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Cell Biology International (2007) 31, 703710 (Printed in Great Britain)
Thiazolidinediones exhibit different effects on preadipocytes isolated from rat mesenteric fat tissue and cell line 3T3-L1 cells derived from mice
Hitoshi Mineoa*, Chikako Odaa, Hideyuki Chijib, Teruo Kawadac, Kyoko Shimizua and Toshio Tairaa
aPrimary Cell Co., Ltd., Center for Advanced Science and Technology 306, Hokkaido University, Kita-21 Nishi-11, Sapporo, Hokkaido 001-0021, Japan
bFaculty of Human Life Science, Fuji Women's University, Hokkaido 061-0067, Japan
cDivision of Food Science and Biochemistry, Graduate School of Agriculture, Kyoto University, Kyoto 105-0001, Japan
The effects of PPAR-γ agonists, thiazolidinediones (TZDs), on preadipocytes isolated from rat mesenteric adipose tissue and murine cell line 3T3-L1 were compared using an in vitro cell culture system. After each cell formed a confluent monolayer under appropriate medial conditions, pioglitazone or troglitazone was applied at 10
Keywords: Thiazolidinediones, TZD, PPAR-γ, Cell culture, Adipose tissue, Fat tissue, Rats, Obesity.
*Corresponding author. Tel./fax: +81 11 706 7325.
The major function of adipose tissue is to store excess energy as neutral fat during periods of nutritional excess (Flier, 1995). These stored fats are used by the organism as metabolic energy during periods of nutritional deficiency, such as starvation. Recently, obesity has become a prevalent health problem in Western countries. Triglyceride (TG) has been shown to be the main fat related to obesity (Ross et al., 1993) and excess accumulation in visceral (omental and mesenteric) adipose tissue was shown to lead to subsequent metabolic syndromes (Matsuzawa et al., 1999; Wajchenberg, 2000). The delivery of free fatty acids to the liver from visceral adipose tissue may contribute to pathological symptoms such as hyperinsulinemia, hypertriglycemia and glucose intolerance (Kissebah, 1991; Bjorntorp, 1997). To reveal the mechanism of differentiation and proliferation of visceral adipocytes is an important problem in the fields of both basic science and applied medicine.
Adipocyte differentiation, which consists of various complex processes regulated by a range of factors, has been reviewed in some reports (Gregoire et al., 1998; Rangwala and Lazar, 2000). The transcription process begins with the binding of endogenous or exogenous ligands to encode peroxisome proliferator-activated receptor-γ (PPAR-γ), which is a member of the ligand-activated nuclear receptor family (Kota et al., 2005). A cascade of gene transcription processes then occurs during differentiation, resulting in the expression of adipocyte-specific genes (Tontonoz et al., 1995; IJpenberg et al., 1997). Thus, PPAR-γ is involved in various adipose tissue functions during the adipocyte maturation process.
Thiazolidinediones (TZDs) are representative exogenous PPAR-γ agonists that induce differentiation and proliferation from preadipocytes to mature adipocytes (Furnsinn and Waldhausl, 2002). During the adipocyte maturation process, the uptake of glucose and fatty acids, storage of TG and production of adiponectin were also found to be promoted via TZD-induced activation of PPAR-γ (Spiegelman, 1998; Yki-Jarvinen, 2004). One TZD, pioglitazone, increases the weight of subcutaneous fat tissue through the up-regulation of genes facilitating adipocyte lipid storage in vivo (Bogacka et al., 2004). TZDs have been used as anti-type 2 diabetes drugs via their direct effect on fatty acid accumulation in adipocytes, disappearance of glucose and/or their indirect effect on adiponectin release, resulting in lowered insulin sensitivity in outside adipose tissue (Spiegelman, 1998; Yki-Jarvinen, 2004). To clarify the effect of TZDs on adipocytes, it is necessary to evaluate the response to TZDs by various cell types and to recognize differences in cell derivation, such as primary culture cells or cell lines.
