Estradiol replacement improves high-fat diet-induced insulin resistance in ovariectomized rats

Abstract

The role of 17β-estradiol (E2) in high-fat diet (HFD)-induced alteration of the protein kinase B (Akt) signaling pathway in ovariectomized (OVX) rats is unclear. Therefore, we examined whether chronic estrogen replacement restores HFD-induced impairment in insulin sensitivity by its effects concomitant with alterations in the Akt isoform 2 (Akt2) and Akt substrate of 160 kDa (AS160) phosphorylation in muscles of OVX rats. Nine-week-old female Wistar rats underwent ovariectomy under anesthesia; after 4 weeks, subcutaneous implantation of either E2 or placebo (PL) pellets was performed, and HFD feeding was initiated. Intravenous glucose tolerance tests were performed to assess insulin sensitivity. Following insulin injection into rats' portal vein, the liver and gastrocnemius muscle were dissected for insulin signaling analysis. We observed that HFD increased energy intake and body weight in the PL group; however, it was temporarily decreased in the E2 group. Adipose tissue accumulation was larger in HFD-fed rats than in normal chow diet (NCD)-fed rats in the PL group; however, this difference was not observed in the E2 group. HFD reduced insulin sensitivity in the PL group only. In vivo insulin stimulation increased Akt2 phosphorylation in the muscles of NCD-fed rats in both groups. In contrast, HFD affected insulin-stimulated phosphorylation of Akt2 and AS160 in the muscles of rats in the PL group but not in the E2 group. Our data suggest that E2 replacement improves HFD-induced insulin resistance, and this effect is accompanied by the alterations in the Akt2 and AS160 phosphorylation in insulin-stimulated muscles of OVX rats.

Keywords: AS160; Akt2; estradiol replacement; high-fat diet; insulin resistance; ovariectomized rat.

FIGURE 1

Changes in food intake (a), energy intake (b), body weight (c), and wet weights of adipose tissues (d) in ovariectomized (OVX) rats. Rats were assigned to the 17β‐estradiol (E2)‐replaced or placebo (PL)‐treated group after a 4‐week recovery period from the OVX operation. These rats were fed a normal chow diet (NCD) or a high‐fat diet (HFD). Values are means ± SE (n = 14 in PL‐NCD; n = 13 in PL‐HFD; n = 14 in E2‐NCD; n = 12 in E2‐HFD). There was an interaction of age or diet and group effects in food intake (p < 0.05: PL‐NCD vs. PL‐HFD; p < 0.001: PL‐HFD vs. E2‐HFD, E2‐NCD vs. E2‐HFD), energy intake (p < 0.01: PL‐NCD vs. PL‐HFD; p < 0.001: PL‐HFD vs. E2‐HFD), body weight (p < 0.01: E2‐NCD vs. E2‐HFD; p < 0.001: PL‐NCD vs. PL‐HFD, PL‐NCD vs. E2‐NCD, PL‐HFD vs. E2‐HFD), and the wet weights of visceral (p < 0.01) and inguinal adipose tissues (p < 0.01). * and ***, significant differences (p < 0.05 and p < 0.001, respectively) between the PL and E2 groups. ^#, ^##, and ^###, significant differences (p < 0.05, p < 0.01, and p < 0.001, respectively) between the NCD and HFD groups

FIGURE 2

Assessments of insulin sensitivity by the intravenous glucose tolerance test in ovariectomized rats. Values are means ± SE [n =7 in placebo (PL)‐normal chow diet (NCD); n = 11 in PL‐ high‐fat diet (HFD); n = 12 in 17β‐estradiol (E2)‐NCD; n = 12 in E2‐HFD]. Line graphs represent the courses of change in plasma glucose (Left side of a) and insulin (Left side of b) concentration after glucose injection in the four groups. There was an interaction of time and group effects in the plasma insulin response (p < 0.05: PL‐NCD vs. E2‐NCD; p < 0.01: PL‐HFD vs. E2‐HFD), but not in the plasma glucose response. Bar graphs represent the area under the curve (AUC) for glucose (Right side of a) and insulin (Right side of b) levels over 60 min after the glucose injection and glucose‐insulin index (c) in each group. * and **, significant differences (p < 0.05 and p < 0.01, respectively) between the PL and E2 groups. ^#, significant differences (p < 0.05) between the NCD and HFD groups

FIGURE 3

Effects of high‐fat diet (HFD) and 17β‐estradiol (E2) replacement on insulin‐stimulated protein kinase B (Akt)/Akt substrate of 160 kDa (AS160) pathway in muscles. Representative blots and relative values of Akt and phospho (p)‐Akt serine (Ser)⁴⁷³ (a), Akt2 and p‐Akt2 Ser⁴⁷⁴ (b), and AS160 and p‐AS160 threonine (Thr)⁶⁴² (c) in the gastrocnemii of ovariectomized rats. Protein levels were normalized with the control β‐actin and were expressed as relative values calculated by dividing the value of the individual rat in each group by the average value for the placebo (PL)‐normal chow diet (NCD) group injected saline. Values are means ± SE, with (+) or without (–) insulin stimulation by injection of 10⁻⁵ mol/l insulin or saline in the portal vein [n =12 in PL‐NCD; n = 12 in PL‐HFD; n = 11 in E2‐NCD; n = 11 in E2‐HFD]. There was an interaction of replacement and insulin injection effects in the insulin‐induced phosphorylation of Akt2 Ser⁴⁷⁴ in the HFD‐fed rats (p < 0.05). *, **, and ***, significant differences (p < 0.05, p < 0.01, and p < 0.001, respectively) between saline and insulin injections. ^# and ^##, significant differences (p < 0.05 and p < 0.01, respectively) between the PL and E2 groups

FIGURE 4

Effects of high‐fat diet (HFD) and 17β‐estradiol (E2) replacement on insulin‐stimulated protein kinase B (Akt) activation in livers. Representative blots and relative values of Akt and phospho (p)‐Akt serine (Ser)⁴⁷³ in the livers of ovariectomized rats. Protein levels were normalized with the control β‐actin and were expressed as relative values calculated by dividing the value of the individual rat in each group by the average value for the placebo (PL)‐normal chow diet (NCD) group injected saline. Values are means ± SE, with (+) or without (–) insulin stimulation by injection of 10⁻⁵ mol/l insulin or saline in the portal vein [n = 12 in PL‐NCD; n = 12 in PL‐HFD; n = 11 in E2‐NCD; n = 11 in E2‐HFD]. * and **, significant differences (p < 0.05 and p < 0.01, respectively) between saline and insulin injections