Báo cáo khoa học: Dictyostelium differentiation-inducing factor-1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells pdf

Dictyostelium differentiation-inducing factor-1 induces glucose transporter translocation and promotes glucose uptake in mammalian cells Waka Omata1, Hiroshi Shibata1, Masahiro Nagasawa1, Itaru Kojima1, Haruhisa Kikuchi2, Yoshiteru Oshima2, Kohei Hosaka3 and Yuzuru Kubohara4 Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Department of Basic Sciences for Medicine, Gunma University School of Health Sciences, Maebashi, Japan Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan Keywords antitumor agent; Dictyostelium; DIF-1; glucose uptake; GLUT1 Correspondence Y Kubohara, Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan Fax: +81 27 2208834 Tel: +81 27 2208831 E-mail: kubohara@showa.gunma-u.ac.jp Website: http://imcr.showa.gunma-u.ac.jp/ index-e.htm (Received 26 February 2007, revised May 2007, accepted May 2007) doi:10.1111/j.1742-4658.2007.05872.x The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF-1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells In the present study, we investigated the effects of DIF-1 and its analogs on normal (nontransformed) mammalian cells Without affecting the cell morphology and cell number, DIF-1 at micromolar levels dose-dependently promoted the glucose uptake in confluent 3T3-L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3-kinase) DIF-1 affected neither the expression level of glucose transporter nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6-phosphate kinase, pyruvate kinase, and glucose 6-phosphate dehydrogenase Most importantly, stimulation with DIF-1 was found to induce the translocation of glucose transporter from intracellular vesicles to the plasma membranes in the cells In differentiated 3T3-L1 adipocytes, DIF-1 induced the translocation of glucose trasporter (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin These results indicate that DIF-1 induces glucose transporter translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3-kinase ⁄ Akt-independent pathway in mammalian cells Furthermore, analogs of DIF-1 that possess stronger antitumor activity than DIF-1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF-1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth Abbreviations DIF-1, differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; DIF-3, differentiation-inducing factor-3 [1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; DMEM-HG, DMEM containing a high concentration of glucose; DMEM-LG, DMEM containing a low concentration of glucose; EtOH, ethanol; GLUT, glucose transporter; LDM, low-density microsome; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one; 8-MIBMX, 8-methoxymethyl-3-isobutyl-1-methylxanthine; 2-MIDIF-1, 2-methoxy isomer of DIF-1; PDE1, calmodulin-dependent cyclic nucleotide phosphodiesterase; PI3K, phosphatidylinositol 3-kinase; PM, plasma membrane; Tg, thapsigargin; THPH, 1-(2,4,6-trihydroxyphenyl)hexan-1-one 3392 FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS W Omata et al The cellular slime mold Dictyostelium discoideum is an excellent model organism in the fields of cell and developmental biology because of its simple pattern of life cycle; this organism forms fruiting bodies, each consisting of spores and a cylindrical multicellular stalk, at the end of its development The differentiation-inducing factor-1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one] (Fig 1) is a lipophilic signal molecule that was identified as a stalk-cell-inducing factor in D discoideum [1,2] DIF-3 [1-(3-chloro-2,6-dihyd- Fig Chemical structure of DIF-1, DIF-3 and their derivatives (A) DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1one DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one 2-MIDIF-1; 2-methoxy isomer of DIF-1 DMPH; 1-(2,6-dihydroxy-4methoxyphenyl)hexan-1-one THPH; 1-(2,4,6-trihydroxyphenyl)hexan-1-one (B) Derivatives of DIF-1 that have alkyl chains of different lengths (C) Derivatives of DIF-1 and DIF-3 that have a different halogen DIF-1 promotes glucose uptake in mammalian cells roxy-4-methoxyphenyl)hexan-1-one] (Fig 1), the initial product in the process of DIF-1 breakdown, is much less active in inducing stalk cell differentiation [2–4] DIF-1 is thought to function at least in part via an increase in the cytoplasmic calcium concentration ([Ca2+]c) [5–7], but the precise signaling system of DIF-1, including the target molecule(s) of DIF-1, is still unknown On the other hand, it has been shown that DIF-1 and DIF-3 (designated DIFs) exhibit strong antiproliferative activity and occasionally induce cell differentiation in vitro in mammalian cells [8–16] and that DIF-3 and some derivatives of DIF-3 are the most potent antitumor agents among the DIF analogs tested to date [12,16,17] As to the mechanism of the actions of DIFs, it has been shown that: (a) DIFs increase [Ca2+]c in some tumor cells [9–12], (b) DIFs activate Akt ⁄ protein kinase B in human leukemia K562 cells [13], (c) DIF-1 inactivates STAT3 in gastric cancer cells [15], and (d) DIFs inhibit the expression of cyclins D ⁄ E and the phosphorylation of the retinoblastoma protein in vascular smooth muscle cells [14] and K562 cells [18] Recently, we have found that calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is a pharmacological and specific target of DIFs in mammalian cells [19] Yet, the mechanisms underlying the actions of DIFs in mammalian cells remain to be elucidated Meanwhile, in the course of a study of the in vitro actions of