Effects of fluorophore structure and hydrophobicity on the uptake and metabolism of fluorescent lipid analogs

Cellular transport and metabolism of fatty acids are integral components of lipid metabolism, but the mechanisms and regulation involved are poorly understood. A variety of commercially available fluorescent analogs of fatty acids, are potentially useful probes for the study of lipid metabolism by such techniques as cell sorting and fluorescence microscopy. We have screened a series of fluorescent fatty acids to identify analogs that would reliably simulate the metabolic behavior of natural fatty acids; i.e., similar kinetics of transport, of intracellular movement, and of metabolic fate. The metabolic behavior of these analogs was compared with those of some naturally occurring fatty acids in HepG2 cells, which are a good model of some aspects of hepatic function. Fluorescent analogs containing polar fluorophores yielded the lowest rates of cellular uptake and conversion to acylated lipid products. Similarly, fluorescent analogs with the fluorophore located near the carboxylic acid group were poorly metabolized. Fatty acid analogs containing anthracene or pyrene at the n-terminus of the acyl chain were the most extensively incorporated into cellular lipids. The types and amounts of labeled lipid products formed from these analogs and from natural fatty acids were similar. Pyrene-labeled analogs have spectral properties that can be measured fluorometrically at very low concentrations. Therefore, we compared the cellular metabolism of 12-(1-pyrenyl)dodecanoic acid with those of palmitic and oleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic flux analysis of cultured hepatocytes exposed to plasma

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.     

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.     

Grazing rates on toxic and non-toxic strains of cyanobacteria by Hypophthalmichthys molitrix and Oreochromis niloticus

The tilapia Oreochromis niloticus and the silver carp Hypophthalmichthys molitrix were exposed to toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa in order to determine if cells of the toxic strain were ingested and, if not, by what mechanism they were excluded. Enumeration of cyanobacterial particles before and after exposure to fish showed that there were no significant differences (P<0.05) at the end of the trial between the toxic treatment and the control consisting of toxic M. aeruginosa with no fish. Fish exposed to the non-toxic strain increased opercular beat rate, elevating the volumes of water and food material passed over the gills whereas those that were held in the toxic strain did not. Of the cyanobacterial toxins (microcystins) presented to the fish, most were in the cyanobacterial cells, toxin levels in the water being below the level of detectability (<250 ng l⁻¹), The ability of the fish to differentiate between toxic and non-toxic cyanobacterial strains may thus be determined by very low levels of extracellular microcystins or/and other features which distinguish toxic from non-toxic M. aeruginosa strains, such as cell surface components.     

Primary and secondary metabolism in the sun‐exposed peel and the shaded peel of apple fruit

The metabolism of carbohydrates, organic acids, amino acids and phenolics was compared between the sun‐exposed peel and the shaded peel of apple fruit. Contents of sorbitol and glucose were higher in the sun‐exposed peel, whereas those of sucrose and fructose were almost the same in the two peel types. This was related to lower sorbitol dehydrogenase activity and higher activities of sorbitol oxidase, neutral invertase and acid invertase in the sun‐exposed peel. The lower starch content in the sun‐exposed peel was related to lower sucrose synthase activity early in fruit development. Dark respiratory metabolism in the sun‐exposed peel was enhanced by the high peel temperature due to high light exposure. Activities of most enzymes in respiratory metabolism were higher in the sun‐exposed peel, but the concentrations of most organic acids were relatively stable, except pyruvate and oxaloacetate. Due to the different availability of carbon skeletons from dark respiration in the two peel types, amino acids with higher C/N ratios are accumulated in the sun‐exposed peel whereas those with lower C/N ratios are accumulated in the shaded peel. Contents of anthocyanins and flavonols and activities of phenylalanine ammonia‐lyase, UDP‐galactose:flavonoid 3‐O‐glucosyltransferase and several other enzymes were higher in the sun‐exposed peel than in the shaded peel, indicating the entire phenylpropanoid pathway is upregulated in the sun‐exposed peel. Comprehensive analyses of the metabolites and activities of enzymes involved in primary metabolism and secondary metabolism have allowed us to gain a full picture of the metabolic network in the two peel types under natural light exposure.     

Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2‐C3A cells were cultured under varying levels of glucose (high, low, and glucose‐free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose‐free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose‐free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2‐C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose‐free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose‐free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2‐C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2‐C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347–356. © 2010 Wiley Periodicals, Inc.     

The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.     

Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: role of culture medium composition

The biochemical mechanisms responsible for the ethanol-induced inhibition of testicular testosterone synthesis were studied in isolated rat Leydig cells in vitro. This inhibition was removed when HAM-F12 nutrient mixture was added to the DME culture medium. The components of HAM-F12, i.e. vitamins, amino acids and other supplements, were tested individually and the amino acids L-glutamate (Glu) and L-aspartate (Asp) were found to potentiate strongly the hCG stimulated testosterone synthesis. None of the other components of HAM-F12 had any effect upon testosterone synthesis or its ethanol-induced inhibition. Moreover, Glu, but not Asp, effectively reversed the acute inhibition of steroidogenesis by ethanol. These results demonstrate the importance of the composition of the culture media and provide the first piece of evidence that the metabolic stress in rat Leydig cells in vitro induced by the metabolism of ethanol can be overcome by proper culture medium supplementation.     

Dynamic Metabolic Disruption in Rats Perinatally Exposed to Low Doses of Bisphenol-A

Along with the well-established effects on fertility and fecundity, perinatal exposure to endocrine disrupting chemicals, and notably to xeno-estrogens, is strongly suspected of modulating general metabolism. The metabolism of a perinatally exposed individual may be durably altered leading to a higher susceptibility of developing metabolic disorders such as obesity and diabetes; however, experimental designs involving the long term study of these dynamic changes in the metabolome raise novel challenges. ¹H-NMR-based metabolomics was applied to study the effects of bisphenol-A (BPA, 0; 0.25; 2.5, 25 and 250 μg/kg BW/day) in rats exposed perinatally. Serum and liver samples of exposed animals were analyzed on days 21, 50, 90, 140 and 200 in order to explore whether maternal exposure to BPA alters metabolism. Partial Least Squares-Discriminant Analysis (PLS-DA) was independently applied to each time point, demonstrating a significant pair-wise discrimination for liver as well as serum samples at all time-points, and highlighting unequivocal metabolic shifts in rats perinatally exposed to BPA, including those exposed to lower doses. In BPA exposed animals, metabolism of glucose, lactate and fatty acids was modified over time. To further explore dynamic variation, ANOVA-Simultaneous Component Analysis (A-SCA) was used to separate data into blocks corresponding to the different sources of variation (Time, Dose and Time*Dose interaction). A-SCA enabled the demonstration of a dynamic, time/age dependent shift of serum metabolome throughout the rats’ lifetimes. Variables responsible for the discrimination between groups clearly indicate that BPA modulates energy metabolism, and suggest alterations of neurotransmitter signaling, the latter finding being compatible with the neurodevelopmental effect of this xenoestrogen. In conclusion, long lasting metabolic effects of BPA could be characterized over 200 days, despite physiological (and thus metabolic) changes connected with sexual maturation and aging.     

Aluminum-induced mitochondrial dysfunction leads to lipid accumulation in human hepatocytes: a link to obesity.

Mitochondrial dysfunction is the cause of a variety of pathologies associated with high energy-requiring tissues like the brain and muscles. Here we show that aluminum (Al) perturbs oxidative-ATP production in human hepatocytes (HepG2 cells). This Al-induced mitochondrial dysfunction promotes enhanced lipogenesis and the accumulation of the very low density lipoprotein (VLDL). Al-stressed HepG2 cells secreted more cholesterol, lipids and proteins than control cells. The level of apolipoprotein B-100 (ApoB-100) was markedly increased in the culture medium of the cells exposed to Al. (13)C-NMR and HPLC studies revealed a metabolic profile favouring lipid production and lowered ATP synthesis in Al-treated cells. Electrophoretic and immunoblot analyses pointed to increased activities and expression of lipogenic enzymes such as glycerol 3-phosphate dehydrogenase (G3PDH), acetyl CoA carboxylase (ACC) and ATP-citrate lyase (CL) in the hepatocytes exposed to Al, and a sharp diminution of enzymes mediating oxidative phosphorylation. D-Fructose elicited the maximal secretion of VLDL in the Al-challenged cells. These results suggest that the Al-evoked metabolic shift favours the accumulation of lipids at the expense of oxidative energy production in hepatocytes.     

