Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice - PubMed (original) (raw)
Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice
Stanley Andrisse et al. Am J Physiol Endocrinol Metab. 2018.
Abstract
Hyperandrogenemia and hyperinsulinemia are believed to play prominent roles in polycystic ovarian syndrome (PCOS). We explored the effects of low-dose dihydrotestosterone (DHT), a model of PCOS, on insulin signaling in metabolic and reproductive tissues in a female mouse model. Insulin resistance in the energy storage tissues is associated with type 2 diabetes. Insulin signaling in the ovaries and pituitary either directly or indirectly stimulates androgen production. Energy storage and reproductive tissues were isolated and molecular assays were performed. Livers and white adipose tissue (WAT) from DHT mice displayed lower mRNA and protein expression of insulin signaling intermediates. However, ovaries and pituitaries of DHT mice exhibited higher expression levels of insulin signaling genes/proteins. Insulin-stimulated p-AKT levels were blunted in the livers and WAT of the DHT mice but increased or remained the same in the ovaries and pituitaries compared with controls. Glucose uptake decreased in liver and WAT but was unchanged in pituitary and ovary of DHT mice. Plasma membrane GLUTs were decreased in liver and WAT but increased in ovary and pituitary of DHT mice. Skeletal muscle insulin-signaling genes were not lowered in DHT mice compared with control. DHT mice did not display skeletal muscle insulin resistance. Insulin-stimulated glucose transport increased in skeletal muscles of DHT mice compared with controls. DHT mice were hyperinsulinemic. However, the differential mRNA and protein expression pattern was independent of hyperinsulinemia in cultured hepatocytes and pituitary cells. These findings demonstrate a differential effect of DHT on the insulin-signaling pathway in energy storage vs. reproductive tissues independent of hyperinsulinemia.
Keywords: DHT; glucose transport; hyperandrogenemia or androgen excess; insulin signaling; reproductive endocrinology.
Figures
Fig. 1.
Mice receiving low-dose dihydrotestosterone (DHT) display differential altered insulin signaling mRNA expression levels in reproductive vs. energy storage tissues. C57BL6 were used in this study. At 3 mo post-original insertion, the livers (A), white adipose tissue (WAT; B), pituitaries (C), and ovaries (D) of control and DHT mice were harvested in the fed state and processed for qRT-PCR analysis using TRIzol for RNA isolation or processed for Western blot analysis after cell lysis: livers (E), WAT (F), pituitaries (G), and ovaries (H). See Table 1 for a list of the abbreviations and functions of qRT-PCR primers and Table 2 for information on antibodies used for Western blotting; n = 10/group for qRT-PCR and n = 4/group for Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2.
Low-dose DHT caused insulin resistance in the liver and WAT but not in the pituitaries and ovaries of fasted mice. At 3 mo post-insertion, control and DHT mice were fasted for 16 h and then injected with saline or 2 U/kg insulin. After 10 min, livers, WAT, pituitaries, and ovaries were harvested and subjected to Western blot analysis A_–_D) and multiplex Luminex assay analysis E_–_H) of p-AKT S473 and AKT. Livers (I) and WAT (J) were harvested from fed control and DHT mice and processed for Western blot analysis using antibodies against p-AKT, AKT, and/or actin; n = 4–7/group. Unpaired 2-tailed _t_-tests were used comparing control with the tested groups and insulin only to the insulin plus DHT group. Different letters indicate a statistically significant difference. Same letters indicate no statistically significant difference. *P < 0.05.
Fig. 3.
Low-dose DHT differential effects on basal and insulin-stimulated glucose uptake in the liver and WAT compared with the pituitary and ovary. At 3 mo post-insertion, control and DHT mice were fasted for 16 h, and livers, WAT, pituitaries, and ovaries were harvested and subjected to ex vivo 3H-2-deoxy-glucose basal glucose transport assays (A_–_D); n = 4–7/group. E_–_H: a subset of samples were analyzed via Western blot analysis with antibodies against GLUT1 and actin; n = 4–7/group. I_–_L: another subset of samples were subjected to ex vivo 3H-2-deoxy-glucose insulin-stimulated glucose transport assays; n = 4–7/group. *P < 0.05.
Fig. 4.
Low-dose DHT did not result in insulin resistance in skeletal muscles. Skeletal muscles extracted from fed control and DHT mice (A) and from control and DHT mice that were fasted for 16 h then injected with saline or 2 U/kg insulin (B) were harvested and subjected to Western blot analysis for p-AKT S473 and AKT. Basal (C) and insulin-stimulated (D) ex vivo 3H-2-deoxy-glucose transport assays were performed in extracted skeletal muscles. E: skeletal muscles of control and DHT mice were harvested and processed for qRT-PCR analysis using TRIzol for RNA isolation. F: skeletal muscles from control and DHT mice were harvested in the fed state and processed for Western blot analysis after cell lysis. See Table 1 for a list of the abbreviations and functions of qRT-PCR primers and Table 2 for information on antibodies used for Western blotting; n = 10/group for qRT-PCR and n = 4/group for Western blotting; *P < 0.05, **P < 0.01, ***P < 0.001. Bas, basal; Ins, insulin; C, control; D, DHT, dihydrotestosterone; n = 4–7/group; different letters indicate significance.
Fig. 5.
Low-dose DHT differentially regulates insulin signaling mRNA expression independent of hyperinsulinemia. A: 3 wk after DHT insertion, fed and fasted, basal and glucose-stimulated insulin levels were determined from serum of tail blood samples of control and DHT mice using an ELISA. B and C: a low-dose DHT cell model was developed to remove the aspect of hyperinsulinemia. The experiments were performed in serum starved and non-serum-starved conditions. The nonstarved data are presented in the graphs. H2.35 female mouse hepatocytes (B) and αT3 female mouse pituitary cell lines (C) were used. mRNA expression was determined via qRT-PCR analysis, and protein expression was determined via Western blot analysis [H2.35 (D) and αT3 (E); n = 6/group. Same letters indicate no statistically significant difference. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6.
DHT mice displayed impaired glucose tolerance, impaired insulin sensitivity, and reproductive dysfunction seen as reduced corpora lutea, increased atretic follicles, and acyclity. A and B: control and low-dose DHT mice fasted for 16 h received intraperitoneal injections of 2 g/kg body wt glucose (GTT) or 0.3 U/kg body wt insulin (ITT; Lilly, Indianapolis, IN), and tail blood was obtained to determine blood glucose at time points between 0 and 120 min using a One Touch Ultra glucometer and One Touch Ultra test strips (Life Scan, Milpitas, CA). C: the estrus cycle was tested by obtaining vaginal smears for 14 consecutive days and analyzing the cytology of the smears. D: at 3 mo post-insertion of DHT, ovaries of control and DHT mice were obtained, sectioned at 5 mm, and morphological analyses were performed. Number of atretic follicles (AF; E) and corpora lutea (CL; F) were determined as previously described (25); n = 6–8/group. *P< 0.05, **P < 0.01, ***P < 0.001.
Fig. 7.
Model depicting the effects of DHT on insulin signaling mRNA expression in energy storage compared to reproductive tissues. Hyperandrogenemia results in differential tissue-specific effects on insulin action that may define metabolic and reproductive dysfunction. DHT lowered several insulin signaling genes in the liver and WAT, but increased several insulin signaling genes in the pituitary and ovary. Dark red = decrease, P < 0.01; light red = decrease, P < 0.05; dark green = increase, P < 0.01; light green = increase, P < 0.05.
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