NMR Metabolomic Analysis of Caco-2 Cell Differentiation Insong James Lee,† Kellie Hom, Guoyun Bai, and Michael Shapiro* Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore Maryland 21201 Received December 16, 2008
The high-resolution 1H NMR spectra as applied to Caco-2 cells during their differentiation into enterocyte like cells are presented. The data clearly reveal differences in the metabolic profiles over time as the Caco-2 cells differentiate. In the 1H NMR spectra, the aliphatic regions from 4.5 to 1.0 ppm are dominated by peaks from myo-inositol, creatine, taurine, glutamine, glutamate, phosphatidylcholine, choline, alanine and lactate. While a majority of metabolites are present at both the early undifferentiated state and the late differentiated states, the levels of certain metabolites are seen to change dramatically, and in particular, the ratio of myo-inositol and taurine. The NMR spectrum from 10 to 5 ppm shows the aromatic amino acids (Phe, Tyr), NAD, ATP and ribose signals. The appearance of glucose resonances in the differentiated cells (30 days old) spectra suggests that these cells become gluconeogenic. Our study represents a novel method to analyze the differentiation of Caco-2 cells using a metabolomic approach. The results indicate, for the first time, that taurine and glucose biosynthesis occurs in these cells and thus by extension may occur in the intestine. This metabolomic approach can therefore be used to detect novel biological pathways as well as yield useful markers for differentiation. Keywords: NMR • Caro-2 • metabolomics
Introduction The human cell line Caco-2, when grown as a monolayer, is often used as a model for intestinal function including drug absorption. These cells, originally isolated from colon carcinoma tissue, possess the ability to spontaneously differentiate into enterocyte-like cells when grown at confluence for approximately 20 days.1,2 Differentiated Caco-2 cells closely resemble enterocytes in their physiological properties such as morphology and biochemical reactions and they express many enterocyte specific proteins such as transporters. The Caco-2 cell line serves as a useful model for the small intestine epithelium, and it is a valuable tool for screening uptake and efflux of per os (po) administered compounds during drug discovery. When grown as a monolayer, differentiated Caco-2 cells resemble columnar epithelial cells and form tight junctions and are polarized with transporters such as P-glycoprotein (Pgp) and multi drug-resistance proteins (MRP) expressed at the appropriate cell domains (apical or basolateral). One challenge with the Caco-2 cell model is the instability of the phenotype which can vary with cell passage and media differences.1 Since transcellular and paracellular permeabilities, measured across Caco-2 monolayers in vitro, are often used for assessing oral drug absorption potential in humans,2 it is important to better understand the process of differentiation of these cells. Our approach toward characterizing Caco-2 cell differentiation was to use the technique of NMR metabolomics * To whom correspondence should be addressed. E-mail: mshapiro@ rx.umaryland.edu. † Current address; Department of Pharmaceutical Sciences, Notre Dame School of Pharmacy, Baltimore Maryland 21210.
4104 Journal of Proteome Research 2009, 8, 4104–4108 Published on Web 05/06/2009
to perform a preliminary study of the biochemistry for these cells as they differentiate. The field of metabol(n)omics has recently emerged as a complementary technology to genomic and proteomic approaches.3,4 Metabolomics can be defined as a measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.5 The metabolome has been defined as the collection of biomolecules and metabolites that reflects the cellular status. The state of the metabolome cumulatively reflects the states of gene expression, protein expression, and the cellular environment as well as multidirectional interactions among these elements. The metabolome has been estimated to be only 2400 compounds and its composition is determined by all metabolic processes. The composite mixture obtained from any organism or fluid can be thought of as a metabolic fingerprint of the system. Metabolic fingerprinting thus provides an efficient tool with which to initiate hypotheses about affected metabolic pathways.3 Traditionally, these metabolites are selected and analyzed one (or few) at a time. Metabolomics, however, focuses on broad identification and analysis of typically 30-50 metabolites simultaneously. These fingerprints are novel measurement tools to evaluate the biochemical status of a living organism by using 1H NMR which provides concurrent detection of all hydrogen-containing molecules in a sample without pretreatment. It is a quantitative and rapid analysis, and its use in metabolomics is well-established.6-8 In a single experiment, it is possible to gain access to information that normally involves more than 30 assays. Prior to any analysis, most metabolomic techniques require some sample preparation. For tissue and cell extracts, both 10.1021/pr8010759 CCC: $40.75
2009 American Chemical Society
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NMR Metabolomic Analysis of Caco-2 Cell Differentiation water- and lipid-soluble metabolites can readily be extracted. With the use of “aqueous” and organic solvent extraction procedures, virtually all cellular and membranous pools of metabolites are extracted, irrespective of their participation in particular metabolic processes in situ. It is a common observation that the aqueous-soluble metabolites represent the cytosolic pool of many metabolites; thus, these metabolite profiles may be regarded as representative of the ‘active metabolome’. What distinguishes metabolomic studies from proteomic or genomic studies is an attempt to consider metabolite changes in context of the global network of metabolic pathways.
