International Journal of Obesity (1997) 21, 261±266 ß 1997 Stockton Press All rights reserved 0307±0565/97 $12.00 in cultured human adipocytes M Maslowska 1, AD Sniderman 1, R Germinario 2 and K Cian one 1 1 McGill Unit for the Prevention of Cardiovascular Disease, Royal Victoria Hospital, McGill University, and 2 Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada OBJECTIVE: The purpose of the present study was to examine the effect of Acylation Stimulating Protein (ASP) on glucose transport in cultured subcutaneous adipocytes. DESIGN AND SUBJECTS: Subcutaneous adipose tissue was obtained from non-obese, healthy females (18±32 y old) undergoing mammoplasty reduction. Preadipocytes were isolated and differentiated into adipocytes. MEASUREMENTS: Following the exposure of preadipocytes and adipocytes to ASP or insulin, glucose transport was assessed as [ 3 H] 2-deoxy glucose uptake. The measurements were normalised per total cell protein. RESULTS: ASP increases speci c membrane glucose transport in both preadipocytes and adipocytes in a time and concentration dependent manner. Stimulation in both cell types is rapid (within minutes), reaching a maximal effect between 1 and 4 h. However, after 24 h exposure to ASP, there is a downregulation in the response. The ASP response is greater following differentiation of preadipocytes to adipocytes and is compared to that of insulin. Dose response studies demonstrated a ve-fold greater sensitivity of adipocytes (half-maximal concentration of ASP on adipocytes ˆ 0.5 mm, preadipocytes ˆ 2.3 mm). CONCLUSION: These results demonstrate that ASP not only stimulates triglyceride synthesis, but also glucose transport in differentiated human adipocytes and is consistent with a physiologically important role for ASP in postprandial energy storage. Keywords: glucose transport; human adipocytes; ASP; C3adesArg Introduction Acylation Stimulating Protein (ASP) is the most potent stimulant of triglyceride synthesis in human adipocytes yet described. 1 The rate at which triglycerides are cleared from the plasma appears to be related not only to the functional activity of LPL but also to the capacity of peripheral tissues to store fatty acid as intracellular triglycerides. The ability of ASP to regulate this process may, therefore, be of physiological importance. 2,3 As human adipocytes differentiate, they become competent to synthesize and secrete the three proteins necessary to generate ASP. These are the third component of complement (C3), factor B, and adipsin. 4 The capacity to produce ASP appears relatively late in differentiation but before the sharp increase in the capacity of adipocytes to synthesize triglyceride. 5 Subsequently, the mass of triglycerides within adipocytes, the rate at which they synthesize triglycerides, and their capacity to generate ASP are closely correlated. 4,5 Moreover, as they differentiate, not only do human adipocytes generate more ASP, they become much more responsive to ASP. 4 Correspondence: Dr K Cian one, Royal Victoria Hospital, Room H7.35, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. Received 18 June 1996, revised 10 October 1996; accepted 4 December 1996 The mechanisms by which ASP increases triglyceride synthesis are under intensive study. Interaction with an apparent membrane receptor appears to be critical 6 and studies of the cell signalling mechanism point to activation of a protein kinase C pathway. 7 ASP increases triglyceride synthesis by two coordinate mechanisms. One is to increase the activity of diacylglycerol acyltransferase, the enzyme which controls the last step in the synthesis of a triglyceride molecule. 8 The other is to increase speci c membrane transport of glucose through speci c effects on translocation of glucose transporters. This second effect of ASP has only been demonstrated in human skin broblasts and recently in L6 myotubes. 9,10 The purpose of the present study was to determine if ASP produced this effect in human adipocytes, a physiologically important tissue in glucose homeostasis, and to compare its potency to that of insulin. Materials and methods All chemicals used were of reagent grade quality and were purchased from Fisher Scienti c (Montreal, Canada). Tissue culture media and supplies were from Gibco (Burlington, Ontario) or Flow Laboratories (Mississauga, Ontario). [ 3 H] 2-deoxy D-glucose (speci c activity 25±50 Ci/mmol) was purchased from
