Downregulation of IRS-1 in adipose tissue of offspring of obese mice is programmed cell-autonomously through post-transcriptional mechanisms

Abstract

We determined the effects of maternal diet-induced obesity on offspring adipose tissue insulin signalling and miRNA expression in the aetiology of insulin resistance in later life. Although body composition and glucose tolerance of 8-week-old male offspring of obese dams were not dysregulated, serum insulin was significantly (p<0.05) elevated. Key insulin signalling proteins in adipose tissue were down-regulated, including the insulin receptor, catalytic (p110β) and regulatory (p85α) subunits of PI3K as well as AKT1 and 2 (all p<0.05). The largest reduction observed was in IRS-1 protein (p<0.001), which was regulated post-transcriptionally. Concurrently, miR-126, which targets IRS-1, was up-regulated (p<0.05). These two features were maintained in isolated primary pre-adipocytes and differentiated adipocytes in-vitro. We have therefore established that maternal diet-induced obesity programs adipose tissue insulin resistance. We hypothesise that maintenance of the phenotype in-vitro strongly suggests that this mechanism is cell autonomous and may drive insulin resistance in later life.

Keywords: Adipose tissue; DEXA, dual energy X-ray absorptiometry; Hyperinsulinemia; IRS1; IRβ, insulin receptor-beta; Maternal obesity; PI3K, phosphotidylinositol 3-kinase; UTR, untranslated region; miRNA, microRNA; microRNAs.

Graphical abstract

Figure 1

Energy expenditure and activity at 8 weeks of age. (A) Energy expenditure of Control (white circle, solid line) and Mat-Ob (black circle, dashed line) offspring. Mice previously trained 1 week prior to the procedure, were singly housed for a 72 h period in a CLAMS chamber with free access to both food and water. Parameters of oxygen and carbon dioxide exchange coupled to food intake enabled quantification of both RER and energy expenditure. No significant differences determined by repeated measures ANOVA. (B) Physical activity as mean counts of horizontal activity (x-tot), ambulation (x-amb) and vertical activity (z-tot) in the Control (white bars, n=6) and Mat-Ob (grey bars, n=5) groups. No significant differences determined by a 2-way ANOVA.

Figure 2

(A) Glucose tolerance following 1 g/kg body weight bolus i.p. glucose injection in Control (open circles) vs. Mat-Ob (closed circles) offspring; and (B) fasting serum insulin concentrations in Control (white bars) and Mat-Ob (grey bars) offspring. n=6 per group. ^⁎=p<0.05 determined by student's t-test.

Figure 3

(A) Protein levels of insulin signalling molecules in adipose tissue at 8 weeks of age. (B) Protein levels of IRS-1 in liver, muscle and heart tissue of 8-week-old males. Signal intensities were quantified from n=6 per group, Control (white bars) and Mat-Ob (grey bars) and data presented as percentage mean expression of the control group±SEM. ^⁎=p<0.05, ^⁎⁎⁎=p<0.001 determined by student's t-test.

Figure 4

miRNA expression in adipose tissue of 8-week-old male offspring. (A) Expression of 5 candidate miRNAs relative to miR-185 and snRNA U6 housekeepers. (B) IRS-1 protein level and parallel miR-126 expression in adipose tissue from 8-week old male offspring. (C) Seed sequence of miR-126 (red) predicted to target the IRS1 3'-UTR. n=8 per group, Control (white bars) and Mat-Ob (grey bars). ^⁎p<0.05 determined by Student's t-test.

Figure 5

(A) Relative luciferase activity in control (white bars) constructs or constructs containing the 3' UTR of IRS1 (grey bars) introduced into HeLa cells in the presence of miR-126 mimic. Firefly luciferase activity for each construct was normalised to the co-transfected Renilla luciferase construct and then normalised to the change in luciferase in the presence of miR-126 (normalised luciferase activity in the absence of miR-126 was set to 1). (B) Relative luciferase activity of the constructs used in (A) in HEK 293 cells upon inhibition of miR-126 with 2-O-Methyl miR-126 antagonist. Normalisation was performed as in (A). ^⁎⁎p<0.01, ^⁎p<0.05.

Figure 6

(A) IRS-1 protein levels and (B) miR-126 expression in adipocytes derived from primary cultures of preadipocytes and differentiated for 11 days in-vitro (from n=4 liters per group, Control (white bars) and Mat-Ob (grey bars)). ⁎⁎ p<0.01 determined by Student's t-test. (C) Adipocytes derived from primary cultures of preadipocytes [from n=4 liters per group, Control (open boxes) and Mat-Ob (closed boxes)] were cultured for 10 days before treatment with cycloheximide. IRS-1 protein was measured by Western blotting and expressed as a percentage of respective protein levels at time 0.