Methods and Materials Cell Culture. Caco-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 µM nonessential amino acids, 1 mM sodium pyruvate, and 50 U/mL penicillin/50 µg/mL streptomycin. Caco-2 cells (5.28 × 106) were cultured as a monolayer on 60 mm plates (Costar, Corning, NY) for 5-30 days. Cells were grown at 37 °C in 5% CO2, and media was changed every other day. Caco-2 cells were used in between passages 30 and 60. Transepithelial resistance (TER) was measured with an EVOM Voltohmmeter (World Precision Instruments, Sarasota, FL). Metabolite Isolation. To isolate metabolites, Caco-2 cells from two different plates were washed twice with 10 mL of icecold PBS with the plates sitting on ice to remove media and other residues. Cells were quickly scrapped off and pelleted in a 15 mL tube using a refrigerated centrifuge. The cell pellet was snap-frozen using dry ice and stored at -80 °C until analysis.9 The cell pellets were thawed on ice and resuspended in 900 µL of a 2:1 chloroform/methanol solution and vigorously mixed for 5 min. This mix was then sonicated 3 times (3 s intervals) at the highest setting for microtip6 using a Sonifier 450 (Branson, Danbury, CT) with the tubes sitting on ice. In between each sonication, the cell mix was vortexed for 3 s to mix the cell lysate. Then, 900 µL of H2O was added and vortexed, to which 600 µL of chloroform was previously added. The mix was then vortexed for an additional 10 s at the highest setting, and then, the cell lysate was moved to a 2 mL centrifuge tube and spun at full speed in a refrigerated microfuge for 20 min. The aqueous and organic phases were collected and the aqueous phase was used for NMR analyses here. NMR Analysis. The lyophilized extracts of the aqueous layer were dissolved in 0.6 mL of phosphate buffered D2O at pH 7.0. The 1H NMR spectra were recorded at 500 MHz on a Varian Instruments (Varian, Inc., Palo Alto, CA) NMR spectrometer at 25 °C using a 60° flip angle, a 6000 Hz sweep width, 4.3 s repetition time, 32K data points, and 256 scans. Solvent suppression was achieved by applying the standard ‘noesypresat’ pulse sequence.10,11 Spectra were processed with ACD NMR software with a pre-exponential factor of 1 Hz. All spectra were manually phased and baseline corrected using a fifth order polynomial baseline fitting routine and the TSP resonance set to 0.00 ppm. For comparison of peak areas, the spectra were integrated after blocking out the region from 6 to 4.5 ppm (area affected due to water suppression) and the total integral value set to 1000 to accommodate different spectral sensitivities.