1H NMR metabolic profiling of human serum associated with benign and malignant gallstone diseases

Proton NMR based metabolic profile of serum associated with different gallbladder pathologies is presented. Quantitative and qualitative variations in the metabolic profile of serum in control samples and three different pathologies of gallbladder, chronic cholecystitis, xanthogranulomatous cholecystitis and carcinoma of gallbladder has been evaluated by use of ¹H NMR based metabonomics and multivariate chemometric methods. Multivariate partial least square discriminant analysis of ¹H NMR spectra showed a clear discrimination between control and diseased groups on the basis of quantitative and qualitative metabolic variations. Increased levels of lactate and pyruvate whereas decreased levels of glucose, some amino acids and low density lipoprotein/very low density lipoprotein (LDL/VLDL) were observed. These metabolites, responsible for class discrimination, from different metabolic pathways could be considered as the signatures of the carcinoma of gallbladder.     

Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.     

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD⁺/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.     

Perfluorooctanoic acid exposure induces endoplasmic reticulum stress in the liver and its effects are ameliorated by 4-phenylbutyrate

Perfluoroalkyl acids (PFAAs) are a group of widely used anthropogenic compounds. As one of the most dominant PFAAs, perfluorooctanoic acid (PFOA) has been suggested to induce hepatotoxicity and several other toxicological effects. However, details on the mechanisms for PFOA-induced hepatotoxicity still need to be elucidated. In this study, we observed the occurrence of endoplasmic reticulum (ER) stress in mouse livers and HepG2 cells after PFOA exposure using several familiar markers for the unfolded protein response (UPR). ER stress in HepG2 cells after PFOA exposure was not significantly influenced by autophagy inhibition or stimulation. The antioxidant defense system was significantly disturbed in mouse livers after PFOA exposure, and reactive oxygen species (ROS) were increased in cells exposed to PFOA for 24 h. However, N-acetyl-L-cysteine (NAC) pretreatment did not satisfactorily alleviate the UPR in cells exposed to PFOA even though the increase of ROS was less evident. Furthermore, exposure of HepG2 cells to PFOA in the presence of sodium 4-phenylbutyrate (4-PBA), a chemical chaperone and ER stress inhibitor, suggested that 4-PBA alleviated the UPR and autophagosome accumulation induced by PFOA in cells. In addition, several toxicological effects attributed to PFOA exposure, including cell cycle arrest, proteolytic activity impairment, and neutral lipid accumulation, were also improved by 4-PBA cotreatment in cells. In vivo study demonstrated that PFOA-induced lipid metabolism perturbation and liver injury were partially ameliorated by 4-PBA in mice after 28 days of exposure. These findings demonstrated that PFOA-induced ER stress leading to UPR might play an important role in PFOA-induced hepatotoxic effects, and chemical chaperone 4-PBA could ameliorate the effects.     

A rational design approach for amino acid supplementation in hepatocyte culture

Improvement of culture media for mammalian cells is conducted via empirical adjustments, sometimes aided by statistical design methodologies. Here, we demonstrate a proof of principle for the use of constraints‐based modeling to achieve enhanced performance of liver‐specific functions of cultured hepatocytes during plasma exposure by adjusting amino acid supplementation and hormone levels in the medium. Flux balance analysis (FBA) is used to determine an amino acid flux profile consistent with a desired output; this is used to design an amino acid supplementation. Under conditions of no supplementation, empirical supplementation, and designed supplementation, hepatocytes were exposed to plasma and their morphology, specific cell functions (urea, albumin production) and lipid metabolism were measured. Urea production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation. Not surprisingly, the urea production attained was less than the theoretical value, indicating the existence of pathways or constraints not present in the current model. Although not an explicit design objective, albumin production was also increased by designed amino acid supplementation, suggesting a functional linkage between these outputs. In conjunction with traditional approaches to improving culture conditions, the rational design approach described herein provides a novel means to tune the metabolic outputs of cultured hepatocytes. Biotechnol. Bioeng. 2009;103: 1176–1191. © 2009 Wiley Periodicals, Inc.     

