Enhanced glucose 6-phosphatase activity in liver of rats exposed to Mg(2+)-deficient diet

Abstract

Total hepatic Mg(2+) content decreases by >25% in animals maintained for 2 weeks on Mg(2+) deficient diet, and results in a >25% increase in glucose 6-phosphatase (G6Pase) activity in isolated liver microsomes in the absence of significant changed in enzyme expression. Incubation of Mg(2+)-deficient microsomes in the presence of 1mM external Mg(2+) returned G6Pase activity to levels measured in microsomes from animals on normal Mg(2+) diet. EDTA addition dynamically reversed the Mg(2+) effect. The effect of Mg(2+) or EDTA persisted in taurocholic acid permeabilized microsomes. An increase in G6Pase activity was also observed in liver microsomes from rats starved overnight, which presented a ~15% decrease in hepatic Mg(2+) content. In this model, G6Pase activity increased to a lesser extent than in Mg(2+)-deficient microsomes, but it could still be dynamically modulated by addition of Mg(2+) or EDTA. Our results indicate that (1) hepatic Mg(2+) content rapidly decreases following starvation or exposure to deficient diet, and (2) the loss of Mg(2+) stimulates G6P transport and hydrolysis as a possible compensatory mechanism to enhance intrahepatic glucose availability. The Mg(2+) effect appears to take place at the level of the substrate binding site of the G6Pase enzymatic complex or the surrounding phospholipid environment.

Figure 1. Time course and net G6P hydrolysis in the presence of varying G6P concentrations

Liver microsomes were isolated from rats maintained for 2 weeks on normal (NMM) or Mg²⁺ deficient diet (MDM) as reported under Materials and Methods. Net hydrolysis rate (Fig. 1A) of glucose 6-phosphatase is reported as nmol Pi/mg protein for both microsomal preparations. Net hydrolysis in the presence of 1mM, 5mM, and 10mM G6P at time 30 min is reported in Fig 1A. Net G6P hydrolysis in MDM in the presence of different protein concentrations in the assay is reported in Fig. 1B. Data are means±S.E. of 6 different preparations for each condition, each tested in duplicate. *Statistically significant vs. corresponding value in NMM.

Figure 2. Glucose 6-phosphatase expression in NM and MD microsomes

Glucose 6-phosphatase expression was assessed in NMM and MDM microsomes as reported under Materials and Methods. A typical Western blot analysis depicting 2 different preparations for each experimental condition is reported in Fig. 2A. Densitometry of 6 different preparations (means±SE), each tested in duplicate is reported in Fig. 2B. For each preparation, MDM expression was normalized using the corresponding NMM expression as 1 (or 100%).

Figure 3. G6P hydrolysis in the presence of extravesicular Mg²⁺ or Mg²⁺ and EDTA in MDM (Fig. 3A) and NMM (Fig. 3B)

NMM and MDM were incubated at the concentration of 0.3 mg protein/ml in the presence of 1mM Mg²⁺. After 15 min incubation, the samples were split into two, one of which received a concentration of EDTA sufficient to chelate all external Mg²⁺ [21], and the incubation continued up to t = 30 min in the absence and in the presence of EDTA. Data are means±S.E. of 6 different preparations. *Statistically significant vs. the corresponding sample in the absence of EDTA.

Figure 4. G6P hydrolysis in the presence of extravesicular Mg²⁺ or Mg²⁺ and EDTA in NMM (Fig. 4A) and MDM (Fig. 4B) permeabilized with taurocholic acid

NMM and MDM were pretreated in ice for 15 min with 1% taurocholic acid. Microsomes were then incubated at the concentration of 0.3 mg protein/ml in the presence of 1mM Mg²⁺ or 1mM Mg²⁺ plus a concentration of EDTA sufficient to chelate all external Mg²⁺ [21] as described in the legend to Fig. 3. The incubation was carried out up to t = 30 min as indicated before. Data are means±S.E. of 6 different preparations. *Statistically significant vs. the corresponding sample in the absence of EDTA.

Figure 5. Mg²⁺ release from NMM and MDM microsomes

NMM and MDM microsomes were passively loaded with 20mM MgCl₂ (Fig. 5A) or 2mM (Fig. 5B) for 40 min at room temperature at the final concentration of 15 mg protein ml as indicated under Materials and Methods. Microsomes were then diluted 50 fold in the incubation medium, and passive Mg²⁺ release from the microsomes was assessed by AAS in the presence or in the absence of 50μM 2-APB as a translocon inhibitor [23]. Data are means±S.E. of 4 experiments for each condition, each performed in duplicate. Fig. 5B: *Statistically significant vs. corresponding values in the absence of 2-APB.

Figure 6. PPi hydrolysis in NMM and MDM

Time course of PPi hydrolysis in the presence of 1mM PPi and 1mM Mg²⁺ added to the incubation system is reported for NMM and MDM. Data, expressed as μmol Pi/mg protein, are means±S.E. of 4 experiments for each condition, each performed in duplicate.

Figure 7. G6P hydrolysis in microsomes isolated from livers of overnight starved animals

Total liver microsomes were isolated from livers of rats starved overnight as described under Materials and Methods. Hydrolysis rate expressed as nmol Pi/mg protein in the presence of various G6P concentrations is reported in Fig. 7A. The effect of 1mM Mg²⁺ plus/minus 2mM EDTA addition to the extramicrosomal milieu on the microsome hydrolysis rate is reported in Fig. 7B. Data are means±S.E. of 4 experiments for each condition, each performed in duplicate. *Statistically significant vs. corresponding values in the absence of EDTA.