1. Volume 23, Issue 5, Pages 837-851 (May 2016)
Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male 
Guadalupe Navarro, Weiwei Xu, David A. Jacobson, Barton Wicksteed, Camille Allard, Guanyi Zhang, Karel De Gendt, Sung Hoon Kim, Hongju Wu, Haitao Zhang, Guido Verhoeven, John A. Katzenellenbogen, Franck Mauvais-Jarvis 
Cell Metabolism 
Volume 23, Issue 5, Pages (May 2016)
DOI: /j.cmet
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Cell Metabolism 2016 23, 837-851DOI: (10.1016/j.cmet.2016.03.015)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Characterization of the βARKO−/y Mouse
(A) PCR showing the recombined 404 bp fragment of the Ar allele in islets from male βARKO−/y.
(B) Pancreas section showing AR immunofluorescent staining (red) in β cells colocalizing with insulin (green) in control mice and confirming successful AR deletion in βARKO−/y islets (the scale bar represents 10 μm).
(C) PCR showing recombination of Ar allele in hypothalamus.
(D–F) Immunohistochemistry staining (D) and quantification of AR immunoreactivity in (E) the ARC and (F) VMH of control and βARKO−/y mice. Results are representative of three to five mice.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2
Male βARKO−/y Mice Are Predisposed to β Cell Dysfunction and Glucose Intolerance
(A) Random fed blood glucose.
(B) Random fed serum insulin.
(C) Fasting blood glucose.
(D) Fasting serum insulin.
(E) i.p. GSIS (3 g/kg) with insulin area under the curve (AUC).
(F) i.p. GTT (2 g/kg) with glucose AUC (n = 12–15).
(G) β Cell mass quantification and representative pictures of islets stained with insulin (green) and glucagon (red) (the scale bar represents 10 μm).
(H) Pancreas insulin content.
(I) Random fed blood glucose following 9 weeks’ Western diet feeding.
(J) Fed insulin levels.
(K) Fed index of insulin deficiency (insulin/glucose).
(L) Fasting blood glucose.
(M) Fasting insulin levels.
(N) Fasting index of insulin deficiency.
(O) i.p. GSIS (3 g/kg) with insulin AUC.
(P) i.p. GTT (2 g/kg) with glucose AUC.
(Q) β Cell mass quantification and representative pictures of islets (the scale bar represents 10 μm).
(R) Pancreatic insulin content.
Data are from mice fed normal chow (A–H) or a Western diet for 9 weeks (I–R). Mice were studied at 12 weeks of age (n = 12–14), and pancreases were removed at 14 weeks of age. Values represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3 AR Deficiency in β Cells but Not in Neurons Alters GSIS
(A) Random fed blood glucose.
(B) Random fed serum insulin.
(C) Fasting blood glucose.
(D) Fasting serum insulin.
(E) i.p. GSIS (3 g/kg).
(F) i.p. GTT (2 g/kg) with glucose AUC.
(G) Random fed blood glucose following 9 weeks’ Western diet feeding.
(H) Fed insulin levels.
(I) Fasting blood glucose.
(J) Fasting insulin levels.
(K) GSIS (3 g/kg).
(L) GTT with glucose AUC. Data are from 20-week old βARKOMIP mice fed a Western diet for 8 weeks.
(M) i.p. GSIS (3 g/kg) with insulin AUC (n = 5–6).
(N) i.p. GTT with glucose AUC (n = 5–6).
Data are from 12 week-old NARKO−/y mice fed normal chow (A–F) or a Western diet for 9 weeks (G–L) (n = 12–15). Values represent the mean ± SEM. ∗p < 0.05.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4 Testosterone Enhances GSIS via AR in Male Islets
(A) GSIS measured in static incubation in islets from the indicated mice fed a normal chow and treated with vehicle or DHT (10−8 M) in vitro for 48 hr prior to static incubation.
(B) GSIS measured in static incubation in islets from mice fed a Western diet for 9 weeks and treated with vehicle or DHT (10−8 M) in vitro prior to static incubation.
(C) GSIS measured in static incubation in male human islets treated with vehicle, DHT, or flutamide.
(D) Islet insulin content from (A).
(E) Islet insulin content from (B).
(F) Islet insulin content from (C). Data in (A)–(F) are from 10 mouse islets or 5 human islet equivalents per condition (n = 6–8 independent wells). Human islet donors were two Caucasian males under 50 years of age with body mass index (BMI) between 25 and 27 kg/m2.
(G and H) GSIS during islet perifusion from (G) Rip-Cre mice and (H) βARKO−/y mice.
(I) Total AUC from GSIS (60–90 min).
(J) AUC from the first-phase GSIS (60–65 min).
(K) AUC from the second-phase GSIS (65–90 min). Islets were isolated from mice 12–14 weeks of age and perifused in batches of 60 islets per group.
