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. Author manuscript; available in PMC: 2017 May 10.
Published in final edited form as: Cell Metab. 2016 Apr 28;23(5):837–851. doi: 10.1016/j.cmet.2016.03.015

Extranuclear actions of the androgen receptor enhance glucose-stimulated insulin secretion in the male

Guadalupe Navarro 2,8, Weiwei Xu 1,8, David A Jacobson 3, Barton Wicksteed 4, Camille Allard 1, Guanyi Zhang 5, Karel De Gendt 6, Sung Hoon Kim 7, Hongju Wu 1, Haitao Zhang 5, Guido Verhoeven 6, John A Katzenellenbogen 7, Franck Mauvais-Jarvis 1,2,*
PMCID: PMC4864089  NIHMSID: NIHMS774737  PMID: 27133133

Abstract

Although men with testosterone deficiency are at increased risk of type 2 diabetes (T2D), previous studies have ignored the role of testosterone and the androgen receptor (AR) in pancreatic β–cells. We show that male mice lacking AR in β-cells (βARKO) exhibit decreased glucose-stimulated insulin secretion (GSIS) leading to glucose intolerance. The AR agonist dihydrotestosterone (DHT) enhances GSIS in cultured male islets, an effect that is abolished in βARKO−/y islets and human islets treated with an AR antagonist. In β-cells, DHT-activated AR is predominantly extranuclear and enhances GSIS by increasing islet cAMP and activating the protein kinase A. In mouse and human islets, the insulinotropic effect of DHT depends on activation of the glucagon like peptide-1 (GLP-1) receptor and accordingly, DHT amplifies the incretin effect of GLP-1. This study identifies AR as a novel receptor that enhances β-cell function, a finding with implications for prevention of T2D in aging men.

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Introduction

Due to the recent dramatic increase in human life expectancy, men will spend a significant proportion of their lives in a state of testosterone deficiency. Against this backdrop, the impact of testosterone deficiency on development of visceral obesity and insulin resistance (IR) in men is well established (Basaria et al., 2006; Khaw and Barrett-Connor, 1992; Mauvais-Jarvis, 2011; Pitteloud et al., 2005; Zitzmann, 2009; Zitzmann et al., 2006). However, the role of testosterone deficiency in β-cell dysfunction remains unknown. This remarkable lack of knowledge is particularly intriguing because previous research has implicated low testosterone levels in the pathogenesis of T2D (Haffner et al., 1996; Oh et al., 2002; Stellato et al., 2000). Recently, an observational study from the Veterans Healthcare Administration showed that among prostate cancer patients, androgen deprivation therapy with GnRH agonists was associated with an increased risk of T2D (Keating et al., 2012). Another study showed marked hyperglycemia and decreased pancreatic β-cell function among prostate cancer patients after androgen-deprivation therapy (Inaba et al., 2005). Notably, testosterone deficiency has also been associated with impaired fasting glucose and glucose intolerance independently of obesity and the metabolic syndrome in men (Ho et al., 2013). Since development of hyperglycemia requires some degree of β-cell dysfunction, these observations, when considered together, raise the possibility that testosterone deficiency predisposes to β-cell failure in men.

Testosterone action is mediated by the androgen receptor (AR), a ligand-activated transcription factor. The extent to which the AR plays a role in β-cell failure in testosterone deficient males is unknown. Remarkably, we currently have no insight on the role of the AR in β-cell function in males. These issues are highly relevant to the health of aging men because novel antidiabetic androgen therapies that do not increase risk of prostate growth could have a substantial public health impact.

We investigated the role of the AR in β-cell function in the male using β-cell-specific AR knockout mice and cultured mouse and human islets. We show that the β-cell AR is important for testosterone potentiation of glucose-stimulated insulin secretion (GSIS) in male mice as well as in human islets. This AR-dependent pathway involves a rise in islet cAMP and the activation of protein kinase A, which amplifies the effect of GLP-1 on GSIS. We identify the AR as a physiological enhancer of β-cell function via the cAMP pathway, a finding that has clinical and pharmacological implications for the prevention of T2D in aging men.

