A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as β-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser³⁰⁷ phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.     

Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.     

Inhibition of lipid synthesis and glucose-6-phosphate dehydrogenase in rat skin by dehydroepiandrosterone

Lipid synthesis from acetate-1-(14)C by rat skin was inhibited 44-56% by 2.5 x 10(-4) m dehydroepiandrosterone (DHA) in vitro with or without addition of glucose in the incubation medium. This inhibition affected all the lipid fractions examined (hydrocarbons, sterols, sterol esters, tri-, di- and monoglycerides, fatty acids, and polar lipids) and could be reversed by NADPH. DHA also inhibited lipid synthesis from glucose-U-(14)C and the formation of (14)CO(2) from glucose-1-(14)C, indicating interference with pentose cycle activity. Experiments with the 105,000 g supernatant fluid of rat skin homogenates demonstrated considerable activities of malic enzyme (ME) (12.6 nmoles of NADPH generated per min per mg of protein), of glucose-6-phosphate dehydrogenase (G6PD), and of 6-phosphogluconate dehydrogenase (6PGD) (17.5 nmoles of NADPH generated per min per mg of protein). G6PD was inhibited 98% by 2.5 x 10(-4) m dehydroepiandrosterone, while 6PGD and ME were not affected. It can be estimated from these data that the pentose cycle may contribute 41-57% of the NADPH needed for lipid synthesis in rat skin; the remainder of the necessary NADPH is presumably supplied by malic enzyme.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate

Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.     

Inactivation of bakers' yeast glucose-6-phosphate dehydrogenase by aluminum

Preincubation of yeast glucose-6-phosphate dehydrogenase (G6PD) with Al(III) produced an inactive enzyme containing 1 mol of Al(III)/mol of enzyme subunit. None of the enzyme-bound Al(III) was dissociated by dialysis against 10 mM Tris-HCl, pH 7.0, containing 0.2 mM EDTA at 4 degrees C for 24 h. Citrate, NADP+, EDTA, or NaF protected the enzyme against the Al(III) inactivation. The Al-(III)-inactivated enzyme, however, was completely reactivated only by citrate and NaF. The dissociation constant for the enzyme-aluminum complex was calculated to be 4 x 10(-6)M with NaF, a known reversible chelator for aluminum. Modification of histidine and lysine residues of the enzyme with diethyl pyrocarbonate and acetylsalicylic acid, respectively, inactivated the enzyme. However, the modified enzyme still bound 1 mol of Al(III)/mol of enzyme subunit. Circular dichroism studies showed that the binding of Al(III) to the enzyme induced a decrease in alpha-helix and beta-sheet and an increase in random coil. Therefore, it is suggested that inactivation of G6PD by Al(III) is due to the conformational change induced by Al(III) binding.