Culturing of HepG2 cells with human serum improve their functionality and suitability in studies of lipid metabolism

Primary human hepatocytes are considered to be the “gold standard” in studies of lipid metabolism despite a number of disadvantages like large inter-donor differences and inability to proliferate. Human hepatoma HepG2 cells retain many hepatocyte-specific functions but do also exhibit disadvantages like secretion of lipoproteins and bile acids that do not emulate human hepatocytes in vivo. The aim of this study was to investigate whether supplementation of the culturing media with human serum could improve the functionality of HepG2 cells and thereby make them more apposite in studies of lipid metabolism. The cells were cultured with human sera (2%) from three healthy individuals or with fetal bovine serum (10%). Lipoprotein, apolipoprotein, bile acid, albumin, and proprotein subtilisin/kexin type 9 (Pcsk9) concentrations in the cell media, as well as gene and protein expressions were then measured. We found apoB-containing LDL-sized but also apoA1-containing HDL-sized particles, increased bile acid and Pcsk9 concentrations in the cell media, as well as increased expression of genes involved in lipid metabolism and differentiation in HepG2 cells cultured with human sera. Thus, supplementation of the culturing media with human serum improves the functionality of HepG2 cells and makes them more apposite in studies of lipid metabolism.     

In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection.     

Characterization of global metabolic responses of glucose-6-phosphate dehydrogenase-deficient hepatoma cells to diamide-induced oxidative stress

Glucose-6-phosphate dehydrogenase (G6PD) is crucial to NADPH generation and redox homeostasis. We have recently shown that G6PD deficiency predisposes cells to oxidant-induced cell death, and it is associated with the impairment of glutathione regeneration. It remains unclear what other metabolic pathways are affected by G6PD deficiency and whether the altered metabolism disturbs cellular redox homeostasis and underlies increased susceptibility to oxidants. In this study, we examined the effects of diamide on global metabolite profiles of SK-Hep1-derived SK-i-Gi and SK-i-Sc cells, which could inducibly express short hairpin RNA (shRNA) against G6PD (Gi) and control shRNA (Sc), respectively. There was no significant difference in their metabolite profiles under uninduced conditions. Doxycycline (Dox) addition resulted in over 70% decrease in G6PD activity in SK-i-Gi cells. This was accompanied by relatively minor changes in the metabolome of SK-i-Gi cells. Upon further diamide treatment, the metabolite profiles of both SK-i-Gi and SK-i-Sc cells changed in a time-dependent manner. A number of metabolic pathways, including those involved in energy metabolism and metabolism of amino acids and glutathione, were affected. However, the changes in the metabolite profile of Dox-treated SK-i-Gi cells were distinct from those of control cells (i.e., Dox-treated SK-i-Sc, SK-i-Gi, and SK-i-Sc cells). Cellular glutathione was depleted, whereas its disulfide form increased significantly in diamide, Dox-treated SK-i-Gi cells. Metabolites related to energy metabolism, such as AMP, ADP, and acetylcarnitine, increased to a greater extent in these cells than in diamide-treated control cells. In contrast, NAD and glutathione dropped to lower levels in SK-i-Gi cells than in control cells. The NAD⁺ depletion in SK-i-Gi cells was accompanied by a significant increase in NAD kinase activity. Targeted analyses revealed that NADP⁺ and NADPH increased significantly in diamide, Dox-treated SK-i-Gi cells compared with similarly treated control cells. Our results suggest that diamide induces oxidation and depletion of glutathione in SK-i-Gi cells under conditions of G6PD shRNA induction and subsequently induces conversion of NAD⁺ to NADP⁺ through enhanced NAD kinase activity. This may represent a compensatory mechanism to restore cellular NADPH reserve in G6PD-deficient cells. It is accompanied by alteration in pathways of cellular energy metabolism, such as glycolysis and β-oxidation.