Results

Generation of βARKO−/y mice

To explore the role of AR in β-cell function, we generated a β-cell-specific AR knockout (βARKO−/y) mouse using the Cre-loxP strategy and crossing ARlox/y mice with RIP-Cre transgenic mice. A previous report showed that mice expressing the RIP-Cre transgene developed glucose intolerance and impaired insulin secretion (Lee JY, 2006). It is therefore recommended that studies involving crosses between floxed mice and RIP-Cre mice use the RIP-Cre as the control group rather than the littermate floxed or wild type mice. We also observed that RIP-Cre mice were hyperglycemic compared to littermate ARlox/y mice and to a lesser extent compared to wild type mice (Fig. S1A & B). Thus, we used RIP-Cre mice as the control group. We confirmed recombination of the AR allele in islets from male βARKO−/y mice by the presence of the excised 404 bp fragment (Fig. 1A). Immunostaining for AR showed cytosolic AR expression in islet β-cells from control mice and confirmed elimination of the AR protein in islets from male βARKO−/y (Fig. 1B). Since gene manipulations using RIP-Cre transgenic mice are reported to promote recombination in nutrient neurons (Lee JY, 2006), we investigated whether recombination of AR had occurred in hypothalamic neurons of βARKO−/y mice. We observed recombination of the AR allele in hypothalamus of male βARKO−/y mice with the presence of the excised 404 bp fragment (Fig. 1C). Accordingly, male βARKO−/y mice displayed a significant decrease in AR protein expression in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) (Fig. 1D–F). We confirmed the presence of the non-recombined 952 bp AR allele in all other tissues of the βARKO−/y mice (Fig. S1C) where AR protein expression was not altered (Fig. S1D). Although AR expression was decreased in hypothalamus, βARKO−/y mice showed no alteration in food intake or body weight between 12 and 20 weeks of age (Fig. S1E–G).

Figure 1. Characterization of the βARKO−/y mouse.

Figure 1

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(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 co-localizing with insulin (green) in control mice and confirming successful AR deletion in βARKO−/y islets (the scale bar represents 10μm). (C) PCR showing recombination of AR allele in hypothalamus. (D) IHC staining and quantification of AR immunoreactivity in (E) the arcuate nucleus (ARC) and (F) ventromedial hypothalamus (VMH) of control and βARKO−/y mice. Results are representative of 3–5 mice.

Male βARKO−/y mice exhibit β-cell dysfunction and glucose intolerance

We assessed glucose homeostasis in male control and βARKO−/y mice at 12 weeks of age. This point is before the development of late onset obesity and insulin resistance observed in mice globally lacking AR (Fan et al., 2005) or selectively in neurons (Yu et al., 2013). On normal chow, βARKO−/y mice showed no alteration in fed or fasting blood glucose or in fed insulin levels (Fig. 2A–C). However, they showed decreased fasting insulin concentrations (Fig. 2D). Following an intraperitoneal (IP) glucose challenge, βARKO−/y mice exhibited decreased basal and GSIS (Fig. 2E) that resulted in glucose intolerance compared to controls (Fig. 2F). Despite their deficient insulin secretion, βARKO−/y mice had similar islet architecture, β-cell mass, and pancreatic insulin concentration as controls (Fig. 2G & H). Control and βARKO−/y mice also exhibited similar insulin sensitivity during an insulin tolerance test (ITT) (Fig. S2A), and had similar serum glucagon concentrations (183±36 and 169±27 pg/ml, controls and βARKO−/y, respectively, mean ±SE). To investigate whether AR deficiency in β-cells synergizes with a second β-cell stress in vivo to alter β-cell function, we induced metabolic stress in male control and βARKO−/y mice by feeding them a western diet for 9 weeks. After this challenge, βARKO−/y mice displayed reduced fasted and fed serum insulin concentrations compared to control mice (Fig. 2J & M) and developed hyperglycemia in both the fed and fasted states (Fig. 2I & L). As a result, the insulin deficiency index (insulin/glucose) was more pronounced in βARKO−/y mice (Fig. 2K & N). It followed that western diet-fed βARKO−/y mice showed decreased GSIS and developed glucose intolerance (Fig. 2O and P) relative to controls. However, following western diet feeding, βARKO−/y mice still showed no alteration in β-cell mass or pancreatic insulin concentrations (Fig. 2Q & R). In addition, βARKO−/y and control mice on western diets had similar insulin sensitivity following an ITT (Fig. S2B). Note that female islets exhibited lower AR expression than males (Fig. S2C & D), and female βARKO−/− mice showed no alteration in GSIS and GTT either on a normal chow or western diet (Fig. S2E–P). We then examined whether AR deficiency in male β-cells synergizes with additional β-cell stress induced by streptozotocin (STZ) to alter β-cell survival. We observed no increased predisposition to STZ in male βARKO−/y mice (Fig. S2Q–T). Thus, βARKO−/y mice developed altered GSIS, which was exacerbated following western diet feeding but without alteration in β-cell mass or increased predisposition to STZ-induced insulin-deficient diabetes.

