* Department of Animal Sciences,
Department of Molecular and Environmental Toxicology, and
Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706
Received May 10, 2003; accepted July 7, 2003
ABSTRACT
Mercury is a common pollutant that alters glucose metabolism in adipocytes; however, the effect of HgCl2 on differentiating adipocytes and their subsequent metabolic function is not well understood. Two adipocyte models, the 3T3-L1 and C3H10T1/2 (10T1/2) cell lines, were differentiated in the presence of HgCl2. To assess the amount of differentiation in a population, markers of differentiation (i.e., PPAR and GLUT 4 expression and lipid accumulation) and functions of adipocytes (i.e., glucose transport and insulin-induced glucose transport) were measured. HgCl2 exposure significantly decreased the number of phenotypic adipocytes and PPAR
expression in both 3T3-L1 and 10T1/2 cells without effects on cell viability. GLUT 4 was significantly reduced by HgCl2 treatment in 10T1/2 but not 3T3-L1 cells. Exposure to HgCl2 during differentiation increased basal glucose uptake in a dose-dependent manner (up to 2.5-fold) and decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. In contrast, HgCl2 had little effect on basal or insulin-induced glucose uptake in 10T1/2 cells, possibly due to their lower insulin responsiveness. We examined the effect of HgCl2 exposure on signaling event involved in differentiation of adipocytes and cellular stress, namely, the phosphorylation of ERK1/2 and JNK, respectively. HgCl2 exposure had no effect on ERK1/2 phosphorylation in either cell line, increased JNK phosphorylation in the 10T1/2, and had no effect on JNK phosphorylation in 3T3-L1 cells. These data indicate HgCl2 exposure can inhibit the differentiation of fibroblasts into adipocytes as well as influence signaling events and the subsequent metabolic activity of differentiated adipocytes.
Key Words: adipocyte; adipogenesis; mercury; differentiation; 3T3-L1; 10T1/2.
Terminal differentiation of precursor cells into adipocytes is an important component of glucose and lipid metabolism throughout the development and life of animals. Thus, disruption of differentiation may result in serious developmental and adult diseases related to glucose/lipid metabolism. In vitro research has shown that adipocyte differentiation and/or metabolism is disrupted by exposure to numerous environmental chemicals, such as dioxin and endrin, and metal contaminants, such as arsenite, vanadate, and the Group IIb metals: Cd2+, Zn2+, and Hg2+ (Alexander et al., 1998; Barnes et al., 1999
; Ezaki, 1989
; Jin et al., 2000
; Liao and Lane, 1995
; Moreno-Aliaga and Matsumura, 2000; Wauson et al., 2002
).
Metals have been shown to have a wide variety of effects on adipocytes. Vanadate inhibits adipocyte differentiation of the preadipocyte 3T3-L1 cell line when added during the first 24 h of differentiation (Jin et al., 2000; Liao and Lane, 1995
). Vanadate also stimulates glucose transport and lipogenesis in rat adipocytes (Dubyak and Kleinzeller, 1980
; Shechter and Ron, 1986
). Arsenite inhibits adipocyte differentiation of the C3HT101/2 fibroblast cell line with both chronic and acute exposure (Trouba et al., 2000
; Wauson et al., 2002
). Zinc increased both glucose transport and phosphorylation of Akt, a kinase involved in the insulin signaling leading to increased glucose transport (May and Contoreggi, 1982
; Tang and Shay, 2001
). Similarly, studies have shown that low micromolar concentrations of cadmium can increase GLUT 1mediated glucose uptake in 3T3-L1 adipocytes (Harrison et al., 1991
). Ezaki (1989)
showed a 4-fold stimulation of glucose transport in primary rat adipocytes due to acute exposure to 100 µM HgCl2, while we have found similar effects in 3T3-L1 adipocytes with chronic exposure to low concentrations of HgCl2 (110 µM; Barnes and Kircher, unpublished data). Despite the clear effect of metals on adipocytes, the specific impact of HgCl2 on cellular differentiation and subsequent metabolic function of adipocytes remains unclear.
