Androgen-induced insulin resistance is ameliorated by deletion of hepatic androgen receptor in females

Abstract

Androgen excess is one of the most common endocrine disorders of reproductive-aged women, affecting up to 20% of this population. Women with elevated androgens often exhibit hyperinsulinemia and insulin resistance. The mechanisms of how elevated androgens affect metabolic function are not clear. Hyperandrogenemia in a dihydrotestosterone (DHT)-treated female mouse model induces whole body insulin resistance possibly through activation of the hepatic androgen receptor (AR). We investigated the role of hepatocyte AR in hyperandrogenemia-induced metabolic dysfunction by using several approaches to delete hepatic AR via animal-, cell-, and clinical-based methodologies. We conditionally disrupted hepatocyte AR in female mice developmentally (LivARKO) or acutely by tail vein injection of an adeno-associated virus with a liver-specific promoter for Cre expression in AR^fl/fl mice (adLivARKO). We observed normal metabolic function in littermate female Control (AR^fl/fl ) and LivARKO (AR^fl/fl ; Cre^+/- ) mice. Following chronic DHT treatment, female Control mice treated with DHT (Con-DHT) developed impaired glucose tolerance, pyruvate tolerance, and insulin tolerance, not observed in LivARKO mice treated with DHT (LivARKO-DHT). Furthermore, during an euglycemic hyperinsulinemic clamp, the glucose infusion rate was improved in LivARKO-DHT mice compared to Con-DHT mice. Liver from LivARKO, and primary hepatocytes derived from LivARKO, and adLivARKO mice were protected from DHT-induced insulin resistance and increased gluconeogenesis. These data support a paradigm in which elevated androgens in females disrupt metabolic function via hepatic AR and insulin sensitivity was restored by deletion of hepatic AR.

Keywords: PCOS; androgen; androgen receptor; glucose; insulin signaling; liver.

FIGURE 1

Androgen receptor (AR) is specifically deleted in liver. (A–C) AR mRNA levels of LivARKO compared to control littermates, measured by quantitative polymerase chain reaction, in livers (A), gonadal fat (B) and skeletal muscles (C). (D–F) AR mRNA levels in livers (D), gonadal fat (E) and skeletal muscles (F) of adLivARKO mice compared to adCon mice. Two-tailed Students t-tests were applied. p-values were stated in the graphs. Values were mean ± SEM. Con, control; Veh, vehicle

FIGURE 2

Conditional KO of Liver androgen receptor prevents dihydrotestosterone (DHT)-induced impaired glucose homeostasis. Control and LivARKO mice, with (6–8 weeks after DHT insertion) and without DHT treatment were subjected to (A,B) glucose tolerance test (GTT) (N = 4–6/group), (C,D) insulin tolerance test (ITT) (N = 4–14/group), or (E,F) Pyruvate tolerance test (PTT) (N = 6–13/group). The area under the curve (AUC) was determined for the GTT, ITT, and PTT. Statistical analysis was performed for the line graph at the same time point between groups and *p < .05; **p < .01. (G) Fat mass and lean mass were determined via echo magnetic resonance imaging 8 weeks after insertion. (H) body weight among groups was recorded (N = 5–10/group). Data were analyzed using two-tailed studenťs t-tests. Values are means ± SEM

FIGURE 3

Insulin and hormone levels. Insulin levels were measured from control and LivARKO mice without dihydrotestosterone (DHT) (A) or at 6–8 weeks after DHT insertion (B) after 16-h fasted conditions at basal (0 min) and upon GSIS (glucose stimulated insulin secretion at 30 min after glucose injection). N = 5–24/group. Hormone levels (C) leptin, (D) IL-6, (E) TNFα, were measured after 7-h fasted conditions. N = 9–12/group. Data were compared by two-tailed studenťs t-tests. Values are means ± SEM. IL-6, interleukin 6; TNFα, tumor necrosis factor alpha