Figure 2. Male βARKO−/y mice are predisposed to β-cell dysfunction and glucose intolerance.

Figure 2

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Data are from mice fed a normal chow (A–H) or a western diet for 9 weeks (I–R). (A) Random fed blood glucose. (B) Random fed serum insulin. (C) Fasting blood glucose. (D) Fasting serum insulin. (E) IP-GSIS (3 g/kg) with insulin area under the curve (AUC). (F) IP-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 10μm). (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) IP-GSIS (3 g/kg) with insulin AUC. (P) IP-GTT (2 g/kg) with glucose AUC. (Q) β-cell mass quantification and representative pictures of islets (the scale bar represents 10μm). (R) Pancreatic insulin content. Mice were studied at 12 weeks of age (n = 12-14) and pancreas were removed at 14 weeks of age. Values represent the mean ± SEM. * P < 0.05, ** P < 0.01.

AR deficiency in neurons does not alter β-cell function in male mice

Innervation of islet β-cells regulates insulin secretion by afferent signals arising from the hypothalamus (Ahren, 2000). To determine whether AR hypothalamic deletion contributes to the metabolic phenotype of male βARKO−/y mice, we generated a neuronal AR knockout mouse (NARKO−/y) by crossing ARlox/y mice with synapsin-Cre transgenic mice that selectively express Cre in neuronal cells (Schoch et al., 1996). We confirmed decreased AR expression in brains of male NARKO−/y mice with normal AR expression in other non-neuronal tissues (Fig. S3A & B). Male NARKO−/y mice showed no alteration in fasting and fed blood glucose or in insulin levels, observations that were similar when the mice were on normal chow (Fig. 3 A–D) or on a western diet (Fig. 3G–J). Importantly, unlike male βARKO−/y, male NARKO−/y mice showed no alteration in GSIS and retained similar glucose tolerance and insulin sensitivity compared to controls on both normal chow and western diet (Fig. 3E–F & 3K–L and Fig. S3C & D). To further eliminate the possibility that the defect in GSIS observed in male βARKO−/y mice derives from the partial AR hypothalamic deletion, we generated a second β-cell specific ARKO model (βARKOMIP) by crossing our ARlox mice with the MIP-CreERT1Lphi (MIP-CreERT) transgenic mouse that lacks Cre activity in the hypothalamus (Wicksteed et al., 2010). The MIP-CreERT transgenic mouse, however, has limitations because of transgene-driven expression of human growth hormone leading to decreased glucagon secretion and improved insulin sensitivity (Oropeza et al., 2015). Therefore, we used ARlox MIP-CreERT mice (without Tam injection) as controls of βARKOMIP. ARlox (with Tam) and ARlox MIP-CreERT (without Tam) displayed similar glucose tolerance (Fig. S3E). Tam-induced recombination was induced in adult ARlox MIP-CreERT mice to obtain βARKOMIP mice. Characterization of the βARKOMIP confirmed the selective AR deletion in β-cells (Fig. S3F), but βARKOMIP β-cells exhibited incomplete recombination leading to a 60% decrease in AR expression (Fig. S3G). Despite these limitations, upon exposure to a western diet, βARKOMIP mice exhibited decreased fasting insulin and blunted GSIS following an IP glucose challenge that resulted in glucose intolerance, compared to controls (Fig. 3M–N). The insulin sensitivity remained similar between the two groups (Figure S3H). Taken together, these observations confirm that elimination of AR in β–cells in mice produces a defect in GSIS leading to glucose intolerance.

Figure 3. AR deficiency in β-cells but not in neurons alters GSIS.

Figure 3

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Data are from 12 week-old NARKO−/y mice fed a normal chow (A–F) or a western diet for 9 weeks (G–L) (n = 12-15): (A) Random fed blood glucose. (B) Random fed serum insulin. (C) Fasting blood glucose. (D) Fasting serum insulin. (E) IP-GSIS (3 g/kg). (F) IP-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) IP-GSIS (3g/kg) with insulin AUC (n= 5-6). (N) IP-GTT with glucose AUC (n= 5-6). Values represent the mean ± SEM. * P < 0.05.

