Full-length adiponectin attenuates insulin signaling and inhibits insulin-stimulated amino Acid transport in human primary trophoblast cells

Abstract

Objective: Maternal adiponectin levels are reduced and placental nutrient transporters are upregulated in obesity and gestational diabetes mellitus; however, the effects of adiponectin on placental function are unknown. We hypothesized that adiponectin regulates placental amino acid transport.

Research design and methods: Human primary trophoblast cells were cultured and incubated with globular adiponectin (gAd) or full-length adiponectin (fAd) alone or in combination with insulin. System A and L amino acid transport and SNAT1, SNAT2, and SNAT4 isoform expression was measured. The activity of the AMP-activated protein kinase (AMPK), phosphatidylinositol 3 kinase-AKT, and peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling pathways was determined.

Results: In the absence of insulin, gAd stimulated AMPK Thr172 phosphorylation, SNAT2 protein expression, and system A activity. This effect appeared to be mediated by interleukin-6 release and signal transducer and activator of transcription 3 (STAT3) signaling because gAd failed to stimulate system A in cells in which STAT3 had been silenced using small interfering RNA. fAd alone had no effect on system A activity or SNAT expression. Insulin increased AKT and insulin receptor substrate 1 (IRS-1) phosphorylation, system A activity, and SNAT2 expression. When combined with insulin, gAd did not affect system A activity or SNAT expression. In contrast, fAd abolished insulin-stimulated AKT Thr308 and IRS-1 Tyr612 phosphorylation, system A activity, and SNAT2 expression. Furthermore, fAd increased PPARalpha expression and PPARalpha (Ser21) phosphorylation.

Conclusions: In contrast to the insulin-sensitizing actions of adiponectin in liver and muscle reported in the literature, fAd attenuates insulin signaling in primary human trophoblast cells. As a result, fAd inhibits insulin-stimulated amino acid transport, which may have important implications for placental nutrient transport and fetal growth in pregnancy complications associated with altered maternal adiponectin levels.

FIG. 1.

Sodium-dependent ¹⁴C-MeAIB (A and B) and BCH-inhibitable ³H-leucine (C and D) uptake after incubation of cultured trophoblast cells in control media, gAd (A and C), or fAd (B and D) for 24 h. Data are mean ± SEM for cells isolated from six different placentas. gAd significantly (P < 0.05) stimulated MeAIB uptake in a dose-dependent manner (RMANOVA with post hoc tests, *P < 0.05, **P < 0.01).

FIG. 2.

Sodium-dependent ¹⁴C-MeAIB uptake after incubation of cultured trophoblast cells in control media, insulin (1 nmol/l), gAd (5 μg/ml) (A), or fAd (5 μg/ml) (B) for 24 h. Subsets of cells were pretreated with insulin (1 nmol/l) for 4 h and then exposed to 5 μg/ml gAd (IgAd) or fAd (IfAd) for an additional 20 h. Data are mean ± SEM for cells isolated from six different placentas. Insulin alone, gAd alone, and insulin + gAd (P < 0.01) significantly stimulated MeAIB uptake; however, insulin + fAd significantly (P < 0.001) reduced MeAIB uptake compared with insulin alone (RMANOVA with post hoc tests, *P < 0.05, **P < 0.01). C: Summary data of real-time PCR of system A amino acid transporter isoforms SNAT1, SNAT2, and SNAT4 after incubation of cultured trophoblast cells as indicated. Data are mean ± SEM for six placentas. Insulin significantly increased SNAT gene expression. In the presence of fAd, the effect of insulin on the mRNA expression of SNAT2 and SNAT4 was significantly reduced compared with both fAd and insulin treatment alone (RMANOVA, P < 0.01; Tukey-Kramer multiple comparisons post tests, *P < 0.05, ***P < 0.01). D: Representative Western blot of SNAT1, SNAT2, and SNAT4 expression after incubation of cultured trophoblast cells as indicated. E: Summary of SNAT2 protein expression levels. n = 6 for each treatment, RMANOVA, P < 0.01; Tukey-Kramer multiple comparisons post tests, *P < 0.05, **P < 0.01.

