Androgen excess in pancreatic β cells and neurons predisposes female mice to type 2 diabetes - PubMed (original) (raw)

. 2018 Jun 21;3(12):e98607.

doi: 10.1172/jci.insight.98607.

Camille Allard 2, Jamie J Morford 2, Weiwei Xu 2, Suhuan Liu 1, Adrien Jr Molinas 3, Sierra M Butcher 3, Nicholas Hf Fine 4 5, Manuel Blandino-Rosano 6, Venkata N Sure 7, Sangho Yu 8, Rui Zhang 8, Heike Münzberg 8, David A Jacobson 9, Prasad V Katakam 7, David J Hodson 4 5, Ernesto Bernal-Mizrachi 6, Andrea Zsombok 3, Franck Mauvais-Jarvis 2 3 10 11

Affiliations

Androgen excess in pancreatic β cells and neurons predisposes female mice to type 2 diabetes

Guadalupe Navarro et al. JCI Insight. 2018.

Abstract

Androgen excess predisposes women to type 2 diabetes (T2D), but the mechanism of this is poorly understood. We report that female mice fed a Western diet and exposed to chronic androgen excess using dihydrotestosterone (DHT) exhibit hyperinsulinemia and insulin resistance associated with secondary pancreatic β cell failure, leading to hyperglycemia. These abnormalities are not observed in mice lacking the androgen receptor (AR) in β cells and partially in neurons of the mediobasal hypothalamus (MBH) as well as in mice lacking AR selectively in neurons. Accordingly, i.c.v. infusion of DHT produces hyperinsulinemia and insulin resistance in female WT mice. We observe that acute DHT produces insulin hypersecretion in response to glucose in cultured female mouse and human pancreatic islets in an AR-dependent manner via a cAMP- and mTOR-dependent pathway. Acute DHT exposure increases mitochondrial respiration and oxygen consumption in female cultured islets. As a result, chronic DHT exposure in vivo promotes islet oxidative damage and susceptibility to additional stress induced by streptozotocin via AR in β cells. This study suggests that excess androgen predisposes female mice to T2D following AR activation in neurons, producing peripheral insulin resistance, and in pancreatic β cells, promoting insulin hypersecretion, oxidative injury, and secondary β cell failure.

Keywords: Beta cells; Diabetes; Endocrinology; Metabolism; Sex hormones.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. AR expression in pancreatic β cells and the hypothalamus of βARKORIP female mice.

(A) Pancreas sections showing AR immunofluorescent staining (red) colocalizing with insulin (green) in β cells of control RIP-Cre female mice and confirming successful AR deletion in representative βARKORIP islets at 12 weeks. Original magnification, ×40. (B) AR expression in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) of RIP-Cre and βARKORIP mice, as assessed by immunostaining. (C) AR-immunoreactive cell number in the ARC. (D) AR-immunoreactive cell number in the VMH. Values represent the mean ± SEM with dot plots (n = 4). *P < 0.05, Student’s t test.

Figure 2

Figure 2. Chronic DHT excess predisposes female controls, but not βARKORIP mice, to diabetes.

Female mice were fed WD for 9 weeks followed by s.c. treatment with vehicle (V) or DHT for 4 weeks. (A) Fasting serum insulin levels. (B) Random fed serum insulin levels. (C) Insulin secretion during an i.p. glucose-stimulated insulin secretion (GSIS) test (3 g/kg) and corresponding AUC. (D) i.p. insulin tolerance test (ITT) with blood glucose represented as percentage decrease from baseline, with corresponding area under baseline (AUB). (E) β Cell mass and representative islets with insulin staining (green). Scale bar: 10 μm. (F) Islet area. (G) Pancreas insulin concentration. (H) Fasting blood glucose. (I) Random fed blood glucose. (J) Blood glucose during an i.p. glucose tolerance test (GTT) (2 g/kg) and AUC. (K) Hepatic pck1 relative expression quantified by qPCR. Values represent the mean ± SEM with dot plots (n = 6–19). *P < 0.05, **P < 0.01, Student’s t test or 2-way ANOVA.

Figure 3

Figure 3. AR expression and activity in hypothalamic neurons of female mice.

(A) AR staining in the MBH. Original magnification, ×40 (scale bar: 100 μm). (B) High-magnification image of the inset in A. (C) AR protein levels in the ARC and (D) the VMH. Data are expressed relative to the number of AR-positive cells in females (n = 3–4). (E) Estrogen receptor α (esr1) and AR relative mRNA expression in the MBH quantified by qPCR following microdissection. (F) Electrophysiological recordings of action potentials of a representative DHT-inhibited neuron or (I) a DHT-activated neuron in the ARC of wild-type females following treatment with DHT. (G and J) Resting membrane potential (RMP) and (H and K) action potential frequency (AP) before and after application of DHT and following washout. (L) Biocytin staining of one representative recorded neuron in the ARC (scale bar: 100 μm) and an illustration of the localization of the recorded neurons. Values represent the mean ± SEM with dot plots (n = 15 mice or n = 10–17 neurons). *P < 0.05, ***P < 0.001, Student’s t test, 1-way ANOVA or Friedman test.