The aim of the present study was to compare the effects of TZDs between primary cultured mesenteric adipocytes derived from rat mesentery and line cell 3T3-L1 cells derived from the murine fetus. The cell line 3T3-L1 cells (Green and Meuth, 1974) have been used widely as a representative experimental model for the effects of TZDs on adipose tissues. We observed morphological changes in each cell, particularly the accumulation of lipid droplets in the cytoplasm, during the culture periods and evaluated cell differentiation and proliferation parameters such as DNA content, TG accumulation in the cytoplasm and adiponectin production. We also determined glycerol-3-phosphate dehydrogenase (GPDH) activity, which promotes the conversion of glycerol-3-phosphate to triacylgricerol in the 2 types of cultured cells in the presence of the two TZD drugs.
2 Materials and methods
Two types of TZD, pioglitazone and troglitazone were purchased from Takeda Pharmaceutical Co. Ltd. (Osaka, Japan) and Sankyo Co. Ltd. (Tokyo, Japan), respectively. Hanks' balanced salt solution (HBSS), Dulbecco's modified Eagle/F-12 medium (DMEM/F12), newborn calf serum (NCS) and Trypsin-EDTA solution were purchased from Invitrogen Corporation (USA). Collagenase (Type II), BSA (Fraction V), 3-Isobutyl-1-Methylxanthine (IBMX), Dexamethasone (DEX), and fetal calf serum (FCS) were obtained from Sigma (USA). Other biochemical grade reagents were purchased from Wako Pure Chemical (Osaka, Japan).
2.2 Animals and feeding conditions
The animals were maintained in accordance with the Hokkaido University guidelines for the care and use of laboratory animals. The cages were placed in a room with controlled temperature (21–23
2.3 Cell culture protocol
Fig. 1 shows the experimental protocols for the culture of mesenteric preadipocytes and 3T3-L1 preadipocytes. To induce cell differentiation, the application of promoters (DEX, insulin and/or IBMX) was required for the 3T3-L1 cultures. On the other hand, no specific promotion treatment was needed to induce cell differentiation in the mesenteric adipocyte culture, since insulin was added to the medium during the experimental period. The TZDs were added to the medium immediately after formation of a confluent monolayer in each cell culture system; mesenteric adipocytes at 4
Experimental protocol for mesenteric adipocyte and 3T3-L1 culture systems in vitro. Mesenteric preadipocytes were harvested on incubation plates at Day 1. Specific treatment for the promotion of cell differentiation was not undertaken for the mesenteric adipocyte culture, since insulin (10
2.3.1 Visceral preadipocytes from mesenteric fat tissue
Rat mesenteric adipocytes were prepared according to the method recently developed in our laboratory (Shimizu et al., 2006). Animals were killed by cervical dislocation, and a midline sterile incision was made to expose the abdominal cavity and its contents. Visceral adipose tissue situated on the mesentery was removed and washed with HBSS. The fat tissue was minced with scissors and added to HBSS containing 0.2% collagenase and 1.0% BSA, and then incubated at 37
2.3.2 Cell line 3T3-L1
Cell line 3T3-L1 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and was preserved at −80
2.4 Maintenance of cell culture and addition of TZDs
The 2 cell types were cultured under appropriate medium conditions at 37
2.5 Determination of DNA content, TG content, and GPDH activity in cultured cells and adiponectin release into the medium
During the specified experimental periods, the cultured medium was collected and stored at −80
2.6 Statistical analysis
All values are expressed as means
3.1 Microscopic observation and photographs during cell growth
In the two cell types, the number of lipid droplets increased, and the size of the particles grew until maximal fat accumulation in the cytoplasm was reached during the experimental periods. Small, multiple particles containing lipids formed initially, later combining to form large mono-particles at the final stage of maturation in each cell type. Photomicrographs show the cultured cells (A, Mesenteric adipocytes; B, 3T3-L1 cells) at the maturation stages in the control experiment (Fig. 2). In the control plates, the amount of lipid in mesenteric adipocytes was larger than that in 3T3-L1 cells. There were no obvious differences between the control cells and those treated with DMSO in the photographs taken of the mesenteric adipocytes and 3T3-L1 cells (data not shown). On the other hand, the shape and size of the TG droplets in the cell cytoplasm differed slightly between the control and those treated with TZDs (Fig. 3). Under TZD treatment, the number of droplets decreased, but the size of droplets was larger in the TZD-treated cells than in the control cells for both mesenteric adipocytes and 3T3-L1 cells.
