Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Abstract

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

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

“…We then compared the effects of DIF-1 and LW6 on glucose uptake in confluent 3T3-L1 cells in vitro (Figure 4E,F). DIF-1 at 10-40 µM significantly promoted glucose uptake, with the strongest effect at 20 µM DIF-1 (Figure 4E), as described previously [16]. LW6 at 2-20 µM promoted glucose uptake, with the strongest effect at 10 µM LW6.…”

“…We thus compared the effects of DIF-1 and the MDH2 inhibitor LW6 [24][25][26][27][28] on the growth of HeLa cells and 3T3-L1 cells in vitro (Figure 4A-D). In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 µM dose-dependently suppressed cell growth (Figure 4A,C), as described previously [16]. LW6 at 20 µM suppressed the growth of these cells but not significantly at 2-10 µM (Figure 4B,D); 20 µM LW6 was rather toxic to HeLa cells, with the cell morphology appearing abnormal (Figure 4B).…”

“…We have previously shown that DIF-1 promotes glucose uptake, at least in part by increasing intracellular cAMP levels and uncoupling mitochondria in 3T3-L1 cells [15,16]. To elucidate the relationship between MDH2, mitochondrial uncoupling, and intracellular cAMP in the action of DIF-1, we examined the effects of LW6, dinitrophenol (DNP; a mitochondrial uncoupler), 8-bromo-cAMP (Br-cAMP; a membrane-permeable analog of cAMP), or their combinations on glucose uptake in confluent 3T3-L1 cells in vitro (Figure 5A).…”

“…DIF-1 promotes glucose uptake by inducing translocation of glucose transporter 1 (GLUT1) from intracellular vesicles to the plasma membrane, at least in part via the mitochondria-and states of mouse 3T3-L1 cells, which are used to study diabetes, obesity, and related disorders [14]. DIF-1 promotes glucose uptake by inducing translocation of glucose transporter 1 (GLUT1) from intracellular vesicles to the plasma membrane, at least in part via the mitochondria-and 5′-AMP-activated kinase (AMPK)-dependent and 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent pathways [15,16]. Oral administration of DIF-1 decreases blood glucose levels in streptozotocin-treated diabetic rats [17].…”

“…In this study, to elucidate the mechanisms underlying the glucose-uptake-promoting effect of DIF-1, we attempted to identify the pharmacological target molecules of DIF-1 in 3T3-L1 fibroblasts, using HeLa cells as a reference. We performed affinity chromatography As for potential pharmacological target molecules of DIF-1 and DIF-3 in mammalian cells, DIFs have been shown to directly inhibit PDE1 [19], a calmodulin-dependent phosphodiesterase that degrades cAMP and cyclic guanosine monophosphate (cGMP) [20][21][22], and might thereby exhibit anticancer activities [16,19,22]. A previous study identified mitochondrial malate dehydrogenase, known as malate dehydrogenase 2 (MDH2), as a DIF-1-binding protein (DBP) in human cervical cancer HeLa cells and further showed that DIF-1 inhibits MDH2 activity in vitro, but not that of MDH1 (cytoplasmic MDH), whereas DIF-3 affects neither MDH1 nor MDH2 activity [23].…”