In vitro experiments have shown that terminal adipocyte differentiation has two distinct phases: a commitment phase and a maturation phase (Shao and Lazar, 1997; Xue et al., 1996
). Cascades of transcription factors, including peroxisome proliferator-activated receptor
(PPAR
) and the CCAAT/enhancer-binding proteins (C/EBPs), control these two phases. Insulin responsiveness of adipocytes is a characteristic that is acquired during the maturation phase of differentiation (Resh, 1982
) and involves the expression of the proteins responsible for the functions of adipocytes (e.g., lipogenesis and insulin-sensitive glucose transport). The ability of adipocytes to respond to insulin is an important component of whole body glucose and lipid metabolism and storage. A decrease in insulin-mediated responses from insulin target tissues is termed insulin resistance and is an important characteristic of Type II diabetes (Zimmet et al., 2001
).
In this study, we used the well-characterized 3T3-L1 preadipocyte and the multipotent C3H10T1/2 fibroblast cell models to determine the effects of inorganic HgCl2 on adipocyte differentiation and function. Our results demonstrate that inorganic HgCl2 exposure inhibits differentiation, but not viability, of both cell lines. In addition, we show that HgCl2 treatment decreases the expression of several adipocyte-specific proteins, affects kinases involved in insulin signaling, and decreases insulin-mediated glucose uptake in the 3T3-L1 cell line. The reduction in adipocyte differentiation and subsequent decrease in insulin-mediated glucose transport following HgCl2 exposure suggest that HgCl2 may contribute to insulin resistance and the onset of diseases related to insulin responsiveness.
MATERIALS AND METHODS
Reagents.
For the culture of 10T1/2 cells, Dulbeccos modified Eagles medium (DMEM) formulated with F12 media was obtained from Gibco/Invitrogen (Carlsbad, CA) and fetal bovine serum (FBS) was purchased from Atlanta Biological (Norcross, GA). For the culture of 3T3-L1 cells, DMEM was obtained from Sigma Chemical Co. (St. Louis, MO) and FBS and new calf serum (NCS) were obtained from Biowhittaker (Walkersville, MD). Other cell culture reagents for both cell lines were the same and include: bovine pancreatic insulin (28 U/mg), mercuric chloride, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX), which were purchased from Sigma Chemical Co. [3H]-deoxyglucose (DG, >30 Ci/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA). Viability of treated and untreated cells was assessed by the release of lactate dehydrogenase using the CytoTox-ONE kit from Promega (Madison, WI) and the manufacturers recommended protocol. All other chemicals were reagent grade or higher and obtained from commercial sources.
Differentiation of 3T3-L1 cells.
3T3-L1 fibroblasts obtained from ATCC (Rockville, MD) were grown to confluence in high-glucose DMEM containing 10% NCS. Differentiation of 3T3-L1 fibroblasts was performed as described previously with minor modifications (Frost and Lane, 1985). Briefly, the confluent cells were incubated 3 days in DMEM containing 10% FBS, 1 µg/ml insulin, 0.25 µM dexamethasone, and 500 µM IBMX (IDM). Cells were then incubated for an additional 2 days in DMEM containing 10% FBS and 1 µg/ml insulin. After differentiation, adipocytes were maintained in DMEM containing 10% FBS. Visual assessment of lipid droplet formation indicated that > 80% of the cells exhibited the adipocyte phenotype.
Differentiation of C3H10T1/2 cells.
C3H10T1/2 cells (ATCC) were maintained in DMEM:F12 media and differentiated as described previously (Phillips et al., 1995). Upon reaching confluence, cells were induced to differentiate in DMEM:F12 supplemented with 10% FBS and 10 µg/ml insulin, 1 µM dexamethasone, 0.5 µM IBMX, and 1 µM BRL49653 (IDM/BRL) for 2 days. Cells were then incubated for an additional 5 days in DMEM containing 10% FBS and 10 µg/ml insulin. Differentiation, as assessed by lipid droplet formation, ranged from 3050%.
Flow cytometry and cell staining.
