Pregnancy represses induction of efflux transporters in livers of type I diabetic mice

Abstract

Purpose: To determine whether down-regulation of transcription factor signaling during pregnancy disrupts the induction of efflux transporters in type I diabetic mice.

Methods: Type I diabetes was induced in female C57BL/6 mice with multiple low dose intraperitoneal injections of streptozotocin (STZ) at least 2 weeks prior to mating with normoglycemic male mice. On gestation day 14, livers were collected from vehicle- and STZ-treated non-pregnant and pregnant mice for quantification of efflux transporter and transcription factor signaling.

Results: STZ treatment up-regulated expression of Mrp1-5, Mdr1, Abcg5, Abcg8, Bcrp, and Bsep mRNA and/or protein in the livers of non-pregnant mice. Interestingly, little to no change in transporter expression was observed in STZ-treated pregnant mice compared to vehicle- and STZ-treated non-pregnant mice.

Conclusions: This study demonstrates the opposing regulation of hepatobiliary efflux transporters in response to diabetes and pregnancy and points to PPARγ, Nrf2, and FXR as candidate pathways underlying the differential expression of transporters.

Figure 1. Hepatic mRNA expression of efflux transporters in diabetic pregnancy

Messenger RNA expression of canalicular (A) and basolateral (B) efflux transporters was quantified using total hepatic RNA from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Black bars represent vehicle-treated mice and light gray bars represent STZ-treated mice. Data were normalized to mRNA levels of ribosomal protein l13A (Rpl13A) and presented as mean ± SE (n=4–11). Asterisks (*) represent statistically significant differences (p < 0.05) compared to vehicle-treated, non-pregnant mice. Daggers (^†) represent statistically significant differences (p < 0.05) compared to vehicle-treated, pregnant mice. Double daggers (^‡) represent statistically significant differences (p < 0.05) compared to STZ-treated, non-pregnant mice.

Figure 2. Hepatic protein expression of efflux transporters in diabetic pregnancy

Liver expression of canalicular (A) and basolateral (B) efflux proteins was quantified by western blot (50 g protein homogenates/lane) from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. β-actin was used as a loading control. The western blot data are presented as individual blots and mean relative protein expression. Black bars represent vehicle-treated mice and light gray bars represent STZ-treated mice. Data are normalized to vehicle-treated, non-pregnant controls and presented as mean ± SE (n=4). Asterisks (*) represent statistically significant differences (p < 0.05) compared to vehicle-treated, non-pregnant mice. Double daggers (^‡) represent statistically significant differences (p < 0.05) compared to STZ-treated, non-pregnant mice.

Figure 3. Immunofluorescent staining of efflux transporters in diabetic pregnancy

Indirect immunofluorescence against canalicular (Mrp2 and Bsep) and sinusoidal (Mrp3, 4, 6) transporters (green) was conducted on liver cryosections (5 m) obtained on gestation day 14 from vehicle- and STZ-treated non-pregnant and pregnant mice. Representative regions are shown. Magnification, x320.

Figure 4. Liver histology in diabetic pregnancy

Livers were collected on gestation day 14 from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Samples were fixed in zinc formalin prior to routine processing and paraffin embedding. Sections (5 μm) of livers were stained with hematoxylin and eosin and examined by light microscopy for histopathological changes. Paraffin-embedded liver sections from non-pregnant mice, regardless of treatment, and vehicle-treated pregnant mice had normal histology. Minimal to mild microvesicular vacuolation was observed in STZ-treated, pregnant mice. Magnification, x400.

Figure 5. Hepatic PPAR transcription factor signaling in diabetic pregnancy

(A) mRNA expression of transcription factors, (B) nuclear binding, and (C) mRNA of target genes were quantified in livers from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Black bars represent vehicle-treated mice and light gray bars represent STZ-treated mice. Data were normalized to mRNA levels of ribosomal protein l13A (Rpl13A) and presented as mean ± SE (n=4–11). Asterisks (*) represent statistically significant differences (p < 0.05) compared to vehicle-treated, non-pregnant mice. Double daggers (^‡) represent statistically significant differences (p < 0.05) compared to STZ-treated, non-pregnant mice.

Figure 6. Hepatic Nrf2 and FXR transcription factor signaling in diabetic pregnancy

(A) mRNA expression of Nrf2 and target gene Nqo1, (B) Nrf2 nuclear binding, and (C) mRNA of FXR and its target gene Shp were quantified in livers from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Black bars represent vehicle-treated mice and light gray bars represent STZ-treated mice. Data were normalized to mRNA levels of ribosomal protein l13A (Rpl13A) and presented as mean ± SE (n=4–11). Asterisks (*) represent statistically significant differences (p < 0.05) compared to vehicle-treated, non-pregnant mice. Daggers (^†) represent statistically significant differences (p < 0.05) compared to vehicle-treated, pregnant mice. Double daggers (^‡) represent statistically significant differences (p < 0.05) compared to STZ-treated, non-pregnant mice.

Figure 7. Proposed hepatic transcription factor and transporter regulation in diabetic pregnancy

STZ treatment induced hyperglycemia and activated PPAR, Nrf2, and FXR signaling pathways by increasing transcription factor and/or target gene mRNA. Potential downstream efflux transporters were induced at the mRNA (M) and/or protein (P) levels. Pregnancy directly or indirectly repressed expression of transcription factor and transporter pathways in maternal livers.