Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells

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1 International Journal of Obesity (1998) 22, 448±453 ß 1998 Stockton Press All rights reserved 0307±0565/98 $ Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells H Hauner, K RoÈhrig, M Spelleken, LS Liu and J Eckel Diabetes Research Institute at the Heinrich-Heine-University DuÈsseldorf, D DuÈsseldorf, Germany OBJECTIVE: In differentiating human preadipocytes glucose uptake in the presence of insulin is a prerequisite for lipid accumulation. The aim of this study was to characterize the insulin-regulated glucose transport system during and after differentiation. DESIGN AND METHODS: Human adipocyte precursor cells kept in primary culture were allowed to differentiate into fat cells under serum-free hormone-supplemented conditions. 2-Deoxy-glucose uptake was measured as a functional parameter of the glucose transport system, the amount of GLUT1 and GLUT4 protein was determined by Western blotting. RESULTS: In the undifferentiated state, cells did not increase 2-deoxy-glucose uptake in response to insulin. On day 16, when cells have acquired the adipocyte phenotype, there was a 3±4-fold stimulation of glucose transport by insulin compared to basal rates, whereas basal glucose uptake was dramatically diminished. Measurement of GLUT4 protein in cell extracts, showed a marked increase in the amount of this insulin-regulated transporter isoform during the differentiation period. On average, the amount of GLUT4 was 16.7-fold greater after than before differentiation. In contrast, the amount of GLUT1 protein decreased during differentiation to almost undetectable levels on day 16. When newly developed adipocytes were maintained in culture for another 14 d, the stimulation of glucose uptake and the amount of GLUT4 remained stable. CONCLUSION: Differentiating human fat cells in primary culture develops an insulin-responsive glucose transport system which exhibits a high stability, thereby providing a valuable model for long-term studies of glucose transport and GLUT4 expression in human adipocytes. Keywords: adipose differentiation; glucose transport; GLUT1; GLUT4; human adipose tissue Introduction Many studies have demonstrated that insulin stimulates glucose transport in adipocytes by rapid translocation of glucose transporters from an intracellular membrane pool to the cell surface. 1±4 It is now established that the insulin effect on glucose uptake in adipose tissue depends on the presence of the glucose transporter isoform GLUT4. 5 In addition, adipocytes also express GLUT1, another glucose transporter which is responsible for basal glucose uptake, but is also partially responsive to insulin at least in 3T3 L1 cells. Kinetic studies in this clonal adipocyte cell line have shown that GLUT4 is not present in the broblastic state, but is progressively expressed during the course of adipose differentiation, whereas GLUT1 is highly expressed in the undifferentiated state. 6±8 Such models of rodent origin have been widely used in studies to investigate the control of glucose transport in general and the regulation of GLUT4 expression in particular. 3,4 Correspondence: Hans Hauner, MD, Diabetes Research Institute, Heinrich-Heine-University DuÈ sseldorf, Auf'm Hennekamp 65, D DuÈ sseldorf, Germany. Received 11 February 1997; revised 3 November 1997; accepted 9 January 1998 However, it is well known that clonal preadipocyte cell lines of rodent origin differ in many aspects from other probably more physiological models, including human adipocyte precursor cells in primary culture. 9 For example, while 3T3 L1 cells require critical cell divisions to enter the differentiation process, 10 human adipocyte precursor cells kept in primary culture are able to develop into fat cells without in vitro mitosis, as these cells are apparently in a later stage of the differentiation programme. 11 At present, a human white adipocyte cell line is not available. Therefore, the only possibility to perform long-term studies in human adipose tissue is to use the model of in vitro differentiating human adipocyte in primary culture. 12 The aim of this study was to characterize the insulinregulated glucose transport system in cultured human preadipocytes during the course of differentiation and to study the usefulness of human fat cells developed in vitro for long-term studies of glucose transport. Materials and methods Triiodothyronine (T 3 ), human transferrin, pantothenate and bovine serum albumin (BSA) were obtained

