Overfeeding in the early postnatal period aggravates inflammation and hepatic insulin sensitivity in the 5α-dihydrotestosterone-induced animal model of PCOS

Abstract

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age and is closely associated with chronic low-grade inflammation and insulin resistance. To clarify the contribution of prepubertal weight gain to the development of insulin resistance in PCOS, we investigated the effects of early postnatal overfeeding on inflammatory and energy-sensing pathways as well as on markers of insulin signaling in the liver of the PCOS rat model.

Methods: Obesity induced by overfeeding was achieved by reducing litter size, while the PCOS-like condition was developed by treatment with 5α-dihydrotestosterone (DHT). Western blot and qPCR were used to analyze the expression of pro-inflammatory transcription factors and cytokines, as well as markers of the energy sensing and insulin signaling pathways.

Results: The results showed that hepatic insulin sensitivity was impaired only in DHT-treated rats raised in small litters, as evidenced by increased phosphorylation of IRS1 on Ser307 and decreased expression of total IRS1. Postnatal overfeeding stimulated JNK1 activation independent of hyperandrogenemia; nevertheless, the synergistic effect of both factors triggered NLRP3 activation and increased IL1β expression in the small litter DHT-treated group. This pro-inflammatory state was accompanied by decreased activatory phosphorylation of AMPK and reduced levels of its protein targets.

Conclusions: Overfeeding in the early postnatal period leads to a decrease in hepatic insulin sensitivity in the rat model of PCOS, which is associated with decreased activation of AMPK and stimulation of the hepatic NLRP3-IL1β signaling pathway. Accordingly, the inhibition of NLRP3 activation could provide a basis for the development of new therapeutic strategies for the treatment of insulin resistance in women with PCOS.

Keywords: inflammation; insulin resistance; obesity; polycystic ovary syndrome; postnatal overfeeding.

Figure 1

The effects of litter size reduction and DHT treatment on weight gain after weaning period. Each point represents the mean ± SEM (n = 6). Comparison between body masses of animals from small and normal litters by time was performed by two-way repeated measures ANOVA followed by Sidak’s post hoc test. Different symbols denote the values that are significantly different from NL-Placebo group (^*p < 0.05, ^**p < 0.01, ^***p < 0.001), NL-DHT group (^#p < 0.05, ^##p < 0.01, ^###p < 0.001) and SL-Placebo group (^$p < 0.05, ^$$$p < 0.001).

Figure 2

The levels of phosphorylated and total IRS1 protein in the liver after postnatal overfeeding and DHT treatment. Representative Western blots and relative quantification of phospho-IRS1 (Ser³⁰⁷) and total IRS1 in the cytosolic fraction. β-actin was used as loading control. All data are presented as mean ± SEM (n = 6). Two-way ANOVA followed by Tukey’s post hoc test was performed to determine the effects of litter size reduction and DHT treatment, as well as their interaction. Different symbols denote significant differences from NL-Placebo (^*p < 0.05, ^**p < 0.01) and SL-Placebo group (^$p < 0.05, ^$$p < 0.01).

Figure 3

The effects of litter size and DHT treatment on inflammatory mediators in the liver. (A) Relative expression of Tlr4 gene and (B) relative quantification with representative Western blots of NLRP3 protein and (C) phosphorylated (Thr183/Tyr185) and total form of JNK in the whole cell extract. Quantification of mRNA levels was done relative to the amount of Hprt, while β-actin was used as protein loading control. All data are presented as mean ± SEM (n = 6). Two-way ANOVA followed by Tukey’s post hoc test was performed to determine the effects of litter size reduction and DHT treatment, as well as their interaction. Different symbols denote significant differences from NL-Placebo group (^*p < 0.05) and NL-DHT group (^##p < 0.01).

Figure 4

The levels of hepatic proinflammatory cytokines after postnatal overfeeding and DHT treatment. Relative expression of Tnfα, Il1β, Il6 and Mif gene. Quantification of mRNA levels was done relative to the amount of Hprt. All data are presented as mean ± SEM (n = 6). Two-way ANOVA followed by Tukey’s post hoc test was performed to determine the effects of litter size reduction and DHT treatment, as well as their interaction. Different symbols denote significant differences from NL-Placebo group (*p < 0.05), NL-DHT group (^#p < 0.05) and SL-Placebo group (^$p < 0.05).

Figure 5

The effects of litter size and DHT treatment on the level and subcellular distribution of transcriptional regulator NFκB in the liver. Representative Western blots and relative quantification of NFκB protein in the cytosolic and nuclear fraction. β-actin and lamin B1 were used as loading controls for cytosolic and nuclear fraction, respectively. All data are presented as mean ± SEM (n = 6). Two-way ANOVA followed by Tukey’s post hoc test was performed to determine the effects of litter size reduction and DHT treatment, as well as their interaction.

Figure 6

The level of energy sensors in the liver after postnatal overfeeding and DHT treatment. Representative Western blots and relative quantification of (A) phospho-AMPK (Thr¹⁷²) and total AMPK protein in the cytosolic fraction, (B) SIRT1 protein in the nuclear fraction and (C) phospho-ACC (Ser⁷⁹) in the cytosolic fraction. β-actin and lamin B1 were used as loading controls for cytosolic and nuclear fraction, respectively. All data are presented as mean ± SEM (n = 6). Two-way ANOVA followed by Tukey’s post hoc test was performed to determine the effects of litter size reduction and DHT treatment, as well as their interaction. Symbols denote significant differences from NL-Placebo group (^*p < 0.05) and NL-DHT group (^#p < 0.05, ^##p < 0.01).