262 ICN Biochemicals Canada (Mississauga, Ontario). Insulin, collagenase Type II and all other tissue culture grade compounds were from Sigma Chemicals (St. Louis, MO). A commercial protein assay kit as well as bovine serum albumin (BSA) used for standard curves were obtained from Bio-Rad Laboratories (Mississauga, Ontario). Scintillation uid was purchased from ICN Biochemicals Canada and scintillation vials from Fisher Scienti c. Acylation Stimulating Protein was isolated and puri ed from frozen human plasma according to the published method. 7 Isolation of human preadipocytes The isolation procedure for stromal vascular cells was modi ed 4,5 from that of Hauner et al. 11 Human adipose tissue was obtained with informed consent from normal weight, healthy, non-diabetic females, aged 18±32 y, undergoing mammoplasty reduction. Fat lobules were excised under sterile conditions immediately after tissue had been removed from the patient. The adipose tissue was then cleaned of any connective and glandular material as well as visible blood vessels. The remaining fat was nely minced with scalpels and digested for 30 min at 37 C in Hank's buffered salt solution containing 1 mg/ml Type II collagenase and 0.5% fatty acid free bovine serum albumin. Stromal-vascular cells containing preadipocytes were separated from mature adipocytes by centrifugation at 2500 rpm for 10 min. The fat cake and the supernatant was discarded and the remaining pellet was resuspended in a lysing buffer consisting of 0.154 M NH 4 Cl, 10 mm KHCO 3 and 0.1 mm EDTA for 10 min to lyse contaminating red blood cells. To remove any remaining undigested connective tissue, the cell suspension was ltered through a 53 mm lter (Spectrum, Houston, Texas) and centrifuged at 2500 rpm for 10 min to pellet cells. The sedimented preadipocytes were resuspended in Minimum Essential Medium (MEM) medium supplemented with 10% fetal bovine serum (FBS). Cells to be differentiated were plated out on 24 well plates at a high density (3 6 10 4 cells/cm 2 ). Cells not to be differentiated were plated out at one tenth the cell density (3 6 10 3 cells/cm 2 ). Preadipocyte and adipocyte cell culture Cells were incubated in MEM medium containing 10% FBS for 24 h. Subsequently, the media was changed and preadipocytes were grown in 10% FBS containing MEM. Cells destined for differentiation to adipocytes were cultured in serum-free Dulbecco's minimum essential medium/ham's F12 (DMEMM/ F12) medium supplemented with 1.25 mm bovine insulin, 1 mm dexamethasone, 0.2 mm triiodothyronine, 33 mm biotin, 17 mm pantothenate, 15 mm NaHCO 3 and 15 mm Hepes. 11 Preadipocytes and differentiating adipocytes were maintained in a 37 C humid incubator with 5% CO 2 with media changes twice a week. By 21 d, preadipocytes formed a con- uent cell layer and the differentiating cells exhibited adipocyte-type morphology of rounded cells containing multilocular droplets as we 5 and others 11 have demonstrated. At this time, cells were used for experiments. Glucose transport assay Cells were grown and differentiated as described above in 35 mm diameter tissue culture dishes. On the 18th d of culture the medium on differentiating adipocytes was changed to serum-free hormone-supplemented DMEM/F12 differentiation medium which did not contain insulin. On the 21st d, the medium on all the cells was changed to serum- and addition-free DMEM/F12 medium (containing 17.5 mm glucose) overnight. The next day, cells were stimulated with ASP, insulin, or both, at speci c concentrations for one hour (or the indicated times) at 37 C. After exactly one hour, glucose transport was assessed by measuring the cellular uptake of [ 3 H] 2-deoxy-glucose ( 3 H 2-DG). In each experiment, cells isolated from a different subject were exposed to each of the incubation conditions (control, insulin-stimulation and ASPstimulation). The medium was removed and the cells were washed once with 2 ml of phosphate-buffered saline (PBS) at 37 C. Next, 0.8 ml of PBS containing 3 H 2-DG (0.05 mm; average speci c, activity 50 dpm/ pmol) was added to each dish and incubated at 37 C in a water bath for exactly 10 min. In all the experiments, zero-time controls were performed to account