Cells were differentiated in the presence or absence of HgCl2. After differentiation, cells were lifted from the plate with trypsin and fixed with paraformaldehyde (1% final concentration). Nile Red dissolved in DMSO was added to a final concentration of 0.25 µg/ml and staining was quantified by flow cytometry. Cellular Nile Red fluorescence, a measure of lipid accumulation within adipocytes, was measured at 530 nm on a FACSCalibur dual-laser flow cytometer (Becton Dickinson, San Jose, CA). The adipocyte population was identified based on Nile Red staining (lipid content) and side scatter (cell complexity). Data was acquired and analyzed with CellQuest software (Becton Dickinson). For Oil Red O staining and extraction, cells were washed one time with 37°C PBS and incubated with formalin (10% formaldehyde, 90% PBS) for 15 min to fix the cells. After fixing, cells were washed three to four times with tap water. Oil Red O solution (5 mg Oil Red O/ml isopropanol) was added to each well and incubated at room temperature for 2 h. Plates were rinsed three to four times with tap water and allowed to air dry. To extract Oil Red O, isopropanol was added to each well and shaken at room temperature for 5 min, and a sample was read at 510 nm. Oil Red O binds neutral lipids and was used to determine relative amounts of lipid in treated and untreated adipocyte populations for confirmation of Nile Red data. Oil Red O measures total lipid content of all cells in the population rather than just the adipocyte population measured by flow cytometry and Nile Red.
Glucose transport.
Glucose uptake was measured 7 days after initiation of the differentiation protocol. Cells were washed twice with KRPH buffer (5 mM Na2HPO4, 20 mM HEPES, 1 mM MgSO4, 1 mM CaCl2, 4.7 mM KCl, 0.1% BSA, pH 7.4) and then incubated for 1 h in KRPH buffer. Glucose uptake was measured in the presence and absence of 10 µM cytochlasin B to determine effects on mediated and nonmediated DG uptake. Glucose uptake was measured during the final 10 min by the addition of [3H]-2-deoxyglucose to a final assay concentration of 50 µM DG containing 0.5 µCi 2-[3H]-deoxyglucose/ml. Cells were washed three times in cold PBS, solubilized in 0.5 N NaOH, and aliquots were subjected to scintillation counting. Total protein levels were determined using the Bradford protein assay (Bio-Rad, Hercules, CA).
Immunoblotting.
Monoclonal anti-PPAR, polyclonal anti-GLUT 4, monoclonal antiphospho-ERK, and the appropriate HRP-conjugated antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific antibodies raised against JNK were purchased from Upstate Biotechnology (Lake Placid, NY). Equal amounts of protein were separated by SDS-PAGE using 10% polyacrylamide gels and then transferred to ECL nitrocellulose membrane (Amersham, Peapack, NJ), per the manufacturers protocol. Immunoblotting was performed and proteins were visualized using the HRP-conjugated secondary antibody and ECL detection reagents, per the manufacturers protocol (Amersham). Blots were scanned and the analysis performed on a Macintosh G4 computer using the public domain NIH Image (version 1.61, developed at the U.S. NIH and available at http://rsb.info.nih.gov/nih-image/).
Statistical analysis.
Data were analyzed by one-way analysis of variance (ANOVA, InStat version 2.03). Differences among data means were assessed using a Student-Newman-Keuls multiple comparison test (p < 0.05). Experiments were replicated at least three times with representative experiments shown in the figures.
RESULTS
HgCl2 Treatment Inhibits Adipocyte Differentiation in a Dose-Dependent Manner
HgCl2 treatment reduced the number of 10T1/2 and 3T3-L1 cells that acquired the adipocyte phenotype when treated with the hormonal mixture required for differentiation (IDM/BRL and IDM, respectively). The number of adipocytes was quantitated using flow cytometric analysis of the size, complexity, and amount of Nile Red staining of cells. The addition of HgCl2 from the initiation of IDM treatment through 7 days reduced the number of 3T3-L1 cells that became adipocytes in a dose-dependent manner. Treatment with 510 µM HgCl2 reduced the number of phenotypic adipocytes by 4060% (Fig. 1A). The addition of HgCl2 reduced the number of 10T1/2 cells that become adipocytes by 6090%, with a maximum effect at 10 µM HgCl2 (Figs. 1A
and 1B
). Lactate dehydrogenase activity was measured to determine the effects of treatment on cell viability. Media LDH was not different from control for 120 µM HgCl2, although 50 µM HgCl2 increased media LDH in both cell lines (data not shown). In other experiments, differentiated adipocytes were incubated with 110 µM HgCl2 for 16 days without an effect on cell number (Barnes and Kircher, unpublished data).