FIG. 3.

Summary of phospho-AMPK and AMPK protein expression after incubation of cultured trophoblast cells with control media, insulin (1 nmol/l), gAd (5 μg/ml), or fAd (5 μg/ml) for 24 h. Subsets of cells were pretreated with insulin (1 nmol/l) for 4 h and then exposed to 5 μg/ml gAd (IgAd) or fAd (IfAd) for an additional 20 h. n = 5 placentas for each treatment. Phospho-AMPK expression was significantly increased by gAd (RMANOVA, P < 0.01). AMPK expression was significantly increased by fAd (RMANOVA, P < 0.002). Tukey-Kramer multiple comparisons post tests, *P < 0.05.

FIG. 4.

A: IL-6 secretion was significantly (Student paired t test, P < 0.01) increased after incubation of cultured trophoblast cells with gAd (1 μg/ml). Data are mean ± SEM for six placentas. B: TNF-α secretion was significantly (Student paired t test, P < 0.01) increased after incubation of cultured trophoblast cells with gAd (1 μg/ml). Data are mean ± SEM for six placentas. C: IL-6 secretion was significantly (RMANOVA, P < 0.01) reduced in a dose-dependent manner after incubation of cultured trophoblast cells with fAd (1 μg/ml). Data are mean ± SEM for six placentas. D: TNF-α secretion was significantly (RMANOVA, P < 0.01) increased in a dose-dependent manner after incubation of cultured trophoblast cells with fAd. Data are mean ± SEM for six placentas. **P < 0.01.

FIG. 5.

A: Representative Western blot showing reduction in protein expression of STAT3 compared with control (C) using three unique siRNAs (1, 2, 3) targeted against STAT3. B: System A activity in control, siRNA STAT3 knockdown, and gAd-treated cells. Data are mean ± SEM for six placentas. Knocking down STAT3 significantly (RMANOVA, P = 0.002) reduced gAd stimulation of system A activity. Tukey-Kramer multiple comparisons post tests, **P < 0.01.

FIG. 6.

Representative Western blots (A) and summary data (B) of phospho-AKT Ser473 and phospho-AKT Thr308 protein expression after incubation of cultured trophoblast cells with control media, insulin (1 nmol/l), gAd (5 μg/ml), or fAd (5 μg/ml) for 24 h. Subsets of cells were pretreated with insulin (1 nmol/l) for 4 h and then exposed to 5 μg/ml gAd (IgAd) or fAd (IfAd) for an additional 20 h. n = 5 placentas for each treatment. Insulin significantly increased expression of both phospho-AKT Ser473 (P < 0.01) and Thr308 (P < 0.01). Addition of fAd to insulin-stimulated cells significantly (P < 0.01) reduced expression of phospho-AKT Thr308 compared with insulin treatment alone. Tukey-Kramer multiple comparisons post tests, *P < 0.05, **P < 0.01.

FIG. 7.

A: Representative Western blot of phospho–IRS-1 Tyr612 protein and β-actin expression after incubation of cultured trophoblast cells with control media (C), insulin (I; 1 nmol/l) for 24 or 4 h pretreatment with insulin (1 nmol/l) followed by fAd (5 μg/ml) for 20 h. B: Summary of phospho–IRS-1 Tyr612 protein expression. RMANOVA, P < 0.01, n = 5 placentas for each treatment; Tukey-Kramer multiple comparisons post tests, **P < 0.01. C: Representative Western blots of PPARα, phospho-PPARα Ser21, and β-actin protein expression after incubation of cultured trophoblast cells with control media (C), insulin (I; 1 nmol/l) for 24 or 4 h pretreatment with insulin (1 nmol/l) followed by fAd (5 μg/ml) for 20 h. D: Summary of PPARα and phospho-PPARα Ser21 protein expression. RMANOVA, P < 0.01, n = 5 placentas for each treatment; Tukey-Kramer multiple comparisons post tests, *P < 0.05.