for background binding of 3 H 2-DG. At exactly 10 min, the radioactive solution was aspirated and the cells were washed twice with 2 ml of ice-cold PBS. Finally, the cell monolayer was dissolved in 0.5 ml of 0.1 N NaOH. Aliquots of 0.2 ml were than transferred into a scintillation vial containing scintillation uid and counted with a scintillation counter. Protein concentration was determined by the method of Bradford 12 using a commercial Bio-Rad protein assay. Results Our rst aim was to compare the effects of ASP and insulin on the stimulation of glucose transport in both preadipocytes and adipocytes. To do so, three days prior to experiments, (on the 18th d of culture) the medium on differentiating adipocytes was changed to differentiation medium which did not contain insulin. This was done because prolonged exposure to insulin during differentiation might result in downregulation of insulin receptors, thus artefactually minimizing true insulin effects. In order to determine the response rate of the preadipocytes and adipocytes to ASP, glucose transport stimulation was assessed following exposure for
different times to a high concentration of ASP (5.5 mm). Glucose transport rates following incubation of adipocytes with ASP are shown in Figure 1, top panel. The ASP effect on glucose uptake was observed by 1 h (5.5 1.7 basal vs 7.55 2.2 with ASP, nmol/mg cell protein, P < 0.025) and reached its maximum at approximately 4 h (8.6 2.6 nmol/mg cell protein, P < 0.025). In a separate set of experiments (using cells from 3 different subjects) a similar ASP response was seen in preadipocytes (Figure 1, bottom panel), where the maximal response was also obtained at 4 h (5.04 0.49 basal vs 13.7 3.1 with ASP, nmol/mg cell protein, P < 0.05). Subsequently, in both adipocytes and preadipocytes there is apparent down-regulation of the ASP response by 24 h at which time the glucose transport was not signi cantly different from the basal values. We then examined the effect of varying concentrations of ASP on glucose transport. Cultured human preadipocytes and adipocytes from the same subjects were exposed for 1 h to increasing concentrations of ASP and the effect of glucose transport was tested (Figure 2, top panel). In differentiated adipocytes, glucose uptake was stimulated by ASP at a concentration as low as 0.3 mm (118% 8% of basal) and reached a maximum of 206% 16% of basal where basal ˆ 100% (2.44 0.39 basal vs 6.54 0.28 with ASP, nmol/mg cell protein, P < 0.0025). Preadipocytes also responded to ASP, reaching a maximal level of 201% 20% of basal (Figure 2, top panel). Note however, that cultured adipocytes are more responsive at the lower ASP concentrations than preadipocytes (range 0.3 mm±2.5 mm). The difference in response between preadipocytes and adipocytes disappears at the higher ASP concentrations (range 3.0 mm±5.5 mm). Thus, the half-maximal dose of ASP for adipocytes is 0.5 mm, whereas it is 2.3 mm for 263 Figure 1 Time-dependent effect of ASP on glucose transport in cultured human preadipocytes and adipocytes. Top panel: Adipocytes (ADIP) were incubated at 37 C with 5.5 m ASP for 15 min to 24 h. Glucose uptake was measured over 10 min with 0.5 mm of [ 3 H] 2-deoxy-glucose ( 3 H 2-DG). Results are expressed as nmols of 3 H 2-DG uptake per mg cell protein per 10 min s.e.m.. Each point represents the average of three experiments from three subjects with each determination performed in duplicate. Bottom panel: Preadipocytes (PREAD) from three different subjects were incubated as described above and glucose transport assessed. *P < 0.05, **P < 0.025, ***P < 0.005, by paired t-test vs basal glucose transport. Figure 2 Concentration-dependent effect of ASP and Insulin on glucose transport in cultured human preadipocytes and adipocytes. Adipocytes (ADIP) and preadipocytes (PREAD) were incubated at 37 C for 1 h with (0.3 mm to 5.5 mm ASP, top panel) or insulin (0.6 nm to 600 nm, bottom panel ). Glucose uptake was then measured for 10 min with 0.5 mm [ 3 H] 2-deoxy-glucose ( 3 H 2-DG). Results are expressed as % stimulation s.e.m. where basal glucose transport is shown as 100%. Each point represents the average of three experiments from three subjects performed in duplicate. *P < 0.05, **P < 0.025, ***P < 0.005, by paired t-test vs basal glucose transport.