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To determine if HgCl2 was effective at a particular stage of differentiation, cells were incubated with HgCl2 for the commitment phase (time exposed to IDM or IDM/BRL), the maturation phase (rest of differentiation), or during the entire differentiation process. Figure 1B shows that 3T3-L1 and 10T1/2 cells differ in their sensitivity to HgCl2 at different phases of differentiation. Mercury inhibited differentiation at all stages, however, the magnitude of inhibition differed among stages. In 3T3-L1 cells, 10 µM HgCl2 treatment was slightly less effective at inhibiting adipogenesis when added during the maturation phase (days 3 to 7) than when HgCl2 was present for the entire differentiation period (Fig. 1B
). In 10T1/2 cells, 10 µM HgCl2 exposure during the commitment phase was as effective at inhibiting adipocyte differentiation as if HgCl2 was present throughout differentiation (Fig. 1B
). For this reason, in the subsequent experiments, 10T1/2 cells were only exposed to HgCl2 during the commitment phase of differentiation (days 02). Exposure of 10T1/2 cells to HgCl2 during only the maturation phase significantly inhibited adipocyte differentiation, although to a lesser degree than the other exposure periods.
HgCl2 Treatment Decreases the Lipid Content of 3T3-L1- and 10T1/2-Derived Adipocytes
The effect of HgCl2 on lipid accumulation (an important component of adipocyte differentiation) in 3T3-L1 and 10T1/2 cells is shown in Figure 2. Cells were exposed to HgCl2 during differentiation and the amount of Oil Red O staining was determined after 8 days. Oil Red O staining can be used to assess the amount of lipid in a population of cells. HgCl2 treatment resulted in a dose-dependent decrease in cellular lipid accumulation in both cell lines (decreased in Oil Red O staining). Oil Red O staining was significantly reduced with 20 µM HgCl2 and 5 µM HgCl2 in 3T3-L1 and 10T1/2 cells, respectively. Using flow cytometry and Nile Red staining, lipid accumulation within individual 3T3-L1 adipocytes was measured. As expected, HgCl2 decreased the average lipid content of differentiated 3T3-L1 adipocytes in a dose-dependent manner (Table 1
). Nile Red staining is a more sensitive method for measuring lipid accumulation. Using this more sensitive analysis of lipid accumulation, we observed a decrease in lipid accumulation with 1 µM HgCl2 as compared to 20 µM HgCl2 with Oil Red O staining. Mean Nile Red fluorescence decreased 43% in 3T3-L1 adipocytes exposed to 10 µM HgCl2.
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Adipogenesis is an intricate, well-controlled process that has been studied extensively in vitro and in vivo. However, microarray analysis reveals distinct transcriptional profiles of adipogenesis in vivo and in vitro (Soukas et al., 2001). Differences in gene expression in vivo and in vitro emphasize the importance of studying multiple in vitro model systems, each of which may shed light on important aspects of in vivo differentiation. Therefore, we examined two cell models to address the hypothesis that HgCl2 inhibits adipogenesis.
HgCl2 dose-dependently inhibits adipogenesis in both 3T3-L1 and C3H10T1/2 cells (Fig. 1). The mechanism through which HgCl2 inhibits adipocyte differentiation has yet to be defined; however, we present evidence that the disruption of transcription factor expression and insulin-signaling pathways may be involved. There are two phases of differentiation: commitment and maturation. Disruption of differentiation can occur during one or both of these phases. For example, previous studies showed that retinoic acid and TCDD decrease adipogenesis when present during the commitment phase but not when present during the maturation phase (Phillips et al., 1995
; Xue et al., 1996
). Similarly we have demonstrated that while the timing of HgCl2 exposure has different effects on adipocyte differentiation in the two cell lines (e.g., HgCl2 is more effective during the commitment phase in 10T1/2), HgCl2 inhibited adipogenesis when present in one or both phases of differentiation. Our data indicate that, while HgCl2 treatment inhibits adipogenesis in both cell lines, different mechanisms are at work in the different cell lines and may explain differences of other end points between the two cell lines (e.g., GLUT 4 expression and insulin-mediated glucose transport).
