Leptin affects system A amino acid transport activity in the human placenta: evidence for STAT3 dependent mechanisms

Abstract

Background: Amino acids are important nutrients during fetal development, and the activity of placental amino acid transporters is crucial in the regulation of fetal growth. Leptin, an adipocyte- and placenta-derived hormone, has been proposed to act as a peripheral signal in reproduction in humans. Leptin is elevated during pregnancy and elevated further in pathologic pregnancies such as preeclampsia. However, the role of leptin in placental function has not been fully elucidated. We hypothesize that leptin plays a role in the regulation of placental amino acid transport by activation of the JAK-STAT pathway.

Methods: Placental amino acid transport, specifically system A transport was studied in placental villous fragments using the amino acid analog, methylaminoisobutyric acid (MeAIB). Specific inhibitors of the JAK-STAT signal transduction pathway were used to further elucidate their role in leptin-mediated effects on amino acid transport activity. Western blotting was performed to identify STAT3 phosphorylation as a measure of leptin receptor activation.

Results: Leptin significantly increased system A amino acid transporter activity by 22-42% after 1h of incubation. Leptin activated JAK-STAT signaling pathway as evidenced by STAT3 phosphorylation, and inhibition of STAT3 or JAK2 resulted in 36-45% reduction in system A amino acid transporter activity. Furthermore, blocking endogenously produced leptin also decreased system A transport by 45% comparable to STAT3 inhibition.

Conclusions: These data demonstrate that leptin stimulates system A by JAK-STAT dependent pathway in placental villous fragments. Our findings support the autocrine/paracrine role of leptin in regulating amino acid transport in the human placenta.

Figure 1

Leptin receptor activation increases STAT3 phosphorylation. A) Stimulation of STAT3 phosphorylation by leptin in placental villous fragments. Villous fragments were treated for 10 min with leptin (1000 ng/ml). Representative western Blot of pSTAT3, STAT3 and β-actin as respective loading control of three experiments on a 10% gel. B) Representative scatter plot showing increased pSTAT3/STAT3 ratio for leptin treated villous fragments as compared to control (n=6 placentas). Data are presented as fold change in pSTAT3/STAT3 ratio of leptin treated fragments compared with that of control (dashed line). The median fold change for each condition is denoted by a solid line.

Figure 2

Leptin effect on placental system A activity. A) Leptin and insulin significantly increase system A amino acid transport activity in placental villous fragments after a 1 h incubation (n=6 placentas). B) After 2 h of incubation leptin does not increase system A amino acid transport activity, but insulin increases and angiotensin II (ANG II) decreases system A activity. (n=9 placentas). Data are presented as fold change in system A activity compared with that of control (dashed line). The median fold change for each condition is denoted by a solid line.

Figure 3

Leptin induced increase of system A activity is dependent on STAT3 phosphorylation. A) Inhibition of JAK2 and STAT3 phosphorylation reduces system A amino acid transport by 36-45% (n=5 placentas). B) The effect of STAT3 inhibitors on system A activity is dose dependent (n=3 placentas). Data are presented as fold change in system A activity compared with that of control (dashed line). The median fold change for each condition is denoted by a solid line.

Figure 4

Western Blot analyses to detect phosphorylated STAT3 after incubation with leptin (500 ng/ml) or STAT3 inhibitor (0.5 μM). A) Representative western blot to demonstrate the relative levels of phosphorylated STAT3 (pSTAT3), STAT3, or β-actin in villous fragments incubated with control, leptin (500 ng/ml) or STAT3 phosphorylation inhibitor (0.5 μM) for 1 h and 2 h (n=5 placentas). B) Leptin treatment of villous fragments increased pSTAT3/STAT3 ratio after 1 h of incubation and STAT3 inhibitor treatment reduced pSTAT3/STAT3 ratio when compared to control at 1 h, (n=5 placentas). The median fold change for each condition is denoted by a solid line.

Figure 5

Effect of binding endogenous leptin on system A activity. Binding of endogenous leptin with an anti-leptin antibody (5 μg/ml) reduces system A amino acid transport within 1 h of incubation by 45%. A nonspecific antibody had no effect on system A activity (n=5 placentas).