DIFs in mammalian normal (nontransformed) cells, we noticed that DIF-1 promotes color conversion (red to yellow) of the incubation media of rat gastric mucosal RGM-1 and rat leptomeningeal cells [20], which suggests that DIF-1 might promote the cellular metabolism in some way In the present study, in order to assess the newly found function of DIF-1, we examined the effects of DIF-1 and its analogs (Fig 1) on glucose consumption in vitro in RGM-1 cells, mouse 3T3-L1 fibroblasts, and 3T3-L1 adipocytes, and investigated the mechanism of the novel function of DIF-1 We show here that DIF-1 induces the translocation of glucose transporter (GLUT) from intracellular vesicles to the plasma membrane and thereby promotes glucose uptake, at least in part, via a phosphatidylinositol 3-kinase (PI3K) ⁄ Akt-independent pathway in the cells We also show that DIF-1 possesses the most potent activity among its analogs tested Results Effect of DIF-1 on glucose consumption in 3T3-L1 fibroblasts and RGM-1 cells We first assessed the cytotoxic effect of DIF-1 on confluent 3T3-L1 cells (Fig 2) Neither ethanol (EtOH) FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS 3393 DIF-1 promotes glucose uptake in mammalian cells Continuous incubation W Omata et al Medium exchange A EtOH D EtOH B THPH E THPH C DIF-1 F DIF-1 0.1 mm Fig Effect of DIF-1 on cell morphology in 3T3-L1 fibroblasts Confluent 3T3-L1 cells were incubated in vitro for days continuously with 0.2% EtOH (vehicle) (A), 20 lM THPH (B), or 20 lM DIF-1 (C), or cells were incubated for days by exchanging the media every 24 h with 0.2% EtOH (vehicle) (D), 20 lM THPH (E), or 20 lM DIF-1 (F) On day 3, the cells were observed by using a phase-contrast microscope Most of the cells under continuous incubation with DIF-1 were dead (C), but cells appeared to be healthy when the incubation medium was exchanged (F) Schematic color images of the culture wells are shown on the photos Scale bar, 0.1 mm (vehicle) at 0.2% (Fig 2A), nor 1-(2,4,6-trihydroxyphenyl)hexan-1-one (THPH) at 20 lm (Fig 2B) affected cell morphology and viability but, as previously reported with RGM-1 cells [20], DIF-1 at 20 lm induced cell death (Fig 2C) where medium acidification (redto-yellow color change) was promoted However, since the apparent cytotoxic effect of DIF-1 was canceled when the incubation medium was exchanged daily with a fresh one containing DIF-1 (Fig 2F), it was likely that DIF-1 would promote cellular metabolism in some way, resulting in medium acidification and subsequent cell death In order to assess what happens in the cells in the presence of DIF-1, we examined the effect of DIF-1 on glucose consumption by monitoring the glucose concentration in the incubation media (Fig 3A) As expected, DIF-1 at the micromolar levels promoted glucose consumption in a dose-dependent manner (Fig 3A,B) without affecting the cell number (Fig 3B) or cell morphology (data not shown) Withdrawal of DIF-1 in 3394 the media resulted in the restoration of the rate of glucose consumption to the normal level (Fig 3A), indicating that the promoting effect of DIF-1 is reversible It should be noted that similar effects of DIF-1 were observed using confluent RGM-1 cells (Fig 4A,B), suggesting that DIF-1 would be a broad promoter for glucose consumption in mammalian cells across species Effect of DIF-1 on glucose uptake in 3T3-L1 fibroblasts In order to confirm that DIF-1 should promote glucose consumption by the cells, we checked the effect of DIF-1 on glucose uptake in confluent 3T3-L1 cells (Fig 3C,D) The cellular activity of the 2-deoxy-glucose uptake was promoted in the presence of 20 lm DIF-1 and reached a saturated level after a 4-h incubation (Fig 3C) DIF-1 at 10–20 lm promoted the 2-deoxyglucose uptake dose-dependently, while the 2-methoxy isomer of DIF-1 (2-MIDIF-1) at 20 lm did not result in such an activity (Fig 3D) The results agree quite well with those shown in Fig 3B, indicating that the glucose-uptake promoting activity of DIF-1 (and possibly its analogs) can be assessed approximately by monitoring the glucose concentration in the incubation media under the conditions In addition, the simple assay system for glucose consumption should be a convenient method for the broad screening of candidate molecules that promote the cellular metabolism Effects of DIF analogs on glucose consumption in 3T3-L1 fibroblasts To assess the chemical structure–effect relationship and specificity of the action of DIF-1, we examined the effects of DIF analogs on glucose consumption in confluent 3T3-L1 cells (Fig 5) While DIF-1 at 20 lm greatly promoted glucose consumption, the same concentration of 2-MIDIF-1, 1-(2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DMPH), and THPH (Fig 1A) showed no effect or a very weak one (Fig 5A) Similar results were obtained with confluent RGM-1 cells (Fig 4C) These results indicate that two chlorines and a methoxy group on the proper position of the benzene ring are important for the promoting effect of DIF-1 on glucose consumption and, in other words, that the action of DIF-1 should be specific to its chemical structure Among DIF-1 derivatives that have different length of alkyl chains (Fig 1B), DIF-1 exhibited the most potent activity for stimulating glucose consumption in 3T3-L1 cells (Fig 5B), indicating that the hexanone chain is most appropriate for the activity On the other FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS W Omata et al DIF-1 promotes glucose uptake in mammalian cells addition of DIF-1 C 2-deoxy-glucose uptake (cpm) 30 000 0.9 