Microscopic appearance of the mesenteric adipocyte and 3T3-L1 cultures at maturation. A, Mesenteric adipocytes at Day 13; B, 3T3-L1 cells at Day 23. Each horizontal bar represents 100
Effect of TZDs on the microscopic appearance of mesenteric adipocyte and 3T3-L1 cell cultures. A, Mesenteric adipocytes at Day 9 (control); B, Mesenteric adipocytes at Day 9 (application of troglitazone at 10
3.2 Comparison of control values for various parameters between visceral adipocytes and 3T3-L1 cells
Table 1 shows the control values for DNA content, TG accumulation, GPDH activity, and adiponectin release into the medium in visceral adipocyte and 3T3-L1 cultures at the end of the experimental period. The DNA and TG contents (μg/well) on the incubation plates were larger for the mesenteric adipocyte culture than for the 3T3-L1 culture (P
Control values for DNA content, TG content, GPDH activity and adiponectin production in mesenteric adipocyte and 3T3-L1 cell cultures
3.3 Effect of TZDs on DNA content, TG accumulation, GPDH activity and adiponectin release
Although the addition of pioglitazone or troglitazone to the medium had no effect on DNA content in the mesenteric adipocyte culture, TZDs significantly increased the DNA content in the 3T3-L1 cells (Fig. 4A). The increase in DNA content induced by troglitazone in the 3T3-L1 cells was significantly larger than that induced by the application of pioglitazone (P
Effect of TZDs on DNA content (A), TG accumulation (B), GPDH activity (C) and adiponectin release (D) in mesenteric adipocyte and 3T3-L1 cell cultures at the end of incubation. C, Control; D, DMSO; P, Pioglitazone; T, Troglitazone. Values are means
3.4 Time course of changes in adiponectin release
Adiponectin release into the medium increased in the control and vehicle-treated mesenteric adipocytes from Day 5 to Day 7 (Fig. 5). Adiponectin production in TZD-treated cells was lower (P
Time course of adiponectin concentrations in the medium of mesenteric adipocyte (MA) and 3T3-L1 cell cultures. Values are means
Microscopic observation and determination of certain parameters indicate that not only the basal characteristics but also the response to TZDs in mesenteric adipocytes was obviously different to those of the 3T3-L1 cells in the in vitro culture system.
The proliferation of (Ohsumi et al., 1994; Tafuri, 1996), and TG accumulation in (Brown et al., 2001), 3T3-L1 preadipocytes was enhanced by TZD application. On the other hand, the administration of TZD did not affect the weight of visceral adipose tissue in humans (Mori et al., 1999; Miyazaki et al., 2002) or rats in vivo (Berthiaume et al., 2004). GPDH activity was increased by TZD application in a dose-dependent manner in 3T3-L1 cells (Mizukami and Taniguchi, 1997; Skurk et al., 2006). On the other hand, GPDH activity was increased in subcutaneous but not omental preadipocytes in the presence of TZD, although PPAR-γ was expressed at comparable levels (Adams et al., 1997). The application of TZDs in 3T3-L1 cells increased adiponectin mRNA levels and medial adiponectin concentration in a dose-dependent manner (Maeda et al., 2001; Grohmann et al., 2005). Although adiponectin release was significantly increased by TZD application in acute experiments using isolated human omental adipocytes (Motoshima et al., 2002), the incubation system and experimental protocol were different to those in the present study.
With regard to the effect of TZDs on preadipocytes, the results of our study using 3T3-L1 cells are almost the same as those of the previous reports. On the other hand, there are few reports on the effects of TZDs on mesenteric adipocytes using an in vitro cell culture technique, since appropriate visceral (mesenteric and omental) adipocyte culture techniques have not yet been developed. The present study is the first to demonstrate that the proliferation and maturation parameters significantly differ between mesenteric adipocytes and 3T3-L1 cells under basal and TZD-treated conditions. We propose the following hypothesis to explain these variations in cell response to TZDs between the various types of preadipocytes in primary and line cell cultures.
