract: To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue-specific or hemodynamic factors, ischemia-induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37°C, 34°C, or 31°C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia-induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.     

Characterization of the Copper-binding of Phosphatidyl Ethanolamine Emulsion in Its Rapid Autoxidation

The copper-catalyzed O₂ uptake of phosphatidyl ethanolamine emulsion was measured by the Warburg’s manometry. When EDTA (ethylenediamine, tetraacetic acid) was added to the emulsion, EDTA inactivated copper stoichiometrically in molar ratio of 1: 1. Mono-ethanolamine, α-glycerophosphoric acid, O-phosphoryl ethanolamine, and glyceryl phosphoryl ethanolamine were not effective. IDA (iminodiacetic acid) depressed the O₂ uptake of phosphatidyl ethanolamine and the affinity of phosphatidyl ethanolamine to copper was estimated as one-thirtieth that of IDA. The emusion diluted with Tween 20 showed lower affinity to copper of one-tenth of the original emulsion. At the interface of the phosphatidyl ethanolamine, its high affinity to copper like chelate effect is assumed.     

The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension

Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O₂). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

Amides of creatine: perspectives of neuroprotection

We evaluated the efficacy of derivatives of creatine and amino acids (CrAA) for decreasing cerebral injury in rats with transient middle cerebral artery occlusion (MCAO). Neuroprotective effects of amides of creatine and glycine (CrGlyOEt), phenylalanine (CrPheNH2), thyrosine (CrTyrNH2), and GABA (CrGABAOEt) were investigated. Brain injury was evaluated on day 2 after transient MCAO using a TTC staining of brain slices. Compared with the MCAO control group, all the CrAms showed decreased cerebral injury (p < 0.05). However CrPheNH2, CrTyrNH2, and CrGABAOEt were toxic after intravenous administration and investigated only after intraperitoneal injection. CrGlyOEt did not show any toxicity at dose of 1 mmol/kg. These data evidenced that creatinyl amides can represent promising candidates for the development of new drugs useful in brain ischemia treatment.     

ACTIONS OF l-GLUTAMATE AND RELATED AMINO ACIDS ON OXYGEN UPTAKE, LACTATE PRODUCTION AND NADH LEVELS OF RAT BRAIN IN VITRO

Abstract— A range of acidic amino acids differing in (i) their potency as neuronal excitants, (ii) their transport properties and (iii) their ability to act as substrates for metabolism have been compared with respect to their effects on energy metabolism of rat cerebral cortex in vitro. l -Glutamate, and d - and l -homocysteate, increased tissue slice NADH levels, and the same three amino acids, together with d -glutamate and kainate, increased oxygen uptake by the slices. It was concluded that these effects were predominantly due to neuronal depolarization and the ensuing activation of ion pump mechanisms. l -Glutamate, d -glutamate and l -homocysteate increased lactate production by the slices, whereas d -homocysteate and kainate did not. Since the two latter amino acids are the strongest neuroexcitants but probably the least rapidly transported, it is suggested that stimulation of lactate production in slices by amino acid excitants is a consequence of the energy requirements of active uptake of the amino acids, and probably occurs mainly in glial cells. Although the metabolism of l -glutamate appeared not to be an essential requirement for the effects observed with this amino acid in the present work, such metabolism may make a proportionately greater contribution under sub-optimal conditions of slice preparation and incubation, where electrical activity of the tissue may be impaired.     

Overexpression, purification, and preliminary X-ray crystallographic analysis of human brain-type creatine kinase

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transferred through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P43212, with cell parameters of a=b=97.963, c= 164.312 A, and alpha=beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (Vm) of 1.80 A3 Da-1 and a solvent content of 31.6%.     

Effects of Amide Creatine Derivatives in Brain Hippocampal Slices, and Their Possible Usefulness for Curing Creatine Transporter Deficiency

The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood–brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.     

EARLY CHANGES IN RESPIRATION, AEROBIC GLYCOLYSIS AND CELLULAR NAD(P)H IN SLICES OF RAT CEREBRAL CORTEX EXPOSED TO ELEVATED CONCENTRATIONS OF POTASSIUM

Abstract— The initial effects of an elevated potassium concentration (30 m m ) on the energy metabolism of incubated slices of rat cerebral cortex have been examined using spectrophotometric and polarographic techniques. Respiratory responses to additions of potassium were found to be definitely limited in time. This response was followed by an increase in the rate of aerobic glycolysis. Slice NAD(P)H and cytochrome b paralleled this metabolic sequence by exhibiting an initial oxidation followed by a net increase in the steady-state levels of reduced intermediates, particularly in the case of NAD(P)H. Substitution of pyruvate (10 m m ) for glucose in the standard incubation media produced significant alterations in the respiratory responses to the addition of potassium. Although the period of increased oxygen consumption was again limited it was somewhat greater in magnitude and significantly prolonged in time relative to changes observed with glucose as substrate. Changes in steady-state levels of NAD(P)H were altered similarly and the net increase of NAD(P)H was not observed with pyruvate as substrate. We suggest that the metabolic responses of brain slices to increased potassium do not involve simultaneous activation of the respiratory and glycolytic pathways as has been previously assumed. Rather, a distinctly biphasic response is observed reminiscent of the Crabtree effect observed in other systems.     

Analysis of metabolome changes in the HepG2 cells of apatinib treatment by using the NMR-based metabolomics

Neovascularization is required for the growth of tumors, vascular endothelial growth factor (VEGF) and related signal pathways are important in tumor angiogenesis. Apatinib is a highly selective and potent antiangiogenesis drug targeting the receptor of VEGFR2, blocking downstream signal transduction and inhibiting angiogenesis of tumor tissue. Apatinib has a wide range of antitumor activities in vitro and in vivo, but its effect on metabolic changes has not deeply research at present. Nowadays, our research first systematically studied the metabolic changes affected by apatinib in the HepG2 cells at the half-maximal inhibitory concentration value. We used the metabolomics by using ¹H nuclear magnetic resonance (¹H-NMR) to analyze the HepG2 cell culture media. Multivariable Statistics was applied to analyze the ¹H-NMR spectra of the cell media, including principal component analysis, partial least squares discriminant analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA). Compared with the uncultured and cultured media (negative/positive control), the metabolic phenotypes were changed in the apatinib treatment with a continuous effect over time. The metabolic pathway analysis is shown that the mainly disturbed metabolic pathways pyruvate metabolism, alanine, aspartate, and glutamate metabolism and amino acid metabolism associated with them in the apatinib treatment. The differential metabolites which were identified from the reconstructed OPLS-DA loading plots also reflected in these disturbed metabolic pathways. Our works could allow us to well understand the therapeutic effect of apatinib, especially in metabolism.