0.8 None EtOH (0.2%) 10 µ M DIF-1 20 µ M DIF-1 20 000 10 000 10 15 20 25 Time of incubation (h) n.s EtOH None 10 2-deoxy-glucose uptake (cpm) 20 D 10 20 (µM) DIF-1 100 80 60 40 20 EtOH ** None 30 * 0.5 Time of incubation (h) 30 120 ** ** 5000 0.5 n.s ** 15 000 10 20 (µM) DIF-1 25 000 p < 0.001 p < 0.001 20 000 15 000 p < 0.05 10 000 5000 EtOH 0.6 ** 25 000 None 0.7 B Approx rate of glucose consumption (µg/ml/h) withdrawal of DIF-1 at 12 h Cell number (% of control) Glucose concentration (mg/ml) A 10 20 20 (µM) DIF-1 2-MID-1 Fig Effect of DIF-1 on glucose consumption, glucose uptake, and cell number in 3T3-L1 fibroblasts (A) Confluent 3T3-L1cells were incubated for 12 h without or with 0.2% EtOH (vehicle) or 10–20 lM DIF-1, the incubation media were discarded, and all the cells were further incubated with fresh media in the absence of EtOH or DIF-1 for 18 h The glucose concentration of each well was measured at the indicated time points, and the mean ± SD (bars) values of the triplicate (n ¼ 3) are presented Note that the rate of glucose consumption by the control cells appear to increase along with the incubation time, which would be the allowable margin of error by the simple assay system (B) Confluent 3T3-L1cells were incubated for 18 h without or with 0.2% EtOH or 10–20 lM DIF-1 The glucose concentration of each well was measured, and the approximate rate of glucose consumption was calculated The cell number of each well was then assessed using a cell-number indicator The mean ± SD (bars) values of the triplicate (n ¼ 3) are presented **P < 0.001 versus others n.s., not significant (C) Confluent 3T3-L1cells were incubated for the indicated period with 20 lM DIF-1, and the 2-deoxy-glucose uptake was assessed as described in Experimental procedures All samples were prepared in quartettes, and the mean ± SD (bars) values of each quartette (n ¼ 4) are presented Note that it takes approximately h for glucose uptake activity to reach a maximal level **P < 0.001 versus *0-time control n.s., not significant (D) Confluent 3T3-L1cells were incubated for h without or with 0.2% EtOH (vehicle), 10–20 lM DIF-1, or 20 lM 2-MIDIF-1 (2-MID-1), and the 2-deoxy-glucose uptake was assessed The mean ± SD (bars) values of the triplicate (n ¼ 3) are presented The results agree well with those of the simple assay in (B) hand, DIF-1 was the only agent that showed strong activity among the halogen-substituted derivatives (Figs 1C and 5C) in spite of the fact that all of them are potent antiproliferative agents in K562 leukemia [17] The results suggest that the mechanism of the action of DIF-1 for stimulating glucose consumption should be different from that for suppressing tumor cell growth Effects of the [Ca2+]i-increasing agents and PDE1 inhibitor on glucose consumption in 3T3-L1 fibroblasts It has been shown that DIF-1 increases [Ca2+]c in mammalian cells [9–11,20] and that DIF-1 is a specific inhibitor for the calmodulin-dependent cyclic nucleotide phophodiesterase, PDE1 [19] We thus examined the effects of two [Ca2+]c-increasing agents, thapsigargin (an inhibitor of Ca2+-ATPase present in endoplasmic reticula) and A23187 (a calcium ionophore), and 8-methoxymethyl-3-isobutyl-1-methylxanhine (8-MIBMX; an inhibitor of PDE1) on glucose consumption in confluent 3T3-L1 cells (Fig 5A) However, these agents did not promote glucose consumption in the cells, suggesting that DIF-1 promotes glucose consumption in a way other than by increasing [Ca2+]c or inhibiting PDE1 To support our hypothesis that an increase in [Ca2+]c is not involved in the DIF-1-promoted glucose consumption, we examined the effects of DIF analogs and thapsigargin (Tg) on [Ca2+]c in 3T3-L1 cells As shown in Fig 6, DIF-1 at 20 lm indeed increased [Ca2+]c, while THPH showed no significant effect on [Ca2+]c Importantly, however, DIF-3 and Tg also increased [Ca2+]c considerably, regardless of the fact that both reagents scarcely promoted glucose consumption (Fig 5A) These results support our hypothesis FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS 3395 DIF-1 promotes glucose uptake in mammalian cells C 0.8 0.7 none 15 µM DIF-1 30 µM DIF-1 0.6 0.5 0.4 0.3 10 15 20 25 Time of incubation (h) 30 ** 40 30 20 10 DMPH T HPH 0.9 50 EtOH DIF -1 DIF -3 No ne withdrawal of DIF-1 at 14 h Ap pr ox rate o f g luco se sum ption (µg /ml/h ) addition of DIF-1 Gluco se centration (m g/m l) A W Omata et al 40 ** 30 * * 20 10 0 15 30 DIF-1 (µM) Cell n u mb er (% of contro l) Ap pr ox rate of gluco se consum ption (µg /ml/h ) B 120 100 80 60 40 20 0 15 30 DIF-1 (µM) Fig Effects of DIF-1 and its analogs on glucose consumption in RGM-1 cells (A) Confluent RGM-1 cells were incubated for 14 h with 15–30 lM DIF-1, the incubation media were discarded, and all the cells were incubated with fresh media in the absence of EtOH or DIF-1 for 12 h The glucose concentration of each well was measured at the indicated time points, and the mean ± SD (bars) values of the triplicate (n ¼ 3) are presented (B) Confluent RGM-1cells were incubated for 12 h without or with 8–30 lM DIF-1 The glucose concentration of each well was measured, and the approximate rate of glucose consumption was calculated The cell number of each well was then assessed using a cell-number indicator The mean ± SD (bars) values of the triplicate (n ¼ 3) are presented *P < 0.02 versus others **P < 0.001 versus others (C) Confluent RGM-1 cells were incubated for 12 h without or with 0.2% EtOH (vehicle) or 20 lM DIF analogs The glucose concentration of each well was measured, and the approximate rate of