First, there is the possibility that the differences in culture cell source are involved in the different responses to TZD among cell types. The preadipocytes of the mesenteric adipocytes were stromal-vascular cells (SVCs) isolated from individual fat tissue in male Sprague–Dawley rats at 3 to 5
In microscopic appearance, TG accumulation was much greater in the mesenteric adipocytes than in the 3T3-L1 cells (Fig. 2). The value for parameters such as TG accumulation, GPDH activity and adiponectin production in the mesenteric adipocytes were higher than in the 3T3-L1 cells (Table 1). Since activation of PPAR-γ is required for the promotion of the adipose cell maturation process (Tontonoz et al., 1995; Kota et al., 2005), timing of the TZD application in terms of the state of cell differentiation is an important problem. There have been reports that down-regulation of PPAR-γ expression can be induced by excess amounts of TZD (Rosenbaum and Greenberg, 1998; Perrey et al., 2001). In the present study, adiponectin release from mesenteric adipocytes was suppressed by 10
Second, there are obvious differences in cell composition between line cell and primary cell culture systems. The cells isolated from fat tissue contain various types of cells such as mature adipocytes, stromal preadipocytes, endothelial cells, macrophages and so on (Hauner, 2005). On the other hand, there was only an extremely low possibility that the culture system for the 3T3-L1 cells contained other cells such as macrophages or vessel endothelial cells. Adipose tissue has been considered to be an endocrine organ that releases various types of adipocytokines into the fat tissues (Trayhurn and Beattie, 2001; Kershaw and Flier, 2004). Many kinds of up- and down-regulation in adipocytes by adipocytokines have been reported (Hauner, 2005). Co-culture of differentiated 3T3-L1 adipocytes and the macrophage cell line RAW264 results in the marked up-regulation of pro-inflammatory cytokines, such as TNF-alpha, and the down-regulation of the anti-inflammatory cytokine, adiponectin (Suganami et al., 2005). In complex components including macrophages (Jiang et al., 1998; Ricote et al., 1998) and vessel endothelial cells (Desmet et al., 2005), the response induced by the activation of PPAR-γ is disordered or obstructed by cytokines or chemokines derived for cells other than adipocytes.
Third, although each cell was cultured under appropriate medium conditions in this study, the difference in medial conditions for each cell culture might be a problem. Only insulin was added to induce cell differentiation and proliferation in the mesenteric adipocyte culture system (Shimizu et al., 2006). On the other hand, for 3T3-L1 cell culture, DEX, IMBX and insulin were used to induce efficient cell growth in the present study. Thus, differences in medial conditions, particularly in specific promoters, may be one reason for the difference in response to the TZDs among the types of cell culture. The mechanisms by which these promoters stimulate cell proliferation and the interactions between these promoters and TZDs in adipocytes are not well understood (Gregoire et al., 1998). These promoters are usually used at supra-physiological doses in cell culture systems. Thus, the function and mechanism of each promoter in adipocyte differentiation needs to be clarified in detail in vitro.
Recently, there has been growing need for information on the function of visceral adipocytes, as excess visceral adipocyte tissue is known to result in various metabolic syndromes (Matsuzawa et al., 1999; Wajchenberg, 2000). On the other hand, 3T3-L1 is a representative adipogenic cell line used for basic biological research or applied studies such as the screening anti-obesity drugs or as a functional food component for the prevention of excess fat accumulation. Clarification of the differences between primary culture cells isolated from the body and established cell lines is needed for the evaluation of the effects of drugs with clinical application, such as TZDs, on adipose tissues. We should pay careful attention to these differences when evaluating the effects of various agents on adipose tissues in a living body.
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Received 1 November 2006/18 December 2006; accepted 9 January 2007doi:10.1016/j.cellbi.2007.01.002