264 preadipocytes, a 4.7 fold increase in sensitivity of adipocytes to ASP in these experiments. Overall the effect of ASP on glucose transport was signi cant over the whole concentration range for adipocytes (P < 0.05). By contrast, it was signi cantly only at high concentrations ( > 2.8 mm) in the preadipocytes (P < 0.05). As shown in Figure 2, bottom panel, experiments with the same cells with increasing concentrations of insulin resulted in an increase in glucose transport in differentiated adipocytes to a maximum of 208% 38 of basal at an insulin concentration of 60 nm, P < 0.005. By contrast, these was only a minor effect of insulin on the preadipocytes (maximum of 118% of basal where basal ˆ 100%.) Thus, in the adipocytes, the ASP stimulation was comparable to that of insulin. The above experiments were performed using cells that were differentiated for three weeks. At this time the cells exhibit adipocyte-type morphology. After longer differentiation times, fat-loaded cells become more fragile and break up and detach from the culture dish and oat up. We thus examined the response of the cells to ASP at earlier stages during differentiation. As shown in Figure 3, basal glucose transport in cultured human differentiated (4.27 0.54 nmol/mg cell protein at week one to 6.54 1.93 nmol/mg cell protein at week two to 9.76 2.55 nmol/mg cell protein at week three). Overall, this is a signi cant increase in basal transport of 190% 39%, P < 0.05 at week three compared to week one. In the rst week of differentiation, little effect of ASP on glucose transport was observed. However, at both two and three weeks of differentiation, ASP stimulated glucose transport signi cantly to 142% 14% of basal in second week (P < 0.0125) and 124% 3% of basal in the third week (P < 0.05). Similar results were obtained with insulin (Figure 3). Although the response of adipocytes to maximally stimulatory ASP concentration was somewhat less than observed previously (Figure 2) one should keep in mind that each set of experiments is performed on cells derived from different subjects. In order to gain some understanding on the mechanisms by which ASP induces glucose uptake in human adipose tissue, the effects of ASP with and without insulin were compared (Figure 4). When preadipocytes were exposed to 6 nm insulin (with no ASP) glucose transport increased to 115% 5% of basal (Figure 4, top panel). Insulin also had an effect on ASP stimulated cells, resulting in an increase in Figure 3 Differentiation-dependent effect of ASP and Insulin on glucose transport in human adipocytes. Preadipocytes were differentiated to adipocytes for a period of 1, 2 or 3 weeks (wk 1, wk 2, wk 3). Glucose uptake was measured over 10 min as [ 3 H] 2-deoxy-glucose ( 3 H 2-DG) uptake at each time following a 1 h stimulation with 5.5 mm ASP (solid bars) or 6 nm insulin (hatched bars). Basal glucose transport is shown in the open bars. Results are expressed as nmols of 3 H 2-DG uptake per mg of cell protein per 10 min s.e.m.. Each point represents the average of four experiments from four subjects with each determination performed in duplicate. * P < 0.05, ** P < 0.01 by paired t-test vs control (basal glucose transport). Figure 4 Effects of ASP and insulin on glucose transport in cultured human adipocytes and preadipocytes. Cultured preadipocytes (PREAD, top panel) were exposed to varying concentrations of ASP alone (solid bars) or supplemented with ASP and insulin (6 nm, hatched bars). Glucose uptake was then measured over 10 min with 0.5 mm [ 3 H] 2-deoxy-glucose ( 3 H 2-DG). Results are expressed as % stimulation s.e.m. where basal glucose transport is shown as 100%. Each point represents the average of ve experiments with from ve subjects with each determination performed in duplicate. * P < 0.05, ** P < 0.01 by paired t-test for ASP and insulin vs ASP alone.
