Defective translocation of PKCepsilon in EtOH-induced inhibition of Mg2+ accumulation in rat hepatocytes

Abstract

Background: Rats chronically fed ethanol for 3 weeks presented a marked decreased in total hepatic Mg(2+) content and required approximately 12 days to restore Mg(2+) homeostasis upon ethanol withdrawal. This study was aimed at investigating the mechanisms responsible for the EtOH-induced delay.

Methods: Hepatocytes from rats fed ethanol for 3 weeks (Lieber-De Carli diet-chronic model), rats re-fed a control diet for varying periods of time following ethanol withdrawal, and age-matched control rats fed a liquid or a pellet diet were used. As acute models, hepatocytes from control animals or HepG2 cells were exposed to varying doses of ethanol in vitro for 8 minutes.

Results: Hepatocytes from ethanol-fed rats presented a marked inhibition of Mg(2+) accumulation and a defective translocation of PKCepsilon to the cell membrane. Upon ethanol withdrawal, 12 days were necessary for PKCepsilon translocation and Mg(2+) accumulation to return to normal levels. Exposure of control hepatocytes or HepG2 cells to a dose of ethanol as low as 0.01% for 8 minutes was already sufficient to inhibit Mg(2+) accumulation and PKCepsilon translocation for more than 60 minutes. Also in this model, recovery of Mg(2+) accumulation was associated with restoration of PKCepsilon translocation. The use of specific antisense in HepG2 cells confirmed the involvement of PKCepsilon in modulating Mg(2+) accumulation.

Conclusions: Translocation of PKCepsilon isoform to the hepatocyte membrane is essential for Mg(2+) accumulation to occur. Both acute and chronic ethanol administrations inhibit Mg(2+) accumulation by specifically altering PKCepsilon translocation to the cell membrane.

Figure 1. Mg²⁺ accumulation in liver cells isolated from rats fed with EtOH in the diet for 3 weeks

Hepatocytes from rats fed EtOH or liquid control diet were stimulated for Mg²⁺ accumulation with VP, OAG or PDBU. Data are means±S.E. of 4 different preparations each tested in duplicate for each experimental condition. *Statistically significant vs. corresponding values in control samples.

Figure 2. Recovery of Mg²⁺ accumulation in liver cells isolated from EtOH-fed rats

Hepatocytes were isolated from EtOH-fed rats prior to or after being switched to control diet for varying periods of time. Net Mg²⁺ accumulation at time 6 min after agonist addition is reported. Data are means±S.E. of 6 different preparations each tested in duplicate for all experimental conditions. *Statistically significant vs. corresponding values at 2 days, 4 days and 7 days.

Figure 3. PKCε and PKCδ distribution in liver cells isolated from rats fed with EtOH in the diet for 3 weeks

Hepatocytes isolated from EtOH-fed rats prior to or after being switched to control diet for varying periods of time underwent stimulation for Mg²⁺ accumulation. PKCε and PKCδ distributions were assessed by Western Blot analysis as described in detail under Materials and Methods. Fig. 3A: a typical Western Blot analysis is reported. T=Total homogenate; TE=Total Homogenate, EtOH sample; M =membrane fraction; ME=membrane fraction, ETOH sample. Densitometry of PKC distribution is reported in Fig. 3B. Fig. 3C reports recovery of PKCε and PKCδ distribution in hepatocytes isolated from EtOH-fed rats following change in diet. Data are means±S.E. of 5 different preparations for all the experimental conditions reported in Figs. 3B and 3C, each tested in duplicate. Fig. 3B: *Statistically significant vs. corresponding values in EtOH-untreated samples; Fig. 3C: *Statistically significant vs. corresponding values at day 0, 2 days, 4 days and 7 days.