Results Cell Differentiation. Under our experimental conditions, Caco-2 cells became confluent 3 days after plating and began
to differentiate and form tight junctions approximately 12 days after confluency. The differentiation of Caco-2 cells was confirmed by parallel experiments performed in transwells in which the transepithelial resistance (TER) of Caco-2 monolayers was measured. A TER value of >300 µΩ is indicative of tight junction formation and differentiation. Plated Caco-2 cells developed TER levels >300 µΩ approximately 12 days after plating and reached levels of approximately 600 µΩ at about 22 days after plating. This level stayed consistent until at least 30 days after plating. The NMR spectra for the Caco-2 cells were analyzed from separate plates at 5, 12, 18, 20, 22, and 30 days after plating. NMR Analyses. We present here the first high-resolution 1H NMR spectra as applied to Caco-2 cells as monitored over time. Metabolites were obtained using the aqueous methanolchloroform-water method which simultaneously extracts both the water-soluble metabolites and the organic-soluble lipid components from the same cells.8,9 Typical broad lipid signals are seen in the spectra of the organic extract and remain the same as the cells were harvested at different time points (data not shown). Meanwhile, the NMR spectra of the aqueous layer samples yielded more information. The first aspect noticed from the NMR spectra is that a wide variety of metabolites can be readily detected (without interference from the broad lipid peaks which were extracted into the organic layer). Thus, extraction of the cells into two layers seems to yield more information. Specific peaks can be assigned, as annotated on the spectrum shown in Figure 1 and provided in Table 1. In the 1H NMR spectra, the aliphatic regions from 4.5 to 1.0 ppm are dominated by peaks from myo-inositol, creatine, taurine, glutamine, glutamate, phosphotidylcholine, choline, alanine and lactate. These metabolites are readily identified from their structural expectations and by comparison of the NMR with literature reports.8,12 The NMR spectrum from 10 to 5 ppm represents chemical shifts of the aromatic nucleoside and ribose signals as well as shows the aromatic amino acids (phenylalanine (Phe), tyrosine (Tyr), nicotinamide dinucleotide (NAD), AMP, and PNPs. Visual inspection of the spectra clearly reveals differences in the metabolic profiles in different time points. Figure 2 shows comparative 1H NMR spectra for the region 3.8 -3.3 ppm normalized to myo-inositol levels obtained for the aqueous phase extracts of Caco-2 cell 5, 12, 18, and 22 days after plating. Alteration in cell contents corresponds to a change in metabolite levels and can be considered as the metabolic fingerprint of the cells. Of particular interest are the taurine levels which are seen to be dramatically increasing relative to the myoinositol which seems to be decreasing throughout the differentiation period. Levels of creatine 3.04 ppm are also seen to be significantly increased. In the aromatic region of the spectrum, resonances assignable to PNPs were also observed to be increasing relative to the AMP signals. Surprisingly, the NMR spectrum of the cells obtained after 30 days of plating (Figure 3), shows the appearance of a glucose signal at δ ) 5.21.
Discussion In the present study, we have examined the metabolic changes that occur as Caco-2 cells differentiate. 1H NMR spectroscopy was used to analyze the metabolic status of Caco-2 cells during cellular differentiation and to identify potential cell biomarkers. Toward that end, Caco-2 cell extracts were studied at distinct stages of differentiation (undifferentiJournal of Proteome Research • Vol. 8, No. 8, 2009 4105
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Figure 1. Proton NMR spectrum of aqueous phase extracts of Caco-2 cells 22 days after plating. Table 1. Major Metabolites Observed in Caco-2 Cells metabolite
chemical shift (ppm)
acetate acid glutamine glutamate creatine phosphocholine glycerophosphocholine taurine myo-Inositol Lactate AMP tyrosine phenylalanine NAD glycine
1.92 2.13, 2.15, 2.45 2.05, 2.36 3.04, 3.94 3.22 3.24 3.27, 3.43 3.55, 3.63, 4.07 1.33, 4.12 6.15, 8.27, 8.67 6.91, 7.20 7.49 8.83, 9.15, 9.35 3.56
ated for day 5 through differentiated at day 30). The NMR spectra are presented in Figures 1-3. Careful comparisons of the NMR spectra at various stages of differentiation indicate that, although most metabolites are present at all time points at similar levels, the levels of some metabolites are seen to change dramatically. This is especially true in the ratio of myoinositol to taurine (see Figure 2). The high initial myo-inositol content observed in the Caco-2 cells probably reflects the tendency of the cells to accumulate this material which is present in the cell culture media used for Caco-2 cells. Thus, it is likely that the transporters responsible for the import of myo-inositol are expressed in both undifferentiated as well as differentiated Caco-2 cells.13-15 Although it has been suggested that the increase in cell volume taking place during the differentiation process could cause an increase in uptake of 4106
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myo-inositol which would act as an osmolyte which is necessary for cell growth and development, we did not see any evidence of this in our experiments.16 Within the small intestine, enterocyte cells are exposed to variety of osmolarity disruptors. Thus, these cells must have mechanisms for the homeostasis of osmolarity. One way enterocytes do this is by maintaining a high intracellular content of nonperturbing osmolytes like myo-inositol, glycerophosphorylcholine (GPC), and taurine.17 The DMEM that we used for culturing Caco-2 cells contains myo-inositol (69.9 mM) but does not contain taurine. Taurine is presumably made from cysteine18 and is thought to behave as an osmolyte in the small intestine. The activity of taurine transport in small intestinal epithelial cells is regulated by extracellular osmolarity.19 It has been shown that mammalian cells, when exposed to an acute hypotonic stimulus, typically release K+, Cl-, and organic osmolytes, such as taurine and myo-inositol in an attempt to counteract the volume increase and to avoid swelling-induced cell lysis.20 The cellular loss of osmolytes drives the osmotic efflux of water, restoring the initial cell volume, a phenomenon which is known as regulatory volume decrease (RVD). In some cell types (e.g., astrocytes, airway and intestinal epithelial cells, and pancreatic acinar cells), hypotonic swelling also provokes the release of ATP, which has been proposed to assist RVD in an autocrine/paracrine way.21 It is not yet understood as to how cells adapt to a change in extracellular osmolarity that leads to the regulation of osmolyte transporters or synthetic osmolyte enzymes through the signal transduction pathway.22 In the absence of taurine in the medium, the NMR spectrum reveals that the Caco-2 cells must synthesize the necessary taurine. Taurine is known to be
NMR Metabolomic Analysis of Caco-2 Cell Differentiation
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Figure 2. Expanded region of NMR spectra of aqueous extracts of Caco-2 cells at days 5 (A), 12 (B), 18 (C), and 22 (D), scaled to myo-inositol levels.
