Developmental programming: Adipose depot-specific changes and thermogenic adipocyte distribution in the female sheep

Abstract

Prenatal testosterone (T)-treated female sheep exhibit an enhanced inflammatory and oxidative stress state in the visceral adipose tissue (VAT) but not in the subcutaneous (SAT), while surprisingly maintaining insulin sensitivity in both depots. In adult sheep, adipose tissue is predominantly composed of white adipocytes which favor lipid storage. Brown/beige adipocytes that make up the brown adipose tissue (BAT) favor lipid utilization due to thermogenic uncoupled protein 1 expression and are interspersed amidst white adipocytes, more so in epicardiac (ECAT) and perirenal (PRAT) depots. The impact of prenatal T-treatment on ECAT and PRAT depots are unknown. As BAT imparts a metabolically healthy phenotype, the depot-specific impact of prenatal T-treatment on inflammation, oxidative stress, differentiation and insulin sensitivity could be dictated by the distribution of brown adipocytes. This hypothesis was tested by assessing markers of oxidative stress, inflammation, adipocyte differentiation, fibrosis and thermogenesis in adipose depots from control and prenatal T (100 mg T propionate twice a week from days 30-90 of gestation) -treated female sheep at 21 months of age. Our results show prenatal T-treatment induces depot-specific changes in inflammation, oxidative stress state, collagen accumulation, and differentiation with changes being more pronounced in the VAT. Prenatal T-treatment also increased thermogenic gene expression in all depots indicative of increased browning with effects being more prominent in VAT and SAT. Considering that inflammatory and oxidative stress are also elevated, the increased brown adipocyte distribution is likely a compensatory response to maintain insulin sensitivity and function of organs in the proximity of respective depots.

Keywords: Adipose tissue; Brown adipose tissue; Inflammation; Insulin sensitivity; Oxidative stress.

Figure 1:

Mean ± SEM of fold change in mRNA expression of inflammatory cytokines and macrophage marker genes in the different adipose depots from control (C) and prenatal T-treated (T) animals. * = p < 0.05 by Student’s t-test and solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 2:

Mean ± SEM of levels of oxidative stress markers in the different adipose depots from control (C) and prenatal T-treated (T) animals. Mean values of oxidized tyrosine concentrations in VAT and SAT depots have been previously published (Puttabyatappa et al., 2017) and means are shown in the grey area for comparison. * = p < 0.05 by Student’s t-test and solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 3:

Mean ± SEM of fold change in mRNA expression of antioxidant genes in the different adipose depots from control (C) and prenatal T-treated (T) animals. * = p < 0.05 by Student’s t-test and solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 4:

Mean ± SEM of fold change in mRNA expression of adipocyte differentiation marker genes in the different adipose depots from control (C) and prenatal T-treated (T) animals. * = p < 0.05 by Student’s t-test and solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 5:

Mean ± SEM of collagen content normalized to total protein in the different adipose depots from control (C) and prenatal T-treated (T) animals. solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 6:

Mean ± SEM of fold change in mRNA expression of insulin signaling genes in the different adipose depots from control (C) and prenatal T-treated (T) animals. solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 7:

Mean ± SEM of fold change in mRNA expression of thermogenic genes in the different adipose depots with VAT depot set as baseline. Gene expression was examined in the adipose depots from control animals. Differing alphabet superscripts indicate statistically significant differences by ANOVA (a vs. b vs. c: p<0.05; ab not different from a or b).

Figure 8:

Mean ± SEM of fold change in mRNA expression of thermogenic gene markers in the different adipose depots from control (C) and prenatal T-treated (T) animals. * = p < 0.05 by Student’s t-test and solid grey circle = large effect size by Cohen’s effect size analysis.

Figure 9:

Schematic showing the depot-specific changes in negative and positive mediators of insulin sensitivity and members of the insulin signaling pathway and their potential contribution to insulin sensitivity of the various AT depots in the prenatal T-treated female sheep. The mediators in the dark blue box represent statistically significant changes while those in the light blue boxes are evident by Cohen’s effect size analysis only. Among the insulin signaling members those in the box shaded yellow represent previously published data (Lu et al., 2016). Note the mRNA expression of members of insulin signaling pathway in VAT examined in this study is consistent with the previous report (Nada et al., 2010). The balance between negative (eg. inflammatory and oxidative state) and positive (eg. antioxidants and distribution of thermogenic adipocytes) mediators may have contributed to the maintenance of the insulin sensitive state in VAT and SAT depots (top 2 panels) as evident from insulin-stimulated increase in phosphoAKT described previously (Lu et al., 2016). Bottom 2 panels show prenatal T-treatment induced changes in both positive and negative mediators and members of insulin signaling pathway in the ECAT and PRAT depots. Whether the balance between the negative and positive mediators of insulin sensitivity is altered to affect insulin sensitivity, and cardiac defects and renal contribution to hypertension, respectively, in ECAT and PRAT depots remains to be determined. Similarly, whether the large magnitude decrease in ECAT and lack of changes in PRAT in the expression of insulin signaling members reflect reduced/unchanged insulin stimulated phosphoAKT / insulin sensitivity in them also needs to be assessed.