2 from Sigma (Munich, Germany); 1-methyl-3-isobutylxanthine (MIX) was purchased from Serva (Heidelberg, Germany); collagenase CLS type 1 was obtained from Worthington (Freehold, NJ, USA); human insulin and cortisol were kindly donated by Hoechst (Frankfurt, Germany); culture media and fetal calf serum were obtained from Gibco (Berlin, Germany); and all other chemicals were from Boehringer (Mannheim, Germany) or Merck (Darmstadt, Germany). Sterile plasticware for tissue culture was purchased from Flow Laboratories (Irvine, Scotland). 2-Deoxy-D-(1-3 H)glucose (15 Ci=mmol) and 125 I- labelled protein A (30 mci=mg) were obtained from Amersham (Braunschweig, Germany); reagent for SDS-PAGE was purchased from Pharmacia (Freiburg, Germany) and Sigma (Munich, Germany), respectively; and the polyclonal GLUT1 and GLUT4 antisera were from Calbiochem (Bad Soden, Germany). Cell culture Adipose tissue samples (20 ±100 g wet tissue) were obtained from mammary adipose tissue of young, normal-weight females (age < 40 y, BMI < 27 kg=m 2 ) undergoing elective mammary reduction surgery. All women were otherwise healthy, in particular free of metabolic or endocrine diseases. The procedure for obtaining human adipose tissue has been approved by the Ethical Committee of the Heinrich-Heine-University DuÈsseldorf. Stromal cells from human adipose tissue were prepared as described previously. 12 Brie y, after removing all brous material and visible blood vessels, adipose tissue samples were cut into small pieces (approximately 10 mg) and digested in 10 mmol=l phosphate buffered saline (PBS) containing 1 mg=ml crude collagenase and 20 mg=ml bovine serum albumin, ph 7.4, for approximately 45 min in a shaking water bath. After short centrifugation at 200 g, the oating fat cells and the incubation solution were aspirated and discarded. The sedimented cells were resuspended in an erythrocyte lyzing buffer consisting of 154 mmol=l NH 4 Cl, 5.7 mmol=l K 2 HPO 4 and 0.1 mmol=l EDTA for 10 min to remove contaminating red blood cells. The dispersed material was ltered through a nylon mesh with a pore size of 150 mm. After two washing steps, the sedimented cells were resuspended in Dulbecco's Modi ed Eagle's=Ham's F-12 medium (v=v, 1:1) supplemented with 10% fetal calf serum and inoculated into culture dishes at a density of approximately 3± =cm 2. After a 16 h incubation period for cell attachment, cells were washed with PBS and refed with a serum-free DME=Ham's F-12 medium supplemented with 15 mmol=l NaHCO 3, 15 mmol=1 Hepes, 33 mmol=l biotin, 17 mmol=l pantothenate, 10 mg=ml human transferrin, 100 U=ml penicillin and 0.1 g=l streptomycin. To induce adipose differentiation, cells were exposed to 66 nmol=l insulin, 100 nmol=l cortisol, 0.2 nmol=l T 3 and for the rst three days 0.25 mmol=l MIX. The medium was changed every 2±3 d. In some experiments, cells were kept in culture for up to 30 d. Glucose transport assay 2-Deoxy-D-glucose uptake was determined as a functional parameter of the glucose transport system. Assays were performed at different time points as indicated in the Results. The glucose concentration was reduced to 5 mmol=l, 24 h prior to the assay, and both insulin and Hepes were completely removed from the medium. For assessment of the stimulatory effect of insulin, some dishes were incubated with human insulin at the concentrations indicated for 15 min immediately before the assay. ( 3 H)-labelled 2-deoxy-D-glucose (1 mci=dish, concentration 4±5 mmol=l) was added to the medium which already contained 5 mmol=l unlabelled glucose. Hexose uptake was measured for 20 min at 37 C. Glucose uptake was terminated by transferring the dishes to an ice-bath. Cells were repeatedly washed with ice-cold PBS and incubated for 20 min with 0.1% SDS. The radioactivity of the cell material was counted in a liquid scintillation counter (Beckman, Munich, Germany). Values were corrected for non-speci c uptake as decribed recently. 