Estrogen Improves Insulin Sensitivity and Suppresses Gluconeogenesis via the Transcription Factor Foxo1

Abstract

Premenopausal women exhibit enhanced insulin sensitivity and reduced incidence of type 2 diabetes (T2D) compared with age-matched men, but this advantage disappears after menopause with disrupted glucose homeostasis, in part owing to a reduction in circulating 17β-estradiol (E₂). Fasting hyperglycemia is a hallmark of T2D derived largely from dysregulation of hepatic glucose production (HGP), in which Foxo1 plays a central role in the regulation of gluconeogenesis. Here, we investigated the action of E₂ on glucose homeostasis in male and ovariectomized (OVX) female control and liver-specific Foxo1 knockout (L-F1KO) mice and sought to understand the mechanism by which E₂ regulates gluconeogenesis via an interaction with hepatic Foxo1. In both male and OVX female control mice, subcutaneous E₂ implant improved insulin sensitivity and suppressed gluconeogenesis; however, these effects of E₂ were abolished in L-F1KO mice of both sexes. In our use of mouse primary hepatocytes, E₂ suppressed HGP and gluconeogenesis in hepatocytes from control mice but failed in hepatocytes from L-F1KO mice, suggesting that Foxo1 is required for E₂ action on the suppression of gluconeogenesis. We further demonstrated that E₂ suppresses hepatic gluconeogenesis through activation of estrogen receptor (ER)α-phosphoinositide 3-kinase-Akt-Foxo1 signaling, which can be independent of insulin receptor substrates 1 and 2 (Irs1 and Irs2), revealing an important mechanism for E₂ in the regulation of glucose homeostasis. These results may help explain why premenopausal women have lower incidence of T2D than age-matched men and suggest that targeting ERα can be a potential approach to modulate glucose metabolism and prevent diabetes.

Figure 1

Estrogen regulates glucose homeostasis in mice. A–C: Blood glucose level in control and L-F1KO mice of both sexes fasted for 16 h overnight. Fasting glucose was measured every week, and the data show the most representative measurement. D–F: Glycogen content in the liver of mice fasted for 16 h overnight. Glycogen content was normalized by weight of liver. G–I: Body weight at 8 weeks after E₂ implantation in control and L-F1KO mice of both sexes fasted overnight. J–L: Serum E₂ level at 8 weeks after E₂ implantation in mice fasted overnight. All data are expressed as the mean ± SEM. For females, n = 4–7, *P < 0.05 vs. OVX control mice with placebo. For males, n = 4–5, *P < 0.05 vs. control mice with placebo. NS, not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Estrogen improves insulin sensitivity and suppresses gluconeogenesis in mice. A–C: GTT was performed in control and L-F1KO mice of both sexes fasted for 16 h overnight. Glucose was administered at 2 g/kg body wt of mice by intraperitoneal injection after 16 h overnight fasting, and glucose level was measured at indicated time points. D–F: Area under the curve (AUC) of the GTT displayed in A–C, respectively. G–I: PTT was performed on mice fasted for 16 hours overnight. Pyruvate was administered at 2 g/kg body wt by intraperitoneal injection after overnight fasting, and glucose level was measured at indicated time points. J–L: Area under the curve of the PTT showed in G–I, respectively. M–O: ITT was performed on mice fasted for 5 h. Insulin was administered at 2 units/kg body wt by intraperitoneal injection, and glucose level was measured at indicated time points. P–R: Area under the curve of the ITT shown in M–O, respectively. All data are expressed as mean ± SEM. For females, n = 4–7, *P < 0.05 vs. OVX control mice with placebo. n = 4–7, #P < 0.05 vs. intact mice. For males, n = 4–5, *P < 0.05 vs. control mice with placebo. NS, not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Estrogen suppresses expression of gluconeogenic genes in liver and influences serum hormone levels associated with glucose metabolism in mice. A–C: Relative mRNA expressions in the liver of control and L-F1KO mice of both sexes fasted for 16 hours overnight after 8 weeks of pellet implantation. D–F: Serum hormone levels in mice fasted for 16 h overnight. Blood samples were collected by cardiac puncture in euthanized mice. All data are expressed as the mean ± SEM. For females, n = 4–7, *P < 0.05 vs. OVX control mice on placebo. For males, n = 4–5, *P < 0.05 vs. control mice on placebo. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Estrogen suppresses HGP and gluconeogenesis depending on hepatic Foxo1 in primary hepatocytes. A: HGP in primary hepatocytes from control and L-F1KO mice. HGP was measured at indicated time points after 0.1 μmol/L E₂ stimulation, and normalized to total protein levels. B: Glucose production in primary hepatocytes from control and L-F1KO mice upon 0.1 μmol/L E₂ stimulation in the presence (HGP) or absence (glycogenolysis) of pyruvate and lactate. The difference between these two values was interpreted to represent gluconeogenesis. C: Relative mRNA levels of gluconeogenic genes in primary hepatocytes from control and L-F1KO mice upon 0.1 μmol/L E₂ stimulation for 3 h. D and E: Western blots (D) and corresponding quantification (E) of insulin signaling protein in hepatocytes from control mice upon 1 h stimulation of 0.1 μmol/L E₂ or 0.1 μmol/L insulin. p-, phosphorylated. F: Glucose production in primary hepatocytes from control and Foxo1 S253A knock-in mice (S253A) upon 0.1 μmol/L E₂ stimulation in the presence (HGP) or absence (glycogenolysis) of pyruvate and lactate (glycogenolysis). All data are expressed as the mean ± SEM. NS, not significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control + vehicle. n = 3 of each group.

