Insulin Modulates the Na+/Mg2+ Exchanger SLC41A1 and Influences Mg2+ Efflux from Intracellular Stores in Transgenic HEK293 Cells.
BACKGROUND
Magnesium deficiency is a common complication of diabetes with an unclear molecular background.


OBJECTIVE
We investigated the effect of the insulin (INS)-signaling pathway (ISP) on the regulation of Mg(2+) efflux (Mg(2+)E) conducted by solute carrier family 41, member A1 (SLC41A1; activated by protein kinase A) in transgenic human embryonic kidney (HEK) 293 cells.


METHODS
HEK293 cells overexpressing SLC41A1 were loaded with the Mg(2+) fluorescent indicator mag-fura-2 and Mg(2+). Measurements of Mg(2+)E were conducted in Mg(2+)-free buffer by using fast-filter fluorescence spectrometry. We examined the effects of INS, inhibitors of ISP or p38 mitogen-activated protein kinase (p38 MAPK), an activator of adenylate cyclase (ADC), and their combinations on SLC41A1-attributed Mg(2+)E.


RESULTS
The application of 400 μU/mL INS inhibited SLC41A1-mediated Mg(2+)E by up to 50.6% compared with INS-untreated cells (P < 0.001). Moreover, INS evoked the early onset of Mg(2+) release from intracellular stores. The application of 0.1 μM wortmannin or 10 μM zardaverine (both ISP inhibitors) restored SLC41A1 Mg(2+)E capacity in the presence of INS to the same levels in INS-untreated cells. The simultaneous application of 10 μM forskolin, an ADC activator, and INS resulted in a reduction of Mg(2+)E of up to 59% compared with untreated cells (P < 0.001), which was comparable to that in cells treated with INS alone. Inhibition of p38 MAPK with 10 μM SB 202190 (SB) in the absence of INS resulted in a decrease (P < 0.001) of SLC41A1-dependent Mg(2+)E (by up to 49%) compared with Mg(2+)E measured in untreated cells. Simultaneous exposure of cells to SB and INS had a stronger inhibitory effect on SLC41A1 activity than INS alone (P < 0.05).


CONCLUSIONS
INS affects intracellular Mg(2+) concentration in transgenic HEK293 cells by regulating SLC41A1 activity (via ISP) and by influencing the compartmentalization and cellular distribution of Mg(2+). In addition, p38 MAPK activates SLC41A1 independently of INS action.
Institute of Veterinary Physiology, Freie Universität, Berlin, Germany; and.
Lucia
L
Mastrototaro
Institute of Veterinary Physiology, Freie Universität, Berlin, Germany; and.
Uwe
U
Tietjen
Institute of Veterinary Physiology, Freie Universität, Berlin, Germany; and.
Gerhard
G
Sponder
Institute for Prevention and Nutrition, Ismaning/Munich, Germany.
Jürgen
J
Vormann
Institute of Veterinary Physiology, Freie Universität, Berlin, Germany; and.
Jörg R
JR
Aschenbach
Institute of Veterinary Physiology, Freie Universität, Berlin, Germany; and martink@zedat.fu-berlin.de.
Martin
M
Kolisek
D000262:Adenylyl Cyclases / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D011724:Pyridazines / Q000494:pharmacology; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000730:Androstadienes; D027682:Cation Transport Proteins; D007328:Insulin; D011724:Pyridazines; C476304:SLC41A1 protein, human; D005576:Colforsin; D048051:p38 Mitogen-Activated Protein Kinases; D000262:Adenylyl Cyclases; D008274:Magnesium; C066436:zardaverine; D000077191:Wortmannin
Mg2+ efflux; Na+/Mg2+ exchanger; diabetes; insulin; magnesium; mitochondria; signaling