glucose consumption was calculated **P < 0.01 versus others Effect of DIF-1 on GLUT1 translocation in 3T3-L1 fibroblasts Insulin and some agents are known to promote glucose uptake by inducing translocation of glucose transporters from cytosolic store vesicles to the plasma membrane [21,22] In 3T3-L1 fibroblasts, the major activity of glucose uptake can be attributed to the glucose transporter isoform GLUT1 [23,24] We therefore examined the effect of DIF-1 on GLUT1 translocation in 3T3-L1 cells and found that DIF-1 at 20 lm stimulated GLUT1 translocation from low-density microsomes (LDMs) to the plasma membrane (PM) by approximately two-fold (Fig 7) These results indicate that DIF-1 promotes glucose uptake, at least in part, by stimulating GLUT1 translocation Effects of DIF-1 and PI3K inhibitors on glucose uptake and GLUT1 expression DIF-1 has been shown to activate the PI3K ⁄ Akt pathway in human leukemia K562 [13,18] and human gastric cancer AGS cells [15] On the other hand, the activation 3396 of PI3K and Akt is involved in insulin actions to promote glucose uptake and glycolysis [25,26] In order to assess whether PI3K and Akt are involved in the action of DIF-1 in 3T3-L1 cells, we examined the effect of DIF-1 on glucose consumption (Fig 8A) and glucose uptake (Fig 8B) in the presence of wortmannin or LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], inhibitors for PI3K However, DIF-1 promoted glucose consumption even in the presence of the inhibitors Western analysis showed that DIF-1 might slightly activate (phosphorylate) Akt, which was perfectly inhibited with wortmannin (Fig 8C) Importantly, DIF-1 did not affect the total amount of GLUT1 present in the cells (Fig 8C), but coaddition of DIF-1 and wortmannin decreased the expression of GLUT1 (Fig 8C), resulting in a due decrease in glucose consumption (uptake) (Fig 8A,B) Nevertheless, DIF-1 promoted glucose consumption (uptake) even in the presence of the inhibitor (Fig 8A,B) Similar results were obtained with confluent RGM-1 cells (data not shown) These results suggest that DIF-1 should promote the glucose uptake in some way, at least in part, via a PI3K ⁄ Akt-independent pathway without FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS W Omata et al 8-MIBMX DMSO A23187 Tg T HPH ** P < 0.01 versus * ** * * DIF -1(+2) DIF -1(+1) DIF -1 * DIF -1(-1) C ** P < 0.01 versus others ** I-DIF-3 I-DIF-1 Br -DIF-3 Br -DIF-1 DIF -3 DIF -1 EtOH Ap pr ox rate o f gluco se co nsump tio n (r atio) DMPH 2-MIDIF-1 EtOH DIF-1 DIF -1(-2) Ap pr ox rate o f gluco se co n su mp tio n (r atio) ** B ** P < 0.01 versus others EtOH Appr ox rate o f g lucose co n su mptio n (r atio) A DIF-1 promotes glucose uptake in mammalian cells Fig Effects of DIF analogs and some reagents on glucose concumption in 3T3-L1 fibroblasts Confluent 3T3-L1cells were incubated for 10–14 h with 0.2% EtOH (vehicle), 20 lM of the indicated DIF derivatives, 20 nM Tg, 0.2 lM A23187, 0.4% dimethylsulfoxide (vehicle), or 0.4 mM 8-MIBMX The glucose concentration of each well was measured, and the approximate rate of glucose consumption was calculated as a ratio of control (EtOH or dimethylsulfoxide) The mean ± SD (bars) values of three independent experiments (n ¼ 3) are presented DMPH, 1-(2,6-dihydroxy-4methoxyphenyl)hexan-1-one; DMSO, dimethylsulfoxide affecting GLUT1 expression in mammalian cells It should be noted that other glucose transporter isoforms, such as GLUT2, GLUT3, and GLUT4, were not detectable in 3T3-L1 cells regardless of the presence of DIF-1 (data not shown) Effect of DIF-1 on the enzymes involved in glucose metabolism in 3T3-L1 fibroblasts To further investigate the mechanism of the action of DIF-1, we examined the effects of DIF-1 on the enzymatic activities of glucose 6-phosphate dehydrogenase and three glycolytic enzymes, such as hexokinase, fluctose 6-phosphate kinase, and pyruvate kinase However, incubation of the cells with 20 lm DIF-1 did not affect the total activities of the enzymes (data not shown) We then examined the direct effects of DIF-1 on the enzyme activities in vitro, but the activities were scarcely affected by DIF-1 (data not shown) Effects of DIF-1 and insulin on the glucose uptake and GLUT translocation in 3T3-L1 adipocytes 3T3-L1 adipocytes are well-established model cells suitable for the analysis of the actions of insulin and other insulinomimetic substances in adipocytes Thus, we examined and compared the effects of DIF-1 and insulin on glucose uptake in 3T3-L1 adipocytes As shown in Fig 9A, DIF-1, as well as insulin, promoted the glucose uptake by two to three-fold However, while the insulin-promoted glucose uptake and the phosphorylation (activation) of Akt were almost completely suppressed by the addition of wortmannin, the DIF-1-promoted glucose uptake was only slightly suppressed by the inhibitor These results indicate that DIF-1 promotes the glucose uptake of 3T3-L1 adipocytes, at least in part, via a PI3K ⁄ Akt-independent pathway We finally examined the effects of DIF-1 and insulin on the translocation of GLUT1 and GLUT4 in 3T3L1 adipocytes (Fig 9B) As expected, insulin induced both GLUT1 and GLUT4 translocation from intracellular stores to the plasma membrane but, in contrast, DIF-1 induced only GLUT1 translocation without affecting the total amount of GLUT1 The total amounts of GLUT4 were reduced after stimulation with DIF-1 or insulin (Fig 9B) for unknown reasons, while the other species of glucose transporters, GLUT2 and GLUT3, were not detectable (data not shown) Our results suggest that DIF-1 may promote