Values represent the mean ± SEM. ∗p < 0.05, ∗∗p = 0.01.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 5 AR Extranuclear Location in Pancreatic β Cells
(A and B) Mouse pancreas section showing an islet with AR immunofluorescent staining (green) in β cells (A and B) colocalizing with insulin (red) (A) and DAPI (blue) merged images (the scale bar represents 10 μm).
(C) Human islet showing AR (green) expression (the scale bar represents 20 μm).
(D and E) Immunofluorescent staining of AR (green) in (D) LNCaP cell and (E) INS-1 cell treated with vehicle or DHT for 40 min and imaged by confocal microscopy (the scale bar represents 15 μm).
(F and G) LNCaP cells (F) and INS-1 cells (G) treated with DHT at the indicated time points, followed by subcellular fractionation. Top: representative immunoblots of AR, GAPDH (cytosolic marker), and Histone H3 (nuclear marker) expression. Middle: AR cytosolic and nuclear localizations were quantified by dividing AR expression by the expression of the respective markers. Bottom: AR relative nuclear translocation was calculated as the ratio of nuclear (N) over cytosolic (C) AR expression. N = 3 independent experiments.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 6 AR Amplifies GSIS in β Cells via cAMP Signaling
(A) Luciferase activity measured in LNCaP cells following a 6 hr treatment by DHT (10−8 M) and ADC (10−7 M) (n = 4 independent wells).
(B) PSA protein expression in LNCaP cells after 2 days of DHT and ADC treatment.
(C) GSIS measured in static incubation in WT male mouse islets treated with vehicle, DHT (10−8 M), flutamide (10−8 M), ADC (10−7 M), and ADC plus flutamide, dendrimer (concentration adjusted to ligand concentration in ADC) in vitro for 40 min. Results from two experiments (n = 6 independent wells).
(D) GSIS measured in static incubation in male human islets treated with vehicle, DHT (10−8 M), flutamide (10−8 M), ADC (10−7 M), and ADC plus flutamide, dendrimer in vitro for 40 min. Results from two experiments (n = 6 independent wells). Human donors were a male Caucasian aged 28 years with BMI 18.6 kg/m2 and a male Latino aged 61 years with BMI 25.8 kg/m2. The results are the average from two different donors and experiments.
(E) Effect of DHT on insulin secretion at low glucose and KCl (30 mM) during a 30 min stimulation (n = 6 independent wells).
(F) ATP concentration measured on lysates from WT male islets following stimulation with glucose and DHT for 30 min (n = 10 independent wells, at least three experiments).
(G) ATP levels measured on islets from male RIP-Cre and βARKO−/y islets following 30 min DHT stimulation and at 11 mM glucose. In (C)–(G), ten mouse islets were used per condition.
(H) Intracellular Ca2+ influx in isolated mouse islets from Rip-Cre and βARKO−/y mice (left) and corresponding AUC (right) from the indicated glucose concentrations.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Figure 7 Mechanism of AR Stimulation of GSIS in β Cells
(A) cAMP concentrations measured in the indicated male mouse islets stimulated with DHT (10−8 M) for 30 min. cAMP was measured by an enzyme-linked immunoassay. Results from three experiments (n = 6 independent wells).
(B) cAMP measured in WT male islets treated with vehicle, DHT (10−8 M), flutamide (10−8 M), and DHT plus flutamide, supplemented with 200 μM IBMX in each condition. Results from two experiments (n = 3 independent wells).
(C) GSIS measured in static incubation in islets from WT male mice treated with vehicle or DHT (10−8 M) in vitro for 48 hr and H89 (10 μM) 30 min prior to stimulation. Results from four experiments (n = 12 independent wells).
(D) Phosphorylation of CREB measured by western blotting in WT male islets treated with DHT for 10 and 30 min. Blots are representative of three experiments. Islets were isolated from mice 12–14 weeks of age. Values represent the mean ± SEM. ∗p < 0.05, ∗∗p = 0.01.
(E) GSIS measured in static incubation in WT male mouse islets (left) and male human islets (right) treated with vehicle, DHT (10−8 M), GLP-1 (10−8 M), or DHT plus GLP-1. Results from two experiments (n = 6 independent wells). Human donors were a male Latino aged 61 years with BMI 25.8 kg/m2 and a male Caucasian aged 53 years with BMI 33 kg/m2. The results are the average from two different donors and experiments.
(F) GSIS measured in static incubation in WT male mouse islets and male human islets treated with vehicle, DHT (10−8 M), exendin (9-39) (10−7 M), or DHT plus exendin (9-39). Results from two experiments (n = 6 independent wells). Human donor was a male Caucasian aged 53 years with BMI 33 kg/m2. The results are a representative experiment from two different donors.
(G) Proposed mechanism of AR stimulation of GSIS in β cells. Testosterone activation of AR in β cell indirectly activates a GPCR coupled with Gαs at the plasma membrane. This stimulates AC and cAMP production, leading to PKA activation, thus amplifying the glucose signal on insulin exocytosis.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions