Implication of low level inflammation in the insulin resistance of adipose tissue at late pregnancy

Abstract

Insulin resistance is a characteristic of late pregnancy, and adipose tissue is one of the tissues that most actively contributes to the reduced maternal insulin sensitivity. There is evidence that pregnancy is a condition of moderate inflammation, although the physiological role of this low-grade inflammation remains unclear. The present study was designed to validate whether low-grade inflammation plays a role in the development of insulin resistance in adipose tissue during late pregnancy. To this end, we analyzed proinflammatory adipokines and kinases in lumbar adipose tissue of nonpregnant and late pregnant rats at d 18 and 20 of gestation. We found that circulating and tissue levels of adipokines, such as IL-1β, plasminogen activator inhibitor-1, and TNF-α, were increased at late pregnancy, which correlated with insulin resistance. The observed increase in adipokines coincided with an enhanced activation of p38 MAPK in adipose tissue. Treatment of pregnant rats with the p38 MAPK inhibitor SB 202190 increased insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and IR substrate-1 in adipose tissue, which was paralleled by a reduction of IR substrate-1 serine phosphorylation and an enhancement of the metabolic actions of insulin. These results indicate that activation of p38 MAPK in adipose tissue contributes to adipose tissue insulin resistance at late pregnancy. Furthermore, the results of the present study support the hypothesis that physiological low-grade inflammation in the maternal organism is relevant to the development of pregnancy-associated insulin resistance.

Fig. 1.

Comparison of circulating concentrations of adipokines in nonpregnant (0) and late pregnant rats (18 and 20 d of gestation). IL-1β (A), PAI-1 (active) (B), and MCP-1 (C).

Fig. 2.

Concentrations of adipokines and immunodetection of ED1, a marker for macrophage infiltration, in lumbar adipose tissue of nonpregnant (0) and late pregnant rats (18 and 20 d of gestation). PAI-1 (total) (A), TNF-α (B), and percentage of ED1-positive cells (C).

Fig. 3.

Correlation analysis between adipokine levels and insulin sensitivity in nonpregnant and pregnant rats at d 18 and 20 of gestation. A, Correlation between concentrations of circulating PAI-1 (active) and QUICKI or FGIR. B, Correlation between concentrations of TNF-α in lumbar adipose tissue and QUICKI or FGIR. Circles, Nonpregnant rats; squares, 18-d-pregnant rats; and diamonds, 20-d-pregnant rats.

Fig. 4.

p38 MAPK activation is increased in adipose tissue at late gestation. A, Phosphorylation of mTOR and JNK1/2. Representative immunoblots for phosphorylated and total proteins of two independent experiments. B, Phosphorylation of p38 MAPK. The graph shows the levels of phosphorylated p38 MAPK, normalized to the total amount of p38 MAPK protein, of six independent experiments. A representative immunoblot (IB) for phosphorylated and total protein is shown in the graph.

Fig. 5.

Inhibition of p38 MAPK at late pregnancy increases insulin signaling in adipose tissue. Starting at d 18 of gestation, rats were treated from d 18 to 20 of gestation, with vehicle or a daily dose of the specific p38 MAPK inhibitor SB 202190. On the morning of d 20 of pregnancy, and 2 h after the last SB 202190 dose, rats were killed, and adipose tissue was used for the analysis of p38 MAPK phosphorylation and pSer IRS-1. A, SB 202190 blocks phosphorylation of p38 MAPK at late pregnancy. The graph shows the levels of phosphorylated p38 MAPK, normalized to the total amount of p38 MAPK protein, of four independent experiments. A representative immunoblot for phosphorylated and total protein is shown in the graph. B, Inhibition of p38 MAPK decreases IRS-1 S307 phosphorylation in adipose tissue of late pregnant rats. The graph shows the levels of Ser-phosphorylated IRS-1, normalized to the total amount of IRS-1 in each sample. For in vivo stimulation of the insulin signaling cascade, after the SB 202190 treatment, some animals were anesthetized, and saline with or without insulin was injected via the portal vein. The graphs show the fold stimulation by insulin of tyrosine phosphorylated IR (C) and IRS-1 (D), normalized to the total amount of IR and IRS-1 protein. Representative immunoblots (IB) for phosphorylated and total IRS-1 are shown above each graph.

Fig. 6.

Inhibition of p38 MAPK in adipose cell of late pregnant rats increases the metabolic actions of insulin. Isolated adipocytes from fasted 20-d-pregnant rats were treated for 30 min in the absence (control) and presence of 15 μm of the specific p38 MAPK inhibitor SB 202190 before incubation for further 30 min in the absence (basal) or presence of 90 nm insulin. A, Incubation supernatants were used for glycerol determination to measure the effect of SB 202190 on the antilipolytic action of insulin. Data represent independent experiments with at least five different rats and are expressed as percentage of decrease of lipolytic activity by insulin as compared with the basal condition (i.e. in the absence of insulin). Glycerol determinations were performed in duplicate. B, Adipocytes were harvested to measure the effect of SB 202190 on the lipogenic action of insulin. Western blot analysis of ACC-Ser79 phosphorylation was used as estimate of lipogenesis. Data represent four independent experiments and are expressed as percentage of decrease of ACC phosphorylation by insulin compared with the basal condition (i.e. in the absence of insulin). A representative immunoblot for phosphorylated ACC (pSer-ACC) is shown in the graph.