Figure 3. PKCε and PKCδ distribution in liver cells isolated from rats fed with EtOH in the diet for 3 weeks

Hepatocytes isolated from EtOH-fed rats prior to or after being switched to control diet for varying periods of time underwent stimulation for Mg²⁺ accumulation. PKCε and PKCδ distributions were assessed by Western Blot analysis as described in detail under Materials and Methods. Fig. 3A: a typical Western Blot analysis is reported. T=Total homogenate; TE=Total Homogenate, EtOH sample; M =membrane fraction; ME=membrane fraction, ETOH sample. Densitometry of PKC distribution is reported in Fig. 3B. Fig. 3C reports recovery of PKCε and PKCδ distribution in hepatocytes isolated from EtOH-fed rats following change in diet. Data are means±S.E. of 5 different preparations for all the experimental conditions reported in Figs. 3B and 3C, each tested in duplicate. Fig. 3B: *Statistically significant vs. corresponding values in EtOH-untreated samples; Fig. 3C: *Statistically significant vs. corresponding values at day 0, 2 days, 4 days and 7 days.

Fig. 4. Mg²⁺ accumulation in liver cells isolated from control rats and treated in vitro with 0.01% EtOH for 8 min prior to the stimulation for Mg²⁺ accumulation

Hepatocytes from control rats were pre-treated with 0.01% EtOH for 8 min. The cells were washed and transferred to an incubation medium containing 10µM or 1.2mM Mg²⁺, and stimulated for Mg²⁺ accumulation (Fig. 4A). Alternatively, hepatocytes were exposed to EtOH in the absence or in the presence of 50µM 4-MP prior to be washed and transferred to a new incubation medium containing 1.2mM Mg²⁺ and stimulated for Mg²⁺ accumulation (Fig. 4B). Net Mg²⁺ accumulation at time 6 min after agonist addition for all the experimental conditions is reported in Figs. 4A and 4B. Data are means±S.E. of 6 different preparations for all the experimental conditions reported in Figs. 4A and 4B, each tested in duplicate. *Statistically significant vs. corresponding values in control samples (Ctrl).

Figure 5. Recovery of Mg²⁺ accumulation in liver cells isolated from control rats upon treatment in vitro with 0.01% EtOH

Hepatocytes isolated from control rats were pre-treated with 0.01% EtOH for 8 min prior to undergoing stimulation for Mg²⁺ accumulation at various time points upon EtOH withdrawal. Net Mg²⁺ accumulation at time 6 min after agonist additions at various time points upon EtOH removal is reported. Data are means±S.E. of 6 different preparations each tested in duplicate for all experimental conditions. *Statistically significant vs. corresponding values at time 0 min, 15 min, 30 min, 45 min, and 60 min; #Statistically significant vs. corresponding values at time 0 min, 15 min, and 30 min.

Figure 6. PKCε and PKCδ distribution in liver cells isolated from control rats and treated in vitro with 0.01% EtOH for 8 min

Hepatocytes from control rats were pre-treated with 0.01% or 1% EtOH for 8 min prior to undergoing stimulation with OAG for Mg²⁺ accumulation at various time points upon EtOH removal. PKCε distribution was assessed by Western Blot analysis (Figs. 6A and 6B). T=Total homogenate; TE=Total Homogenate, EtOH sample; M=membrane fraction; ME=membrane fraction, ETOH sample. Recovery of PKCε and PKCδ distribution in hepatocytes isolated from control rats upon pre-treatment with 0.01% EtOH and stimulation with OAG is reported in Fig. 6C. Data are means±S.E. of 4 different preparations for all the experimental conditions reported in Figs. 6B and 6C. Fig. 6B: *Statistically significant vs. corresponding values in EtOH-untreated samples. Fig. 6C: *Statistically significant vs. corresponding values at time 0 min(EtOH), 15 min, 30 min, 45 min, and 60 min after EtOH-removal; # Statistically significant vs. corresponding values at time 0 min(EtOH).

