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Development of Insulin-Responsive Glucose Uptake and GLUT4 Expression in Differentiating Human Adipocyte Precursor Cells

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Development of Insulin-Responsive Glucose Uptake and GLUT4 Expression in Differentiating Human Adipocyte Precursor Cells International Journal of Obesity (1998) 22, 448±453 ß 1998 Stockton Press All rights reserved 0307±0565/98 $12.00 http://www.stockton-press.co.uk/ijo 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-40225 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 However, it is well known that clonal preadipocyte cell lines of rodent origin differ in many aspects from other probably more physiological models, including Many studies have demonstrated that insulin stimu- human adipocyte precursor cells in primary culture.9 lates glucose transport in adipocytes by rapid trans- For example, while 3T3 L1 cells require critical cell location of glucose transporters from an intracellular divisions to enter the differentiation process,10 human membrane pool to the cell surface.1±4 It is now adipocyte precursor cells kept in primary culture are established that the insulin effect on glucose uptake able to develop into fat cells without in vitro mitosis, in adipose tissue depends on the presence of the as these cells are apparently in a later stage of the glucose transporter isoform GLUT4.5 In addition, differentiation programme.11 At present, a human adipocytes also express GLUT1, another glucose white adipocyte cell line is not available. Therefore, transporter which is responsible for basal glucose the only possibility to perform long-term studies in uptake, but is also partially responsive to insulin at human adipose tissue is to use the model of in vitro least in 3T3 L1 cells. Kinetic studies in this clonal differentiating human adipocyte in primary culture.12 adipocyte cell line have shown that GLUT4 is not The aim of this study was to characterize the insulin- present in the ®broblastic state, but is progressively regulated glucose transport system in cultured human expressed during the course of adipose differentiation, preadipocytes during the course of differentiation whereas GLUT1 is highly expressed in the undiffer- and to study the usefulness of human fat cells entiated state.6±8 Such models of rodent origin have developed in vitro for long-term studies of glucose been widely used in studies to investigate the control transport. of glucose transport in general and the regulation of GLUT4 expression in particular.3,4 Materials and methods Correspondence: Hans Hauner, MD, Diabetes Research Institute, Heinrich-Heine-University DuÈ sseldorf, Auf'm Hennekamp 65, D-40225 DuÈ sseldorf, Germany. Received 11 February 1997; revised 3 November 1997; accepted Triiodothyronine (T3), human transferrin, pantothe- 9 January 1998 nate and bovine serum albumin (BSA) were obtained Insulin responses in adipocyte precursor cells H Hauner et al 449 from Sigma (Munich, Germany); 1-methyl-3-iso- 2±3 d. In some experiments, cells were kept in culture butylxanthine (MIX) was purchased from Serva (Hei- for up to 30 d. delberg, 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 Glucose transport assay 2-Deoxy-D-glucose uptake was determined as a func- fetal calf serum were obtained from Gibco (Berlin, tional parameter of the glucose transport system. Germany); and all other chemicals were from Boeh- Assays were performed at different time points as ringer (Mannheim, Germany) or Merck (Darmstadt, indicated in the Results. The glucose concentration Germany). Sterile plasticware for tissue culture was was reduced to 5 mmol=l, 24 h prior to the assay, and purchased from Flow Laboratories (Irvine, Scotland). both insulin and Hepes were completely removed 2-Deoxy-D-(1-3H)glucose (15 Ci=mmol) and 125I- from the medium. For assessment of the stimulatory labelled protein A (30 mCi=mg) were obtained from effect of insulin, some dishes were incubated with Amersham (Braunschweig, Germany); reagent