Figure 5

Activation of Akt signaling is required for estrogen to suppress gluconeogenesis. A: Glucose release in primary hepatocytes from control mice in the presence (HGP) or absence (glycogenolysis) of pyruvate and lactate. Cells were treated with 10 μmol/L Akti or 0.2 μmol/L Wort 30 min prior to 0.1 μmol/L E₂ stimulation. B: Relative mRNA levels of gluconeogenic genes in primary hepatocytes from control mice upon stimulation of 10 μmol/L Akti and 0.2 μmol/L Wort 30 min prior to 0.1 μmol/L E₂. C and D: Western blots (C) and corresponding quantification (D) of insulin-signaling protein in hepatocytes from control mice upon stimulation of 10 μmol/L Akti and 0.2 μmol/L Wort 30 min prior to 0.1 μmol/L E₂. p-, phosphorylated. E: Blood glucose levels of control and L-DKO mice in random-fed or fasted state upon OVX surgery and E₂ implant. Blood glucose levels were measured in littermates of male, intact female, and OVX female mice at 12 weeks of age random fed or after 16 h overnight fast. F: Glucose production in primary hepatocytes from control and L-DKO mice upon 0.1 μmol/L E₂ or 0.1 μmol/L ERα agonist PPT stimulation in the presence (HGP) or absence (glycogenolysis) of pyruvate and lactate. All data are expressed as the mean ± SEM. NS, not significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control + vehicle. n = 3 of each group.

Figure 6

ERα is required and sufficient to activate Akt-Foxo1 signaling and suppress gluconeogenesis in primary hepatocytes. A: Glucose production in primary hepatocytes from control mice in the presence (HGP) or absence (glycogenolysis) of pyruvate and lactate. Cells were treated with 1 μmol/L ERα antagonist MPP prior to stimulation of 0.1 μmol/L E₂ or 0.1 μmol/L ERα agonist PPT. B: Relative mRNA levels of gluconeogenic genes in primary hepatocytes from control mice upon stimulation of 0.1 μmol/L E₂, 1 μmol/L MPP, or 0.1 μmol/L PPT. C: Western blots and corresponding quantification of insulin-signaling protein in hepatocytes from control mice upon stimulation of 0.1 μmol/L E₂, 1 μmol/L MPP, or 0.1 μmol/L PPT. D: Western blots and corresponding quantification of Foxo1 protein in HepG2 cells upon overexpression of ERα and stimulation of E₂ or insulin. HepG2 cells were transfected with 10 µg plasmid DNA expressing green fluorescent protein (GFP) or ERα for 30 h, followed by 6 h starvation prior to 0.1 μmol/L E₂ or 0.1 μmol/L insulin stimulation for 1 h. All data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control + vehicle. ###P < 0.001 vs. control + ERα overexpression. n = 3 of each group. p- or p, phosphorylated.

Figure 7

Schematic diagram represents the role of estrogen in the regulation of glucose metabolism. Estrogen suppresses hepatic gluconeogenesis and lowers blood glucose through interaction with ERα in a Foxo1-dependent manner. E₂ inhibits Foxo1 and its target G6pc expression indirectly, depending on the activation of PI3K-Akt signaling. IR, insulin receptor; p, phosphorylated; PIP₂, phosphatidylinositol (4,5)-bisphosphate; PIP₃, phosphatidylinositol (3,4,5)-trisphosphate; PDK1, 3-phosphoinositide-dependent protein kinase 1; Y, tyrosin; T, threonine; S, serine.