membrane glucose transport above that observed with ASP alone. This effect, however, was small and was seen only at the highest concentrations of ASP tested ( > 2.4 mm, P < 0.05). In contrast, in cultured differentiated adipocytes (Figure 4, bottom panel), although the cells were responsive to ASP-induced glucose transport, and were responsive to insulin alone, no additional stimulation with insulin was observed. These results suggest that insulin and ASP may be acting via the same mechanism for glucose transport stimulation. Discussion The data from the present study demonstrates (1) that ASP stimulates speci c membrane transport of glucose in human preadipocytes and adipocytes in a time and concentration dependent manner, (2) that differentiated adipocytes are more responsive to ASP than preadipocytes, and (3) that ASP is as potent as insulin in inducing speci c membrane transport of glucose in adipocytes. These data extend our knowledge as to the mechanisms by which ASP causes triglyceride synthesis to increase in human adipocytes. Our previous work focused primarily on triglyceride synthesis. Experimental data demonstrated that both omental and subcutaneous adipose tissue (primary adipocytes or cultured adipocytes) are responsive to ASP 13,14 although the stimulation is greater in subcutaneous tissue suggesting regional speci city. 13 The physiological signi cance of these effects is becoming increasingly apparent. Until recently, triglyceride clearance from plasma was thought to be determined exclusively by lipoprotein lipase activity, greater triglyceridehydrolytic capacity resulting in more rapid removal of triglyceride from plasma. 15,16 However, the correlation between lipoprotein lipase activity and triglyceride clearance is poor 17,18 and studies have shown that, in fact, lipoprotein lipase would appear to be present in excess. 15 There is direct evidence in humans that the rate of fatty acid uptake from triglyceride-rich particles is limited. 19 A major portion of the fatty acids released from chylomicrons are not immediately taken up by adipocytes 20 but rather continue to circulate. Thus, the rate of chylomicron triglyceride hydrolysis by lipoprotein lipase is not a direct function of the mass of this enzyme present on the endothelial surface, but the increase in ambient circulating fatty acids can also result in product inhibition of lipoprotein lipase and in uence triglyceride clearance. 19,21±23 Our hypothesis has been that adipocyte triglyceride synthesis determines the rate at which fatty acids are taken up by adipocytes from the adjacent capillary space. This rate will in uence the proportion of fatty acids which enter adipocytes directly after lipolysis as opposed to the proportion which exit the adipocyte capillary space and pass within the circulation to the liver. A decreased rate of adipocyte fatty acid uptake results in increased delivery of fatty acids to the liver and subsequently, increased VLDL production. 24 Any factor which increases the rate of fatty acid uptake and triglyceride synthesis will enhance the ef ciency of adipocyte triglyceride storage. Normal plasma ASP in a group of healthy control subjects (35±65) is 32.0 2.6 nm. 25 Plasma ASP increases postprandially 26 up to two-fold and is a potent in vitro stimulator of triglyceride synthesis in human adipocytes 4 and may thus enhance adipose tissue ef ciency. However, fatty acids are not the only building block required for triglyceride synthesis and storage. Glucose is the source of the glycerol-3-phosphate backbone, and it is well known that glucose transport increases postprandially in response to hormone stimuli. 27,28 The present study adds importantly to the documentation of this pathway in humans. That ASP causes speci c membrane transport of glucose to increase in adipocytes is clear, although we have not in this instance directly demonstrated the mechanism responsible for this effect. Based on our previous results in cultured human skin broblasts and L6 myotubes, ASP likely induces translocation of glucose transporters. 9,10 In the broblast model, ASP induced translocation of glut-1 transporters to the cell membrane whereas in the L6 myotube model, ASP stimulates translocation of glut-1, glut-3 and glut-4 transporters, all to the same extent as insulin. As well, the fact that lower ASP concentrations are more effective at increasing glucose transport in the differentiated adipocytes is consistent with our previous observation that the effects of ASP on triglyceride synthesis become more pronounced during the process of adipocyte differentiation. 4 Although higher concentrations of ASP were necessary to achieve the same stimulation as insulin, it should be noted that the physiological levels of plasma ASP are also higher than insulin: 32.0 2.6 nm ASP 25 vs 36±180 pm insulin. 29 The effects of ASP and insulin on glucose transport were not additive in the differentiated adipocytes whereas they are in human skin broblast and the L6 myotube models. 9,10 This difference may be consequent to the differentiation induced changes in the level of expression of the various glucose transporters. In the preadipocytes, although there was a trend towards additivity of the ASP and insulin effects, because the insulin effect (although signi cant) was modest, this was dif cult to assess. Nevertheless, demonstration that ASP directly induces speci c membrane transport of glucose is critical to documenting the mechanisms by which it increases triglyceride synthesis in adipocytes. Acknowledgements This work was supported by a grant to K. Cian one from the Medical Research Council of Canada (MA12462). K. Cian one is the recipient of a Scho- 265
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