glucose uptake, at least in main part, via GLUT1 translocation Discussion A novel function of DIF-1 in mammalian cells DIF-1 was originally identified as a stalk-cell differentiation-inducing factor in the cellular slime mold FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS 3397 DIF-1 promotes glucose uptake in mammalian cells 200 400 600 800 200 400 600 n.s * * 1.6 * 1.4 1.2 0.8 T HPH THPH n.s 1.8 DIF -3 1.3 1.2 1.1 0.9 Tg DIF-3 DIF -1 1.5 1.4 1.3 1.2 1.1 0.9 B 1.7 1.6 1.5 1.4 1.3 1.2 1.1 0.9 DIF-1 Basal 1.5 1.4 1.3 1.2 1.1 0.9 Relative in ten sity o f fluo rescen ce Relative in ten sity of flu or escence (Starti ng fluo rescence rati o: R340/380 =1) A W Omata et al Tg 800 Time of monitoring (s) Fig Effects of DIF analogs on [Ca2+]c in 3T3-L1 fibroblasts (A) Fura-2-loaded 3T3-L1 cells were stimulated with 20 lM of DIF-1, DIF-3, or THPH, or 20 nM Tg, and the changes in fluorescence ratio (R340 ⁄ 380) were monitored The representative traces of three independent cells are presented in each graph (B) The intensities of the maximal fluorescence ratio (R340 ⁄ 380) induced by the reagents are presented as the ratio of the basal fluorescence ratio The mean ± SD (bars) values of five independent cells (n ¼ 5) are presented *P < 0.001 n.s.; not significant Mechanism of the action of DIF-1 As described already, since DIF-1 activates PI3K and Akt in K562 cells [13] and human gastric cancer AGS cells [15], we first expected that DIF-1 may have the insulinomimetic function of promoting glucose metabolism However, DIF-1 activated Akt slightly, if at all, in 3T3-L1 fibroblasts (Fig 8C), adipocytes (Fig 9A), and RGM-1 cells (data not shown) Moreover, because DIF-1 promoted the glucose uptake in the cells even in the presence of wortmannin or LY294002 (PI3K inhibitors) (Figs and 9A), we 3398 A Syntaxin4 PM LDM Relative am ou nt o f Glu t1 B Glut1DIF-1: - + - + PM LDM C Relative am ou nt of Glut1 in PM D discoideum [1], and DIF-1 and its analogs were found to possess antiproliferative activity, occasionally inducing cell differentiation in mammalian cells [8–19] In the present study, we have found for the first time that DIF-1 induces the translocation of GLUT1 from intracellular stores to the plasma membrane, at least in part, via a PI3K ⁄ Akt-independent pathway and thereby promotes glucose uptake in 3T3-L1 fibroblasts (Fig 8) and 3T3-L1 adipocytes (Fig 9) Furthermore, we have shown here that DIF-1 possesses the most potent activity among its analogs tested (Fig 5) Because DIF-3 and its derivatives, rather than DIF-1, exhibit stronger antitumor activities [12,16,17,19], the present results suggest that the mechanism of the action of DIF-1 for stimulating glucose uptake is different from that for suppressing tumor cell growth If this is the case, we may be able to develop some DIF-1 derivatives that possess strong glucose uptake-promoting activity but little antitumor activity DIF-1 and such derivatives may have therapeutic potential in the treatment of obesity and diabetes ** EtOH DIF-1 Fig Effect of DIF-1 on GLUT1 translocation in 3T3-L1 fibroblasts (A) Crude membrane fractions of confluent 3T3-L1 cells were applied to sucrose density-gradient centrifugation, and the PM and LDM thus obtained were analyzed by western blotting for syntaxin4 (a marker for PM) (B) Confluent 3T3-L1 cells were incubated for h with 0.2% EtOH (–) or 20 lM DIF-1 (+), and crude membrane fractions of the cells were applied to sucrose density-gradient centrifugation PM and LDM were then analyzed by western blotting for GLUT1, and the relative amounts of GLUT1 in the blot are shown (C) The relative amounts of GLUT1 present in PM were assessed after stimulation with 0.2% EtOH or 20 lM DIF-1, as described in (B) The mean ± SD (bars) values of three independent (n ¼ 3) experiments are presented **P < 0.01 concluded that DIF-1 promotes glucose uptake, at least in part, via a PI3K ⁄ Akt-independent pathway It should also be noted that DIF-1 did not affect the FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS W Omata et al Rate of g lu cose sum ption (r atio ) A ** ** ** 2-d eoxy-glu co se up take (r atio ) ** DIF-1 wort B DIF-1 promotes glucose uptake in mammalian cells - + - + - - + + C * ** Akt p-Akt GLUT1 DIF-1 wort DIF-1 wort DIF-1 LY - + - + - - + + - - + + - + - + - + - + - - + + Fig Effects of DIF-1 and PI3K inhibitors on glucose consumption in 3T3-L1 fibroblasts (A) Confluent 3T3-L1 cells were incubated for h without or with 20 lM DIF-1 and ⁄ or 0.1 lM wortmannin (wort) (left panel) or 30 lM LY294002 (LY) (right panel) All the incubation media contained 0.2% EtOH (left panel) or both 0.3% dimethylsulfoxide and 0.1% EtOH as vehicles (right panel) The glucose concentration of each well was measured, and the approximate rate of glucose consumption was calculated as a ratio for the control All samples were prepared in triplicate, and the mean ± SD (bars) values of the triplicate (n ¼ 3) are presented **P < 0.001 (B) Confluent 3T3-L1cells were incubated for h without or with 20 lM DIF-1 and ⁄ or 0.1 lM wortmannin (wort) (all the incubation media contained 0.2% EtOH as vehicle), and the 2-deoxy-glucose uptake was assessed as described in Experimental procedures Values are the means ± SD (bars) of three independent experiments (n ¼ 3) in which four-well determination was performed for each sample *P < 0.01 **P < 0.05 (C) Confluent 3T3-L1cells were incubated for h without or with 20 lM DIF-1 and ⁄ or 0.1 lM wortmannin (wort) (all the incubation media contained 0.2% EtOH as vehicle), and the cell proteins were analyzed by western blotting for Akt, phospho-Akt (p-Akt), and GLUT1 Representative results of several similar experiments are presented Note that wortmannin can inhibit the phosphorylation of Akt at least for h of incubation (data not shown) activities of four key enzymes involved in the glucose metabolism (data not shown) Most importantly, in the present study, we have shown that DIF-1 induces the translocation of GLUT1 in 3T3-L1 fibroblasts (Fig 7) and adipocytes (but not that of GLUT4) (Fig 9B), without affecting the amounts of GLUT1 (Figs 8C and 9B) GLUTs2–4 in 3T3-L1 fibroblasts and GLUTs2–3 in adipocytes were undetectable in western bolts (data not shown) In addition, since the rates of DIF-1-promoted glucose uptake (Figs and 9A) correlate well with the amounts of GLUT1 in the plasma membranes (Figs and 9B), the major glucose-uptaking activity of DIF-1 should be attributed to GLUT1 translocated to the plasma membrane in the cells, although we cannot exclude the possibility of the involvement of some GLUT isoform(s) other than GLUTs1–4 Glucose uptake and GLUT1 expression are enhanced in cancer cells and especially in malignant cells [27–30], indicating that GLUT activity should be associated with cell proliferation However, in regard to some colorectal adenocarcinomas, the highest level of GLUT1 was found in the most slowly proliferating cell line and the induction of cell cycle arrest increased both GLUT1 expression and glucose consumption [31] Although a simple comparison should be avoided and further research in this area is awaited, the action of DIF-1 is different from that in the above cases in that DIF-1 never increased the expression levels of GLUTs as far as we are aware It has been shown that DIF-1 increases [Ca2+]c in mammalian cells [9–11,20] and that DIF-1 is a direct inhibitor of PDE1 [19] In addition, increases in [Ca2+]c have been shown to stimulate glucose uptake via GLUT1 in avian erythrocytes [32], Swiss 3T3 fibroblasts [33], and rat epithelial cells [34] However, neither Tg, nor A23187 ([Ca2+]c-increasing agents), nor 8-MIBMX (PDE1 inhibitor) promoted glucose consumption in 3T3-L1 fibroblasts (Fig 5A) Furthermore, we have shown that the [Ca2+]c-increasing activities of DIF analogs (Fig 6) hardly correlate with the glucose consumption-promoting effects of the analogs (Fig 5A) but correlate well with the antileukemic effects of the analogs [12] These results strongly suggest that DIF-1 induces GLUT1 translocation and thereby promotes glucose consumption (uptake) in a way other than by increasing [Ca2+]c or inhibiting PDE1 The natural coenzyme a-lipoic acid, a small lipophilic substance that may be similar to DIF-1, can also promote glucose uptake in 3T3-L1 adipocytes [22,35] However, a-lipoic acid stimulates glucose uptake, at least in part, via PI3K ⁄ Akt-dependent GLUT4 translocation [22], which is different from the actions of DIF-1 in several aspects (Figs 7–9) Finally, it is noteworthy that DIF-1 did in fact promote glucose consumption in all the mammalian cell lines tested to date Therefore, although the precise mechanism of DIF-1-stimulated GLUT1 translocation and glucose uptake is unclear at present, DIF-1 and its derivatives appear to be good tools for the study of glucose metabolism and cell biology in general FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS 3399 DIF-1 promotes glucose uptake in mammalian cells A W Omata et al B DIF ± wort or vehicle (EtOH) DMEM (LG, -Serum) BA EtOH Ins ± wort 3T3-L1 adipocytes Assay for 2-deoxy-Glu uptake & Western blot D LDM I GLUT4 Cell lysates C D I ** ** C GLUT1 4.5 (h) GLUT1 C: Control D: DIF-1 I :insulin GLUT4 ** EtOH DIF wort wort Ins wort +DIF +Ins Akt p-Akt GLUT1 & in PM (ratio) 2-deoxy-glucose uptake (ratio) PM C ** ** n.s C D I GLUT1 C GLUT4 Fig Effects of DIF-1 and insulin on glucose uptake (A) and the translocation of GLUT1 and GLUT4 (B) in 3T3-L1 adipocytes (A) As described schematically, adipocytes were stimulated for h with 0.2% EtOH (vehicle), 10 lM DIF-1 and ⁄ or 0.1 lM wortmannin (wort) in DMEM-LG (serum-free) and then for 30 in Buffer A (BA) containing the same reagents Alternatively, 3T3-L1 adipocytes were stimulated for 30 with 100 nM insulin (Ins) and ⁄ or 0.1 lM wortmannin (wort) in BA All samples were then assayed for 2-deoxy-glucose uptake Note that the maximal effect was attained with 10 lM of DIF-1 under the conditions probably because the active concentration of DIF-1 was high due to the absence of calf serum in the incubation media (calf serum reduces the effects of DIF-1; data not shown) The values in the graph are the mean ± SD (bars) values of three independent experiments (n ¼ 3) **P < 0.0001 The adipocyte cell proteins stimulated with the reagents were analyzed by western blotting for Akt and phospho-Akt (p-Akt) (B) 3T3-L1 adipocytes were incubated for 4.5 h with 0.2% EtOH (C) or 10 lM DIF-1 (D) or for 30 with 100 nM insulin (I), and the crude membrane fractions of the cells were applied to sucrose density-gradient centrifugation The PM and LDM thus obtained were analyzed by western blotting for GLUT1 and GLUT4 (representative blots are shown; upper panel) The relative amounts of GLUT1 and GLUT4 present in PM were assessed, and the mean ± SD (bars) values of three independent experiments (n ¼ 3) are shown **P < 0.01 The total cell lysates of the three samples were also analyzed by western blotting for GLUT1 and GLUT4 Experimental procedures Reagents DIF-1, DIF-3, and their derivatives were synthesized as previously described [17] and stored at )20 °C as 5–10 mm solutions