Figure 7. Mg²⁺ accumulation in HepG2 cells treated with EtOH, antisense and cycloheximide prior to stimulation for Mg²⁺ accumulation

HepG2 cells were pre-treated with 0.01% EtOH for 8 min. The cells were washed and transferred to a new incubation medium containing 1.2mM Mg²⁺, and stimulated for Mg²⁺ accumulation. Net Mg²⁺ accumulation at time 6 min after agonist addition is reported in Fig. 7A. Attenuation of PKCε or PKCδ expression in HepG2 cells treated with the corresponding antisense for 72 hours is reported in Fig. 7B. Net Mg²⁺ accumulation at time 6 min following OAG or PMA addition to HepG2 cells treated for 72 hours with PKCε or PKCδ antisense is reported in Fig. 7C. HepG2 cells were treated for 72 hours with 10µM or 50µM cycloheximide prior to undergoing OAG stimulation to induced Mg²⁺ accumulation. Changes in the basal expression of PKCε upon cycloheximide treatment are reported in Fig. 7D. Changes in cellular basal and stimulated Mg²⁺ accumulation by OAG are reported in Fig. 7E. Data are means±S.E. of 4 different preparations each tested in duplicate for all the experimental conditions reported in Figs. 7A, and 7C. A typical experiment out of three is reported in Figs. 7B and 7D. Data are means±S.E. of 3 different preparations each tested in duplicate for all the experimental conditions reported in Fig. 7E. *Statistically significant vs. corresponding values in control, antisense-treated and cycloheximide-treated samples.

Figure 7. Mg²⁺ accumulation in HepG2 cells treated with EtOH, antisense and cycloheximide prior to stimulation for Mg²⁺ accumulation

HepG2 cells were pre-treated with 0.01% EtOH for 8 min. The cells were washed and transferred to a new incubation medium containing 1.2mM Mg²⁺, and stimulated for Mg²⁺ accumulation. Net Mg²⁺ accumulation at time 6 min after agonist addition is reported in Fig. 7A. Attenuation of PKCε or PKCδ expression in HepG2 cells treated with the corresponding antisense for 72 hours is reported in Fig. 7B. Net Mg²⁺ accumulation at time 6 min following OAG or PMA addition to HepG2 cells treated for 72 hours with PKCε or PKCδ antisense is reported in Fig. 7C. HepG2 cells were treated for 72 hours with 10µM or 50µM cycloheximide prior to undergoing OAG stimulation to induced Mg²⁺ accumulation. Changes in the basal expression of PKCε upon cycloheximide treatment are reported in Fig. 7D. Changes in cellular basal and stimulated Mg²⁺ accumulation by OAG are reported in Fig. 7E. Data are means±S.E. of 4 different preparations each tested in duplicate for all the experimental conditions reported in Figs. 7A, and 7C. A typical experiment out of three is reported in Figs. 7B and 7D. Data are means±S.E. of 3 different preparations each tested in duplicate for all the experimental conditions reported in Fig. 7E. *Statistically significant vs. corresponding values in control, antisense-treated and cycloheximide-treated samples.

Figure 8. Effect of DTT or NAC pre-treatment on Mg²⁺ accumulation

HepG2 cells were pre-treated with 100µM DTT or 100µM NAC for 15 min at 37°C prior to undergoing treatment with 0.01% EtOH for 8 min. Following EtOH exposure the cells were washed and transferred in a medium containing 1.2mM Mg²⁺ and stimulated for Mg²⁺ accumulation by addition of 20nM PDBU. PKCε translocation upon PDBU addition is reported in Fig. 8A (one typical experiment out of three is reported). Net Mg²⁺ accumulation at time=6 min after PDBU addition is reported in Fig. 8B (data are means±S.E. of 4 different preparations for all experimental conditions, each tested in duplicate). Modification of protein carbonyls is reported in Fig. 8C (one typical experiment out of three is reported). Similar results were obtained for NAC-pretreated cells. Formation of HNE/protein adducts is reported in Fig. 8D (one typical experiment out of three is reported). *Statistically significant form Control, DTT and NAC-pretreated samples.