Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells

Abstract

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Keywords: AMPK; DIF-1; Dictyostelium discoideum; MDH2; diabetes; obesity.

Figure 1

(A) Chemical structures of DIF-1, DIF-3, and THPH (a nonbioactive derivative of DIF-1). DIF-1, 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one; DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one; THPH, 1-(2,4,6-trihydroxyphenyl)hexan-1-one. (B) Schematic diagram of the coupling reaction between Affi-Gel 10 resin and DIF-1-NH₂ [6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one hydrochloride] to make DIF beads [18].

Figure 2

(A,B) Effects of DIF-1, DIF-3, and THPH on the growth of HeLa and 3T3-L1 cells. Cells were incubated for 3 days in the presence of 0.1% dimethylsulfoxide (DMSO) or ethanol (EtOH) (vehicles) or 20 μM of DIF-1, DIF-3, or THPH, and the relative cell number was determined. The data are mean ± SD of five (A) or four (B) independent experiments. ** p < 0.01 versus control (by t-test). Representative photos of the cells after incubation with the indicated compounds are also shown. Bars: 100 μm. (C) Effects of DIF-1, DIF-3, and THPH on glucose uptake in 3T3-L1 cells. Confluent 3T3-L1 cells were incubated for 16–20 h in the presence of 0.1% DMSO or EtOH (vehicles) or 20 μM of DIF-1, DIF-3, or THPH, and the rate of glucose consumption was assessed. The data are mean ± SD of three independent experiments. ** p < 0.01 versus control (by t-test). Representative photos of the cells after 17 h incubation with the indicated compounds are also shown. All the cells look healthy. Bar: 100 μm.

Figure 3

(A) Flow diagram for the identification of DIF-binding proteins (DBPs) in HeLa and 3T3-L1 cells. Cells were lysed in 0.05% Tween 20 in Tris-buffered saline (TBS-T) and incubated with control beads or DIF beads in the presence of 0.1 mM THPH to reduce nonspecific protein binding to the beads. The beads were washed several times with TBS-T containing THPH, and the bound proteins were eluted with an SDS-sample buffer. The affinity-purified proteins were separated by SDS-PAGE and stained with silver staining. The proteins that specifically bound to the DIF beads were identified by using LC–MS/MS. (B,C) Photos of the silver-stained SDS-PAGE gels. Total cell extracts (1/200 input), the proteins bound to control and DIF beads, and molecular weight standards (MW Std) were subjected to SDS-PAGE, and the resulting gel was stained. The protein bands present in the DIF beads lane but not in the control beads lane were collected, and one of the bands was identified as MDH2.

Figure 4

(A–D) Comparison of the effects of DIF-1 and LW6 (an MDH2 inhibitor) on the growth of HeLa and 3T3-L1 cells. Cells were incubated for 3 days in the presence of 0.2% DMSO (vehicle) or the indicated concentrations of DIF-1 or LW6, and then the relative cell number was determined. The data are the mean ± SD of four (A) or three (B–D) independent experiments. * p < 0.05, ** p < 0.01 versus control (by t-test). Representative photos of the cells after incubation with the indicated concentrations of LW6 are also shown. Bars: 100 μm. (E,F) Comparison of the effects of DIF-1 and LW6 on glucose uptake in 3T3-L1 cells. Confluent 3T3-L1 cells were incubated for 16–20 h in the presence of 0.2% DMSO (vehicle) or the indicated concentrations of DIF-1 or LW6, and the rate of glucose consumption was assessed. The data are the mean ± SD of four (E) or three (F) independent experiments. ** p < 0.01 versus control (by t-test). Representative photos of the cells after 17 h incubation with the indicated concentrations of LW6 are also shown. All the cells look healthy. Bar: 100 μm.

Figure 5

(A) Combinatorial effects of an MDH2 inhibitor (LW6), a mitochondrial uncoupler (dinitrophenol; DNP), and a membrane-permeable cAMP analog (8-bromo-cAMP; Br-cAMP) on glucose uptake in 3T3-L1 cells compared to that of DIF-1. Cells were incubated for 16–20 h in the presence of 0.2% DMSO (vehicle) and 10 μM LW6, 0.1 mM DNP, and/or 0.3 mM Br-cAMP, or 20 μM DIF-1, and the rate of glucose consumption was assessed. The data are the mean ± SD of six independent experiments. ** p < 0.01 versus control (by t-test); ^## p < 0.01 (by ANOVA). (B) Proposed scheme for the action of DIF-1 in 3T3-L1 cells. DIF-1 promotes glucose uptake by uncoupling mitochondria [14,15], increasing intracellular cAMP levels [16], and inhibiting MDH2 (this study). Uncoupling mitochondria and inhibiting MDH2 would decrease cellular ATP levels and activate AMPK, which would promote glucose uptake [15]. MDH2 inhibitor LW6 (with the indicated chemical structure), mitochondrial uncoupler DNP, and membrane-permeable cAMP analog Br-cAMP can mimic the action of DIF-1. The pathways suggested for the first time in this study are shown in green, except that the effects of DIF-1 and LW6 on cellular ATP levels have been shown in other cells [23,25]. (C) Effects of LW6 on AMPK activity in 3T3-L1 cells. Cells were incubated for the indicated time periods in the presence of 10 μM LW6, 20 μM DIF-1, or 0.2 mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), and cell lysates were assessed by Western blotting for AMPK, phospho-AMPK (p-AMPK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an internal control protein. (D) Effects of LW6, DNP, and/or Br-cAMP on AMPK activity in 3T3-L1 cells. The cells were incubated for 3 h in the presence of 0.2% DMSO (vehicle), 0.2 mM AICAR, 20 μM DIF-1, 10 μM LW6, 0.1 mM DNP, and/or 0.3 mM Br-cAMP. Cell lysates were assessed by Western blotting for AMPK, p-AMPK, and GAPDH. The graph shows the ratio of p-AMPK/AMPK of each sample to that of the DMSO control; the means ± SD of four independent experiments are presented. * p < 0.01 versus DMSO control (by t-test).