in EtOH Tg and A23187 were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan) and stored at )20 °C as 10 lm and 0.1 mm solutions in EtOH, respectively 8-MIBMX was obtained from Calbiochem (Darmstadt, Germany) and was stored at °C as 25–100 mm solutions in dimethylsulfoxide LY294002 was purchased from the Sigma-Aldrich Corp (St Louis, MO, USA) and stored at °C as a 10 mm solution in dimethylsulfoxide Sheep anti-syntaxin4 serum, a generous gift from J E Pessin (Stony Brook University, NY, USA), was used for western blotting (1 : 1000 dilution) Rabbit anti(GLUT1 serum) (FabGennix, Inc., Shreveport, LA, USA) and rabbit anti-(GLUT4 serum) were used as described previously [36] Rabbit anti-(Akt serum) and anti-(phosphoAkt serum) (Ser473) were obtained from New England 3400 BioLabs (Beverly, MA, USA), and horseradish peroxidaseconjugated anti-(rabbit serum) and anti-(mouse serum) were obtained from Amersham (Little Chalfont, UK) The antibodies were used for western blotting according to the manufacturer’s instructions Cells Mouse 3T3-L1 fibroblast cells, kindly provided by O Ezaki (National Institute of Health and Nutrition, Tokyo, Japan), differentiated 3T3-L1 adipocytes, and rat gastric mucosal RGM-1 cells [20] were used in this study 3T3-L1 fibroblast cells were maintained in vitro at 37 °C (5% CO2) in DMEM containing a high concentration (4500 mgỈL)1) of glucose (DMEM-HG) (Sigma, D5796) supplemented with 75 lgỈmL)1 penicillin, 50 lgỈmL)1 streptomycin and 10% (v ⁄ v) fetal bovine serum RGM-1 cells were maintained in vitro at 37 °C (5% CO2) in DMEM containing a low concentration (1000 mgỈL)1) of glucose (DMEM-LG) (Sigma, D6046) supplemented with the antibiotics, 10% fetal bovine serum and 10 mm Hepes-NaOH FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS W Omata et al (pH 7.4) Unless otherwise mentioned, cultures were fed every 2–3 days during growth and every days after confluence 3T3-L1 fibroblasts (preadipocytes) were differentiated into adipocytes essentially as described by Student et al [37] Briefly, days after confluence, the medium was removed and replaced with fresh DMEM-HG containing 0.5 mm 3-isobutyl-1-methylxanhine, lm dexamethasone, and 1.7 mm insulin Forty-eight hours later, the medium was withdrawn and replaced with fresh DMEM-HG containing 1.7 mm insulin After 48 h, insulin was withdrawn from the culture medium and cells were maintained in DMEM-HG until use Measurement of the glucose concentration in the incubation media of 3T3-L1 fibroblasts, a simple assay system for glucose consumption For the assay for glucose consumption, 3T3-L1 cells were transferred into 12-well plates, each containing mL of DMEM-HG, and incubated for 5–6 days until they became a confluent state with exchanging DMEM-HG every 2–3 days The cells were then preincubated for 2–3 days with mL of DMEM-LG with exchanging the medium every day The confluent cells thus obtained were treated for the appropriate number of hours with mL of DMEM-LG in the presence or absence of EtOH (usually 0.2%; vehicle), DIF-1 (5–20 lm), DIF analogs (20 lm), or some reagents Aliquots of the incubation media were then assayed for glucose concentration using a blood glucose test meter and sensor chips for it (Sanwa Chemical Institute, Nagoya, Japan), and the approximate rate of glucose consumption was calculated It should be noted that the glucose consumption by 3T3-L1 fibroblasts was assessed in DMEM-LG because DMEM-LG rather than DMEM-HG was suitable for the precise measurement of the glucose concentration by the use of a blood glucose test meter Therefore, to standardize the experimental conditions, we used the DMEM-LG culture system in other experiments (i.e confluent 3T3-L1 cells were preincubated for 2–3 days in DMEM-LG and used to assess the actions of DIF-1 and other reagents in DMEM-LG unless otherwise mentioned) RGM-1 cells were also transferred into 12-well plates, each containing mL of DMEM-LG, and incubated for several days until they reached a confluent state with exchanging DMEM-LG every 2–3 days The confluent cells were used for the assay for glucose consumption as described above It should be noted here that the simple assessment for the medium glucose concentration (Fig 3A,B) can provide only approximate rates of glucose consumption because the measurement of the glucose concentration by the test meter and the sensor chips is bound to have some degree of error (data not shown) and there is a lag for the glucose uptake promoted by DIF-1 to reach a maximum rate (Fig 3C) DIF-1 promotes glucose uptake in mammalian cells Thus, to obtain reliable and comparable data for the rates of glucose consumption, we usually incubated the cells with the drugs for more than h Assay for cell number Confluent 3T3-L1 or RGM-1 cells in 12-well plates were treated for the appropriate number of hours with mL of DMEM-LG in the presence or absence of EtOH (vehicle) or DIF-1 After the glucose concentration was measured, the incubation media were discarded, and the cells were incubated with mL of a fresh DMEM-LG containing 5% (v ⁄ v) of Alamar blue (a cell number indicator; Wako Pure Chemical Industries, Osaka, Japan) until the color changed The relative cell number was assessed as described previously [12,19] Assay for glucose uptake Confluent 3T3-L1 fibroblasts in a 12-well plate were incubated for h in DMEM-LG with or without reagents Cells were then washed twice with Buffer A [Krebs-Ringer Hepes (25 mm), pH 7.4 containing 0.4% BSA and mm sodium pyruvate] and incubated for 30 in the same buffer with the reagents Then, 