13 Western blotting For the preparation of crude membrane fractions, cells were washed in TES buffer (225 mmol=l saccharose, 1 mmol=l EDTA, 20 mmol=l Tris, 2.5 mg=ml leupeptin, 2.5 mg=ml pepstatin, 2.5 mg=ml aprotinin and 0.2 mmol=l phenylmethylsulfonyl uoride, ph 7.4). Cells were scraped from the dishes using a rubber policeman, immediately frozen in liquid nitrogen and stored at 770 C. The cell material was homogenized in 10 ml TES buffer using a te on glass homogenizer at 4 C. The rst centrifugation was performed for 10 min at 1000 g, the supernatant was centrifuged again for 90 min at g. The membrane pellet was resuspended in TES buffer. After a second homogenization step, the membrane fraction was stored at 770 C. Membrane protein samples of 30 ±50 mg, but within one experiment, identical amounts were subjected to SDS-PAGE and transferred to nitrocellulose lters in a semi-dry blotting apparatus. Filters were blocked for 10 min in Tris-buffered saline containing 0.05% Tween-20 and 5% BSA. Then, lters were incubated for 16 h at 4 C with a 1:500 dilution of a polyclonal GLUT1 or GLUT4 antiserum. After extensive washing with Tris-buffered saline containing 0.05 Tween- 20 and 5% BSA, lters were incubated for 21 h with 125 I-protein A (0.3 mci=ml) at room temperature. Filters were again extensively washed, air-dried and exposed to Hyper lm-mp lms using intensifying screens. Autoradiograms were quanti ed by laser scanning densitometry (LKB, GraÈfel ng, Germany). 449

3 450 Other biochemical assays Glycerol-3-phosphate dehydrogenase (GPDH) was determined as a marker enzyme of adipose differentiation and fat cell function, according to an established method. 14 A protein precipitation technique to avoid lipid interference was used to measure the protein content of the culture. 15 Speci c GPDH activities are presented. Statistics Results are expressed as mean s.d. of at least three experiments in triplicate. For comparison, analysis of variance was used. P-values of <0.05 were considered as statistically signi cant. Results Glucose transport during adipose differentiation Stromal cells isolated from human adipose tissue samples and kept in culture for 72 h did not respond to insulin stimulation of 2-deoxy-glucose uptake (Table 1). However, when cells have undergone adipose differentiation in a serum-free medium containing insulin, triiodothyronine and cortisol as adipogenic agents, a clear increase in the rate of glucose uptake after a 15 min preincubation with or mol=l insulin was observed. Compared to basal uptake rates, insulin induced a 3± 4-fold increase in 2-deoxy-glucose uptake. The stimulation of hexose uptake was comparable at the two insulin concentrations, indicating that the maximum insulin effect was already obtained at mol=l (Table 1). In contrast, basal glucose uptake markedly decreased during the differentiation process, by almost 90% from on day 3 to fmol=mg protein 620 min on day 16 when cells have acquired adipocyte morphology (Table 1). Development of GLUT4 and GLUT1 protein As insulin stimulation of glucose transport in adipose tissue depends on the presence of GLUT4 protein, it was important to measure the cellular amount of GLUT4 during adipose differentiation. In crude membrane fractions of undifferentiated cells, the amount of detectable GLUT4 protein was very low. On day 8, when cells have already started to accumulate lipid droplets, GLUT4 protein level increased approximately 4.4-fold and on day 16, after terminal differentiation, the amount of GLUT4 protein was 16.7-fold above the level in the undifferentiated state (Figure 1). Thereby, this rapid and dramatic increase of GLUT4 was apparently responsible for the emergence of insulin responsiveness. On the other hand, a large amount of GLUT1 protein was detectable in undifferentiated cells. Similar