Previous studies have shown that the inhibition of PPAR expression inhibits differentiation (Gurnell et al., 2000
). Figure 3
shows that, at 48 h (peak PPAR
expression), HgCl2 dose-dependently reduced PPAR
expression in both 3T3-L1 and 10T1/2 cells. However, decreased expression of PPAR
during the commitment phase alone does not appear to be responsible for the actions of HgCl2 because the addition of HgCl2 after 48 h (after commitment phase) still reduced differentiation of both cell types.
Insulin is the only component of the differentiation cocktail that is present throughout adipocyte commitment and maturation. Since HgCl2 inhibited adipocyte differentiation when added during either phase of differentiation, the interactions with insulin responses were closely examined. Insulin activates kinases important for adipocyte differentiation and function including extracellular receptor kinase (ERK), which is phosphorylated in response to insulin and activated by metals such as zinc (Tang and Shay, 2000), vanadate, and arsenic (Wu et al., 1999). The action of HgCl2 on the ERK phosphorylation is not well documented. Figure 5
shows that ERK phosphorylation is associated with the initiation of differentiation in both cell lines. HgCl2, however, had no effect on ERK phosphorylation, suggesting that ERK is not a target of HgCl2 in the inhibition of adipogenesis. While there is some controversy as to whether ERK activity is required for adipocyte differentiation (De Mora et al., 1997
; Qiu et al., 2001
), ERK activity is required for the environmental contaminant TCDD to inhibit adipocyte differentiation (Hanlon et al., 2003
).
SAPK family member c-Jun N-terminal kinase (JNK) regulates adipocyte differentiation and function of mature adipocytes (Camp et al., 1999). Although it is not directly activated by insulin, it can contribute to the downregulation of the insulin signal by serine phosphorylation of insulin receptor substrate 1 (IRS-1; Aguirre et al., 2000
). Serine phosphorylation of IRS-1 results in decreased phosphorylation of IRS-1 downstream targets, which has been linked to insulin resistance (Zick, 2001
). In addition to JNK action on insulin-signaling pathways, active JNK also phosphorylates PPAR
, thereby inhibiting its transcriptional activity (Camp et al., 1999
). Reduced activity would lead to decreased adipocyte differentiation and, while we observed a decrease in PPAR
expression, reduced activity may also contribute to the HgCl2-mediated inhibition of differentiation. HgCl2 treatment has been shown to stimulate JNK activity in other cell systems including rabbit renal cortical slices (Turney et al., 1999
) and LLC-PK cells (Matsuoka et al., 2000
), although the physiological consequences of JNK phosphorylation in these cells were not examined. We report here that HgCl2 treatment had no effect on JNK phosphorylation in 3T3-L1 cells. However, in 10T1/2 cells, HgCl2 exposure led to a prolonged phosphorylation of JNK, which occurred independently of the presence of any hormones. If this prolonged JNK phosphorylation leads to serine phosphorylation of IRS-1 and subsequent inactivation, insulin signaling would be disrupted. Since insulin signaling is required for both the commitment to and maturation of adipocytes, downregulation of insulin signaling via JNK activation is a possible mechanism through which HgCl2 inhibits adipogenesis.