for human insulin at the concentrations indicated for SDS-PAGE was purchased from Pharmacia (Freiburg, 15 min immediately before the assay. (3H)-labelled Germany) and Sigma (Munich, Germany), respec- 2-deoxy-D-glucose (1 mCi=dish, concentration 4±5 tively; and the polyclonal GLUT1 and GLUT4 anti- mmol=l) was added to the medium which already sera were from Calbiochem (Bad Soden, Germany). contained 5 mmol=l unlabelled glucose. Hexose uptake was measured for 20 min at 37C. Glucose uptake was terminated by transferring the dishes to an Cell culture ice-bath. Cells were repeatedly washed with ice-cold Adipose tissue samples (20 ±100 g wet tissue) PBS and incubated for 20 min with 0.1% SDS. The were obtained from mammary adipose tissue of radioactivity of the cell material was counted in a young, normal-weight females (age < 40 y, BMI liquid scintillation counter (Beckman, Munich, Ger- < 27 kg=m2) undergoing elective mammary reduction many). Values were corrected for non-speci®c uptake surgery. All women were otherwise healthy, in parti- as decribed recently.13 cular 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 Western blotting from human adipose tissue were prepared as described For the preparation of crude membrane fractions, cells previously.12 Brie¯y, after removing all ®brous mate- were washed in TES buffer (225 mmol=l saccharose, rial and visible blood vessels, adipose tissue samples 1 mmol=l EDTA, 20 mmol=l Tris, 2.5 mg=ml leupep- were cut into small pieces (approximately 10 mg) and tin, 2.5 mg=ml pepstatin, 2.5 mg=ml aprotinin and digested in 10 mmol=l phosphate buffered saline 0.2 mmol=l phenylmethylsulfonyl¯uoride, pH 7.4). (PBS) containing 1 mg=ml crude collagenase and Cells were scraped from the dishes using a rubber 20 mg=ml bovine serum albumin, pH 7.4, for approxi- policeman, immediately frozen in liquid nitrogen and mately 45 min in a shaking water bath. After short stored at 770C. The cell material was homogenized centrifugation at 200 g, the ¯oating fat cells and the in 10 ml TES buffer using a te¯on glass homogenizer incubation solution were aspirated and discarded. The at 4C. The ®rst centrifugation was performed for sedimented cells were resuspended in an erythrocyte 10 min at 1000 g, the supernatant was centrifuged lyzing buffer consisting of 154 mmol=lNH4Cl, again for 90 min at 100 000 g. The membrane pellet 5.7 mmol=lK2HPO4 and 0.1 mmol=l EDTA for was resuspended in TES buffer. After a second 10 min to remove contaminating red blood cells. The homogenization step, the membrane fraction was dispersed material was ®ltered through a nylon mesh stored at 770C. with a pore size of 150 mm. After two washing steps, Membrane protein samples of 30 ±50 mg, but within the sedimented cells were resuspended in Dulbecco's one experiment, identical amounts were subjected to Modi®ed Eagle's=Ham's F-12 medium (v=v, 1:1) SDS-PAGE and transferred to nitrocellulose ®lters in supplemented with 10% fetal calf serum and inocu- a semi-dry blotting apparatus. Filters were blocked for lated into culture dishes at a density of approximately 10 min in Tris-buffered saline containing 0.05% 3±56104=cm2. After a 16 h incubation period for cell Tween-20 and 5% BSA. Then, ®lters were incubated attachment, cells were washed with PBS and refed for 16 h at 4C with a 1:500 dilution of a polyclonal with a serum-free DME=Ham's F-12 medium supple- GLUT1 or GLUT4 antiserum. After extensive wash- mented with 15 mmol=l NaHCO3, 15 mmol=1 Hepes, ing with Tris-buffered saline containing 0.05 Tween- 33 mmol=l biotin, 17 mmol=l pantothenate, 10 mg=ml 20 and 5% BSA, ®lters were incubated for 21 h with human transferrin, 100 U=ml penicillin and 0.1 g=l 125I-protein A (0.3 mCi=ml) at room temperature. streptomycin. To induce adipose differentiation, Filters were again extensively washed, air-dried and cells were exposed to 66 nmol=l insulin, 100 nmol=l exposed to Hyper®lm-MP ®lms using intensifying cortisol, 0.2 nmol=lT3 and for the ®rst three days screens.
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