levels of GLUT1 were still found on day 8, whereas GLUT1 protein was decreased to almost undetectable levels on day 16 (Figure 2). Glucose transport in long-term cultured human adipocytes To examine the suitability of this human cell culture model for long-term studies of glucose transport, we then determined glucose transport capacity for two consecutive weeks in newly developed fat cells. Day 16 was de ned as the starting point when cells have expressed full adipocyte function. Measurement of the activity of GPDH, which is an established marker enzyme for adipose differentiation, revealed that enzyme activity has reached a plateau and remained unchanged for the subsequent 14 d (Table 2). Although fat cell number was stable and no delayed differentiation was occurring (data not shown), there was still a signi cant progression in fat cell morphology as demonstrated in Figure 3. Most cells developed a more spherical shape and the lipid vacuoles became larger probably due to the con uence of small droplets. Finally, many cells have obtained the characteristic signet-ring morphology. The results of 2-deoxy-glucose uptake between day 16 and day 30 are also presented in Table 1. Compared to day 16 there was a moderate decrease in the basal uptake rate that was not statistically signi cant. Likewise, insulin-stimulated glucose transport also slightly decreased. However, when expressed as stimulation above basal rates, insulin responsiveness remained unchanged during the 14 additional days in culture. There was also no signi cant variation in the stimulation of glucose transport by either or mol=l insulin (Table 1). Measurement of the total cellular amount of GLUT4 protein by Western blotting did not reveal any signi cant change over time between day 16 and day 30, although there was a trend to a decrease in transporter number (Table 2). Even at near-physiological insulin concentrations Table 1 Basal and insulin-stimulated 2-deoxy-D-glucose uptake in cultured human adipocytes before and after differentiation. Glucose transport was determined as described under Methods. Mean s.d. of three experiments in triplicate ( 3 H)-2-deoxy-glucose uptake (fmol/mg protein/20 min) Day basal M insulin fold increase above basal M insulin fold increase above basal ± ±

4 ( mol=l), starting from day 16, the relative expression of GLUT4 remained unchanged during the following 14 days (data not shown). 451 Discussion Figure 1 Development of GLUT4 protein in differentiating human adipocyte precursor cells. GLUT4 protein levels were determined by Western blotting as described under Methods. Panel A shoes mean s.d. of three experiments in duplicate, panel B demonstrates one representative autoradiogramm. Figure 2 Course of GLUT1 protein in differentiating human adipocyte precursor cells. GLUT1 was determined by Western blotting as described under Methods. One representative autoradiogram out of four experiments is demonstrated. The results of this study show many similarities in the pattern of insulin-stimulated glucose uptake between human adipocyte precursor cells in primary culture and clonal preadipocytes from rodent origin, both before and after differentiation. In the undifferentiated state, basal glucose uptake was high and decreased during the course of differentiation. Whereas human preadipocytes did not respond to insulin stimulation, fully differentiated cells demonstrated a rapid increase in glucose transport upon insulin stimulation. This was apparently due to a marked increase in GLUT4 protein, which represents the main insulin-regulated glucose transporter isoform in adipose tissue. 3 5 In our experiments, the amount of GLUT4 protein increased from very low levels by more than 16- fold, which is in the same magnitude as ndings reported by other groups in rodent cell lines. 6±8 It has been demonstrated in 3T3 L1 cells that basal rates of 2-deoxy-glucose uptake are higher in growing, than in con uent, cells and newly developed fat Figure 3 Photomicrographs of in vitro differentiated human fat cells from one representative cultre. Cells on A) day 16 and B) day 30.