In addition to the molecular data showing HgCl2 disrupts signaling pathways used by insulin, we present physiological data that shows there is a decrease in insulin-mediated glucose transport in cells differentiated in the presence of HgCl2. Figure 6 shows that 3T3-L1 cells treated with HgCl2 during differentiation reduced insulin-mediated glucose transport dose-dependently. This may be due to fewer adipocytes, reduced expression of the insulin-responsive glucose transporter, disruption of normal insulin-signaling pathways, or some combination of these events. The reduction in adipocyte number was about 50% for 3T3-L1 cells treated with 5 µM HgCl2 (Fig. 1A
). HgCl2 treatment decreased insulin-mediated glucose uptake 2.8-fold (700 to 250 pmole/well for control and 5 µM HgCl2 respectively), suggesting adipocyte number alone would not account for the reduced insulin responsiveness. Although GLUT 4 expression decreased in HgCl2-treated 10T1/2 cells, no difference was observed in 3T3-L1 cells; yet, HgCl2-treated 3T3-L1 adipocytes exhibited insulin resistance. Insulin resistance, characterized as a decreased response of insulin-sensitive tissues to the hormonal stimulus, is a metabolic condition contributing to Type 2 diabetes. Insulin resistance can be induced by numerous factors including elevated levels of TNF-
and free fatty acids (Zick, 2001
). Ryden and coworkers (2002)
showed that, in the adipocyte, TNF-
induces insulin resistance, in part, through the activation of the MAPK pathway including rapid phosphorylation of JNK, suggesting that exposure to chemicals that induce phosphorylation of JNK may contribute to the onset of insulin resistance.
Insulin resistance is a condition that contributes to the onset of noninsulin-dependent diabetes mellitus (NIDDM or Type II diabetes; Zimmet et al., 2001). Many conditions have been shown to induce insulin resistance in in vitro cell culture models including oxidative stress (Rudich et al., 1997
) and exposure to TNF-
(Engelman et al., 2000
). Additionally, exposure to two environmental toxins, arsenic and TCDD, has been linked to an increased incidence of insulin resistance in epidemiological studies (Lai et al., 1994
; Longnecker and Michalek, 2000
). TCDD and arsenic have also been shown to inhibit adipogenesis in cell culture models (Hanlon et al., 2003
; Trouba et al., 2000
). The data presented in this report demonstrate that HgCl2 inhibits adipogenesis in two models of adipocyte differentiation, raising the question of whether exposure to HgCl2 contributes to the onset of insulin resistance. Epidemiological studies of people living in methyl mercury (MeHg-) contaminated areas indicate no correlation of exposure to MeHg and the onset of diabetes (Futatasuka et al., 1996, 2000). However, comparisons between the action of MeHg and HgCl2, forms of mercury with different chemical characteristics and metabolic actions, on glucose homeostasis and adipogenesis have not been examined. Previous research has demonstrated that the effects of MeHg and HgCl2 differ in neuronal cell differentiation (Parran et al., 2001
) and in calcium regulation in T lymphocytes (Tan et al., 1993
). Further comparisons among different forms of mercury are necessary to determine their potential contribution to pathologies associated with glucose homeostasis.
Mercury exposure does not appear to be a contributing factor in the less common form of diabetes, insulin-dependent diabetes mellitus (IDDM). Exposure to HgCl2 was shown to have no effect on the incidence of IDDM in diabetes-prone rats (Kosuda et al., 1997). However, exposure to HgCl2 delays the onset of IDDM in a nonobese diabetic mouse model of diabetes as measured by glucosuria (Brenden et al., 2001
). Thus, there are recognized effects of HgCl2 on systems contributing to diabetes, although effects on the most prevalent from, NIDDM, remain unclear. HgCl2 is a prevalent environmental toxicant that has been recognized to have serious impacts on human health (U.S. EPA, 1997
); however, little research has been done on the effects of HgCl2 on adipocyte function. This study provides evidence that environmental levels of HgCl2 may be a contributing factor to the onset of insulin resistance and, potentially, Type II diabetes mellitus.
ACKNOWLEDGMENTS
This study was supported by grant R825218 from the U.S. EPA (D. M. B.), NIH grant RO1 DK55302 (C. R. Jefcoate), and by grant number T32 ES07015 from the National Institute of Environmental Health Sciences (NIEHS), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS/NIH.
NOTES
1 To whom correspondence should be addressed at Department of Animal Sciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706. Fax: (608) 262-5157. E-mail: barnes{at}calshp.cals.wisc.edu.
2 Authors contributed equally to this work.
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