Figure 4

Figure 4. Chronic DHT excess predisposes female controls, but not NARKO–/– mice, to diabetes.

ARlox/lox or NARKO–/– mice were fed WD for 9 weeks followed by s.c. treatment with vehicle (V) or DHT for 4 weeks. (A) Fasting serum insulin levels. (B) Fed serum insulin levels. (C) Blood glucose represented as percentage decrease from baseline during an i.p. insulin tolerance test (ITT), with corresponding area under baseline (AUB). (D) Fasting blood glucose. (E) Random fed blood glucose. (F) Blood glucose during an i.p. glucose tolerance test (GTT) and corresponding AUC. Values represent the mean ± SEM with dot plots (n = 4–15). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test or 1-way or 2-way ANOVA.

Figure 5

Figure 5. Chronic DHT perfusion in the brain produces insulin resistance in female mice.

Female mice were fed WD for 9 weeks followed by i.c.v. infusion of vehicle (V) or DHT for 4 weeks. (A) Fasting serum insulin levels. (B) Fed serum insulin levels. (C) Blood glucose represented as an initial percentage decrease during an i.p. insulin tolerance test (ITT), with corresponding area under baseline (AUB). (D) Random fed blood glucose. (E) Fasting blood glucose. (F) Blood glucose during an i.p. glucose tolerance test (GTT) and corresponding AUC. Values represent the mean ± SEM with dot plots (n = 10–21). *P < 0.05, **P < 0.01, Student’s t test or 2-way ANOVA.

Figure 6

Figure 6. DHT enhances GSIS via an AR/cAMP/mTORC1 pathway.

(A) GSIS measured in static incubation in islets from mice fed a WD for 8 weeks and treated with vehicle (V) or DHT (10 nM) for 48 hours ex vivo. (B) Islet insulin content from A. Results represent 10 islets/condition in triplicate from n ≥ 3 independent experiments. (C) Glucose-stimulated insulin secretion (GSIS) measured after static incubation in islets from female human donors. (D) Islet insulin content from C. Islets were treated V, DHT (10 nM), and or flutamide (Flut, 10 μM) in vitro for 48 hours. Results represent 5 islets per condition of n = 6 independent experiments. (E) Basal and GSIS at the indicated glucose concentrations during a perifusion in WT female islets (vehicle, ethanol; DHT, 10 nM). Islets were isolated from mice at 14 weeks and perifused in batches of 60 per group. (F) Intracellular Ca2+ influx and corresponding AUC from the indicated glucose concentrations in isolated female mouse islets (n = 397 cells for V, n = 360 cells for DHT). (G) 10 nM DHT- and 10 nM Ex-stimulated cAMP concentrations monitored in female mouse islets infected with adenovirus harboring the FRET probe Epac2 camps, with corresponding AUC and representative live cell imaging. Scale bar: 20 μm. (H) GSIS measured in static incubation in islets from WT female mice fed chow or WD, treated with V, DHT (10 nM), rapamycin (RAPA, 10 nM), or DHT+RAPA for 48 hours (n = 12–14 batches of 10 islets/condition, 4 independent experiments). (I) GSIS measured in static incubation in islets from female WT and KD-mTOR mice, treated as in A. Results represent 10 islets per condition of n = 4 independent experiments. (J) WT female mice were exposed to WD for 4 weeks followed by 4 weeks of treatment with V, DHT, or DHT+RAPA (0.5 mg/kg/d; n = 7–8/group). Results represent the mean ± SEM, with dot plots of fasting and fed serum insulin as well as fasting and fed blood glucose. **P < 0.01, ***P < 0.001, 1-way ANOVA.

Figure 7

Figure 7. Chronic AR activation in β cells predisposes to oxidative stress.

(A) Oxygen consumption rate (OCR) measured from islets isolated from 10 female mice treated with vehicle (V) or DHT (10 nM) (n = 10 wells/condition). (B) Islet TBARS from 13-week-old mice from Figure 2 (n = 7–12). (C–H) Mice were treated s.c. with V or DHT for 1 week followed by induction of diabetes with a single high dose of streptozotocin (STZ) (n = 9–20). (C) Blood glucose measured for 8 days following STZ-induced diabetes and corresponding AUC. (D) Random-fed blood glucose (day 8). (E) Random fed insulin levels (day 8). (F) Insulin/glucose ratio. (G) β Cell mass quantification and IHC images of representative islets. Scale bar: 10 μm. (H) Pancreatic insulin concentration. Values represent the mean ± SEM with dot plots. *P < 0.05, **P < 0.01, 1-way ANOVA.

Figure 8

Figure 8. Proposed mechanism for the development of diabetes in female mice with chronic androgen excess.

In female mice exposed to a Western diet, chronic androgen excess predisposes to T2D through the combined activation of AR in the hypothalamus, producing hepatic insulin resistance, and activation of AR in β cells, increasing oxygen consumption and producing insulin hypersecretion, oxidative injury, and secondary failure.

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