5 452 Table 2 GLUT4 protein levels and GPDH activity in newly developed human adipocytes during a 14 d culture period. Day 16 was de ned as the time point when cells have obtained full adipocyte function. The amount of GLUT4 protein on day 16 was de ned as 100% and the levels of GLUT4 at the other time points expressed in relation to day 16. Results are mean s.d. of three experiments Day GLUT4 (%) GPDH (mu/mg protein) M insulin M insulin M insulin cells exhibited lower transport rates relative to undifferentiated cells. 8 The decline in basal glucose uptake during the course of differentiation was found to occur in parallel with the decrease of GLUT1 level, 6 although in one of these studies the decline was only moderate. 7 In our study, the marked decrease in basal glucose uptake was due to a dramatic reduction in the number of GLUT1 transporters to almost undetectable levels. In contrast to the 3T3 L1 cell line, human adipocytes exhibit a clearly more pronounced loss of GLUT1 during the course of differentiation. An interesting nding was the observation that glucose transport and GLUT4 protein levels remained stable once cells have acquired the adipocyte morphology. Similarly, cultured human adipocytes exhibited stable high activities of the lipogenic marker enzyme GPDH and a high production and release of leptin. 16 This is in contrast to a recent study in isolated rat adipocytes in suspension culture, where a rapid decrease of GLUT4 mrna was reported that was not prevented by treatment of the cells with insulin. 17 In our studies, newly developed fat cells retained their ability to respond to insulin and to express GLUT4 expression for at least 14 d. However, with longer culture periods, cells became more and more spherical and got easily detached from the culture dishes. Therefore, studies on day 30 were to some degree critical and, in single experiments, signi cant cell loss was already observed before day 30 in culture (data not shown). This culture system may provide a number of advantages. Generally, a primary culture model is probably more physiological than clonal cell lines, which may have undergone spontaneous transformation and may differ in their proliferation and differentiation pattern in many aspects. 9 As cells were of human origin, they may better resemble the conditions in human diseases. However, the main advantage is that this primary culture model is also suitable for long-term studies of the regulation of glucose metabolism. In our experience, freshly isolated fat cells remain viable for only a few hours in suspension culture and can be used only for short-term studies. During this short life span, freshly prepared cells are invariably stressed or damaged by the collagenase digestion procedure. In contrast, using the culture system described here, we recently studied the effect of a 3 d incubation, with tumor necrosis factor alpha on glucose and lipid metabolism in human fat cells and found a highly reproducible suppression of GLUT4 protein and mrna levels. 13 Drawbacks of this culture system of human fat cells are that the culture of cells is expensive and time-consuming, and that the number of available cells is limited. The lack of insulin responsiveness of undifferentiated cells found in the present study could also be due to the insulin receptor status, as studies in mouse preadipocyte cell lines indicated that the number of insulin receptors is low in the broblastic stage and increases dramatically up to 35-fold during differentiation. These studies also described a clear correlation between the development of increased insulin binding capacity and the increased sensitivity of glucose transport to insulin. 18±20 On the other hand, human adipocyte precursor cells express high levels of IGF-I receptors, 21 as do rodent adipocyte precursors 22 and 3T3 L1 cells 23 and, therefore, supraphysiologically high concentrations of insulin should also activate glucose transport via IGF-I receptors due to a high sequence and functional homology with the insulin receptor. 23 However, at lower near-physiological insulin concentrations, a similar responsiveness was observed with regard to glucose uptake and GLUT4 expression, arguing against a possible interference by IGF-I. In conclusion, the results of this study indicate that human adipocyte precursor cells are not able to increase glucose uptake upon insulin stimulation, probably due to a lack, or only very low expression, of GLUT4, whereas basal glucose transport is approximately 10-fold higher than in differentiated cells. After acquisition of the adipocyte phenotype, cells exhibit high GLUT4 levels which may confer the marked insulin responsiveness at this stage, whereas GLUT1 levels are substantially decreased after differentiation. The glucose transport system of newly differentiated human fat cells remains stable for at least 14 days. Therefore, this culture technique may provide an excellent model to study the long-term regulation of GLUT4 expression in human adipose tissue. Acknowledgements We wish to thank Prof. R. Olbrisch and his team from the Department of Plastic Surgery at the Florence Nightingale Hospital DuÈsseldorf-Kaiserswerth for their kind support. This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft (SFB 351, Teilprojekt C2).

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