Figure 3. Expanded NMR spectrum of Caco-2 cell extract at day 30. R-Glucose signal apparent at δ ) 5.21 (J ) 3.5).
endogenously synthesized from methionine (Met) and cysteine (Cys) in the liver, brain, and kidney.23 The physiological functions of taurine are diverse and include membrane protection, antioxidation, and osmoregu-
lation.24 Thus, activation of de novo synthesis of taurine may play an important role in the Caco-2 cells (intestine) adaptation to changes in osmotic conditions and could be a useful biomarker for Caco-2 cell (intestine) differentiation and ability Journal of Proteome Research • Vol. 8, No. 8, 2009 4107
research articles to survive osmolarity changes. Thus, since taurine is detected at much higher levels in differentiated state, it is possible that one of these enzymes involved in the biosynthesis of taurine is expressed only in differentiated Caco-2 cells. Our results, therefore, suggest an intriguing and novel finding that enterocytes are capable of biosynthesizing taurine which is then used as an osmolyte to regulate cell volume. Previous to this observation, it has been thought that the liver and brain were the only sites of taurine biosynthesis.23 In the only other report in the literature describing NMR analysis of Caco-2 cells, myoinositol and taurine were not mentioned.25 Unfortunately, the NMR spectra of the cell extracts were unavailable, precluding a direct comparison of the NMR spectra. We have also observed that, as Caco-2 cells differentiate and age, there is an increase in glucose levels in these cells. This may be due to an increase in expression or activity or glucose transporters in Caco-2 cell or, alternatively, Caco-2 cell may become gluconeogenic. In this regard, it is worthy of note that the liver and kidney are often considered to be the only organs capable of gluconeogenesis. However, recent hypotheses concerning glutamine and glucose metabolism suggested that the release of glucose might also occur in the small intestine after fasting.15 Furthermore, it has been shown that intestinal gluconeogenesis is induced during the postabsorptive time in rats which were fed a protein enriched diet.26 Thus, our finding indicate that Caco-2 cells recapitulate, at least some component of this potentially important physiological role of intestinal cells. Further experiments, for example, using stable 13C isotopes to measure the rates of gluconeogenesis, would indicate whether Caco-2 cells can be used as a model system to study gluconeogenesis and controlled release of glucose for energy regulation. Our study has yielded critical information on two novel and important physiological pathways that can be further investigated. In addition, our study suggests the potential of studying enterocyte mediated drug metabolism by characterizing the drug metabolites in Caco-2 cells subsequent to exposure to drugs. Because absorption potential has become a more important criterion in the discovery process, there is a need to better understand the biochemistry of the Caco-2 cell in order to produce a more reliable screening method to assess compound permeability.
Conclusions Our study represents the first NMR metabolomic approach to characterize cell differentiation of Caco-2, the most commonly used cells for drug transport studies. The 1H NMR spectroscopic cell assay allows for up to 20-30 distinct metabolic species to be identified. The obtained metabolic fingerprints of Caco-2 cells appear rather simple and can be viewed as a simplified reflection of the end result of interplay
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Lee et al. between up-regulated (and down-regulated) genes and their RNA translation. The results reveal that taurine and glucose can potentially be used as biomarkers for cell differentiation and that osmolyte composition may play an important role in the differentiation process.
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