FIGURE 4

Adult-onset KO of Liver androgen receptor (adLivARKO) prevents dihydrotestosterone (DHT)-induced impaired glucose homeostasis. DHT (adCon-DHT) and empty pellets (adCon-veh) were inserted in conjunction with tail vein AAV injections. After 6 weeks post insertion, adCon-veh, adCon-DHT and adLivARKO-DHT were subjected to (A) glucose tolerance test (GTT) (N = 3–4/group), (C) Pyruvate tolerance test (PTT) (N = 4–6/group), and (D) insulin tolerance test (ITT) (N = 3–4/group); Area under curve (AUC) was determined for the (B) GTT, (D) PTT, and (F) ITT. (G). Livers were subjected to ex vivo H-2-deoxy-glucose glucose transport assays (N = 3–4/group). Statistical analysis was performed for the line graph at the same time point using two-way ANOVA followed by Tukey’s multiple tests. *p < .05; **p < .01; ***p < .001 (A,C, E: adCon-DHT vs. adLivARKO-DHT and adCon-veh groups; E: adCon-DHT vs. adCon-veh groups). Data (B,D,F,G) were analyzed using two-tailed studenťs t-tests (adCon-DHT vs. adCon-veh or adLivARKO-DHT). Values are mean ± SEM

FIGURE 5

Conditional KO of Liver androgen receptor prevents dihydrotestosterone (DHT)-induced increased expression of gluconeogenic mRNA and proteins. At 10–12 weeks post insertion of pellet, livers of fasted control and LivARKO, with and without DHT mice were taken and processed. (A,B) qRT-PCR analysis to determine mRNA expression of Pck1, G6pc, Foxo1 was performed, where (A) shows Con-veh versus LivARKO-veh (N = 4–6) and (B) shows Con-DHT versus LivARKO-DHT (N = 3–5). Western blot analysis probing for antibodies against PEPCK and G6Pase and graphical representations of the densitometry, where (C–E) show Con-veh versus LivARKO-veh (N = 4–6) and (F–H) show Con-DHT versus LivARKO-DHT (N = 4–6) data are relative fold change to Con-veh). Two-tailed studenťs t-tests were used to analyze the data. Values are means ± SEM. Foxo1, Forkhead box protein O1; G6pc, glucose-6-phosphatase catalytic subunit; Pck1, PEPCK, phosphoenolpyruvate carboxykinase

FIGURE 6

Conditional KO of Liver androgen receptor prevents dihydrotestosterone (DHT)-induced impaired insulin signaling in liver. After 10 weeks post insertion, control and LivARKO mice with or without DHT were fasted for 16 h and then injected with saline or 0.5 U/kg insulin. After 10 min injection, liver samples were collected and subjected to (A) immunoprecipitation and/or western blot analysis. Graphical representations of densitometry for control and LivARKO are shown: (B) p-AKT (S473)/AKT. (C) IP: AR, IB: p85 (N = 3–4/ group). (D) Liver lysates from LivARKO mice were immunoprecipitated using p-Y antibodies and the IP-pY was subjected to a PI3K activity assay. (N = 6/group). A different subset of liver tissues from fasted mice were subjected to (E) qRT-PCR analysis for Pi3kca mRNA expression, n = 3–4 per group. Data in this figure were assessed by two-way ANOVA with Tukey’s multiple comparisons tests. Values are means ± SEM. Different letters represent statistical difference, p < .05. D, DHT; I, insulin; IB, immunoblot; IP, immunoprecipitation; Veh, vehicle

FIGURE 7

Prevention of dihydrotestosterone (DHT)-induced insulin resistance was recapitulated in primary hepatocytes from Control and LivARKO female mice. Adult primary hepatocytes were extracted from (A) Control and (B) LivARKO female mice, cultured for 24 h, and subjected to a cell culture as detailed in the methods. Western blot analysis was performed using antibodies against p-AKT, AKT, p-Foxo1, Foxo1, and Actin. Graphical figures for the densitometry of the blot images are shown below the images. Data were assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. Values are means ± SEM. Different letter represents statistical difference, p < .05

FIGURE 8

The effects of physiological and pathophysiological levels of androgen in female liver. Under physiological levels of dihydrotestosterone (DHT; black line), insulin signaling and gluconeogenesis proceed normally. Under pathophysiological levels of DHT (green line), cytosolic liver AR impairs insulin signaling and nuclear AR directly regulates gluconeogenesis resulting in enhanced gluconeogenesis. This is associated with hyperinsulinemia and systemic insulin resistance. AKT, protein kinase B; G6Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphoinositide 3-kinases