2-[1,2-3H]deoxy-d-glucose (PerkinElmer, Fremont, CA, USA) (0.8 lCiỈwell)1) was added to the cells to the final concentration of 0.1 mm and incubated for 20 at 37 °C The nonspecific uptake was measured in the presence of lm cytochalasin B At the end of incubation, cells were washed twice with ice-cold Buffer A and lysed in 0.4 mL of 0.4% SDS The radioactivity in the lysate was counted with a scintillation counter The net values for glucose uptake stimulated with the reagents were obtained by subtracting the nonspecific uptake in the presence of cytochalasin B As shown in Fig 9A, 3T3-L1 adipocytes in a 12-well plate were incubated for h in serum-free DMEM-LG with or without the reagents (cells were incubated under serumfree conditions to establish a basal condition for the stimulation with insulin) Cells were then washed twice with Buffer A and incubated for 30 in the same buffer containing the reagents They were assayed for glucose uptake as described above, except for the incubation time (10 min) with 2-[1,2-3H]deoxy-d-glucose Subcellular membrane fractionation Confluent 3T3-L1 fibroblasts in 10-cm dishes were stimulated for h in 10 mL of DMEM-LG with 0.2% EtOH (vehicle) or 20 lm DIF-1 Differentiated 3T3-L1 adipocytes in 10-cm dishes were stimulated for h in serum-free DMEM-LG with 0.2% EtOH or 10 lm DIF-1 and then for 30 in Buffer A with the same reagents, or 3T3-L1 adipocytes were incubated for h in DMEM-LG (serum-free) FEBS Journal 274 (2007) 3392–3404 ª 2007 The Authors Journal compilation ª 2007 FEBS 3401 DIF-1 promotes glucose uptake in mammalian cells W Omata et al and then stimulated for 30 in Buffer A with 100 nm insulin All cells were rinsed three times with an ice-cold STE buffer (250 mm sucrose, 10 mm Tris ⁄ CL, pH 7.4 and mm EDTA), scraped in an STE buffer, and homogenized using a Dounce glass homogenizer (Kimble ⁄ Kontes Glass, Deerfield, IL, USA) Subcellular fractionation was carried out by differential and sucrose density gradient centrifugation as described previously [36] to obtain the PM and LDM fractions Western blotting For western blot analysis of the total cell lysates, cells in 12-well plates were incubated for the appropriate number of hours with mL of DMEM-LG in the presence of 0.2% EtOH (vehicle) or reagents After the glucose concentration was measured, the incubation media were aspirated, and the cells were lysed with an SDS-sample buffer Western blotting was performed as described previously [18] The subcellular membrane fractions obtained as described above were also analyzed by western blotting Imaging analysis of [Ca2+]c 3T3-L1 cells were grown on a glass coverslip dipped in DMEM-HG and then DMEM-LG for several days and loaded with lm Fura-2 ⁄ AM (acetoxymethyl ester) (Dojindo, Tokyo, Japan) in Hank’s balanced salt solution (Nissui, Tokyo, Japan) for 30 at room temperature The Fura2-loaded cells were set up in an assay chamber filled with Hank’s balanced salt solution, and Fura-2 fluorometry was carried out using an inverted microscope (IX-71, Olympus, Tokyo, Japan) equipped with a · 40 objective (Olympus) and a cooled CCD camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan) as described previously [38] A short path filter of 330–495 nm was used to reduce background fluorescence in the light path between a diachronic mirror of 505 nm and an emission filter of 535 ⁄ 45 nm band path The fluorescence ratio of the emissions by 340 nm excitation and 380 nm excitation (designated R340 ⁄ 380) was recorded and analyzed with the Aqua Cosmos imaging software (Hamamatsu Photonics) It should be noted here that the Fura-2 signals were not calibrated in terms of the absolute values of [Ca2+]c because the accuracy of such estimates is debatable and calibration was not necessary for the analysis in this study Assay for enzymatic activity Confluent 3T3-L1 cells in 10-cm dishes were treated for h with 10 mL of DMEM-LG containing 0.2% EtOH or 20 lm DIF-1; the incubation media were aspirated, and the cells were washed with 10 mL (per dish) of ice-cold phosphate buffered saline The cells were harvested with 3402 0.5 mL of a Hepes buffer (50 mm Hepes-NaOH, pH 7.5) (per dish) using a cell scraper, transferred into an Eppendorf tube on ice, and disrupted by sonication The soluble fraction was obtained by centrifugation at 10 000 g (MX-150, rotor type TMP-11, TOMY, Tokyo) for 20 at °C and used for the enzyme assay; Hexokinase (EC 2.7.1.1), phosphofructokinase (fructose phosphate kinase; EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), and glucose phosphate dehydrogenase (EC 1.1.1.49) were assayed as described previously [39], except that the total volume of the reaction mixture was mL in our experiment In addition, to examine the direct effect of DIF-1 on the enzyme activity, the control cell lysates were assayed for the enzyme assay in the presence or absence of 20 lm DIF-1 Statistical analysis 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DIF -1 promotes glucose uptake in mammalian cells 200 400 600 800 200 400 600 n.s * * 1. 6 * 1. 4 1. 2 0.8 T HPH THPH n.s 1. 8 DIF -3 1. 3 1. 2 1. 1 0.9 Tg DIF-3 DIF -1 1.5 1. 4 1. 3 1. 2 1. 1 0.9 B 1. 7 1. 6... DIF -1 increases [Ca2+]c in mammalian cells [9? ?11 ,20] and that DIF -1 is a direct inhibitor of PDE1 [19 ] In addition, increases in [Ca2+]c have been shown to stimulate glucose uptake via GLUT1 in. .. [Ca2+]i-increasing agents and PDE1 inhibitor on glucose consumption in 3T3-L1 fibroblasts It has been shown that DIF -1 increases [Ca2+]c in mammalian cells [9? ?11 ,20] and that DIF -1 is a specific inhibitor