AR deficiency in β-cells alters GSIS from male islets

To determine whether the altered insulin secretion of βARKO−/y mice was an islet-cell autonomous effect, we studied GSIS in static incubation in cultured islets from male control and βARKO−/y mice that were fed normal chow. Consistent with the importance of AR in GSIS observed in vivo, at 16.7 mM glucose, control islets exposed to the natural AR agonist dihydrotestosterone (DHT) showed increased GSIS compared to those exposed to vehicle. The stimulatory effect of DHT on GSIS was abolished in βARKO−/y islets (Fig. 4A). Notably, in all groups of mice, only a minor increase in GSIS was observed when glucose was increased from 2.8 to 16.7 mM, probably as a consequence of the deleterious effect of the RIP-Cre transgene on islet function (Lee JY, 2006). Islets from control and βARKO−/y mice fed a western diet showed a modest and non-significant increase in GSIS over basal, which probably resulted from the combined deleterious effect of the RIP-cre transgene and western diet on islet function (Fig. 4B). However, at 16.7 mM glucose, control islets exposed to DHT showed increased GSIS compared to vehicle-exposed islets (Fig. 4B). Importantly, compared to control islets, βARKO−/y islets exhibited decreased insulin secretion at both 2.8 and 16.7 mM glucose (Fig. 4B).

Figure 4. Testosterone enhances GSIS via AR in male islets.

Figure 4

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(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 hours 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 IEQ per condition (n = 6–8 independent wells). Human islet donors were two Caucasian males under 50 years of age and with a BMI between 25 and 27. (G) GSIS during islet perifusion from Rip-Cre mice and (H) βARKO−/y mice. (I) Total AUC from GSIS (60 to 90min). (J) AUC from the 1st phase GSIS (60 to 65min). (K) AUC from the 2st phase GSIS (65 to 90min). Islets were isolated from 12-14 weeks old mice and perifused in batches of 60 islets per group. Values represent the mean ± SEM. * P < 0.05, **P = 0.01.

We translated these findings to male human islets. Exposure of human islets to a physiological concentration of DHT enhanced insulin secretion in the presence of 16.7 mM glucose, an effect that was abolished by antagonizing AR with flutamide (Fig. 4C). Neither DHT, nor AR genetic elimination, nor pharmacological inhibition affected islet insulin content in mouse or human islets (Fig. 4D–F). To study dynamic insulin secretion in vitro, male control and βARKO−/y islets were placed in a perifusion system. In this setting, insulin secretion in response to glucose is characterized by a bi-phasic pattern (Lacy et al., 1972). The first phase is a rapid and marked, but transient elevation in the secretory rate. The second phase is characterized by a gradual increase in secretion that lasts as long as the glucose stimulus is present. Following DHT stimulation, control islets exhibited enhanced first and second phase insulin secretion compared to vehicle treatment (Fig.4 G and I–K). In contrast, the increased first phase was not observed in βARKO−/y islets (Fig. 4H and I–K). Further, βARKO−/y islets exhibited aberrant early second phase-insulin secretion (Fig.4H) and lower global insulin secretion than control islets (Fig. 4I–K).

Extranuclear AR actions enhance GSIS

The AR is a classical ligand-activated nuclear receptor that regulates the expression of target genes through binding to an androgen response element on the promoter of target genes (Chang et al., 1988; Lubahn et al., 1988; Tilley et al., 1989). Nongenomic actions of AR are thought to account for rapid, transcription-independent effects of androgens (Matsumoto et al., 2013). However, these nongenomic androgen effects have been observed only in vitro and await functional validation in animal models. In mouse prostate – a classical androgen target tissue – AR immunohistochemical staining showed nuclear localization (Fig S4A). In male mouse pancreatic islets and in male human islets; however, AR colocalized with insulin in a predominant extranuclear localization (Fig. 5A & C and Fig S4B) and was not observed in α-cells (Fig. 5B). We studied AR subcellular localization by confocal microscopy following binding of DHT in human prostate adenocarcinoma LNCaP cells — a classical model of AR nuclear actions — and in INS-1 rat insulin-secreting cells. In the absence of DHT, AR signal was predominantly extranuclear in LNCaP and INS-1 cells. As expected, upon DHT stimulation, AR underwent nuclear translocation in LNCaP cells (Fig. 5D). In contrast, following DHT stimulation, AR remained predominantly in the extranuclear compartment of INS-1 cells (Fig. 5E). Similar findings were observed in the MIN-6 mouse insulin-secreting cells (Fig S4C). We next studied AR subcellular localization by subcellular fractionation. Confirming results obtained by microscopy, in LNCaP cells, DHT produced a robust nuclear translocation of AR starting at 20 min and sustained for at least 8h (Fig. 5F). In contrast, in INS-1 cells, DHT produced a weak nuclear translocation of AR and the activated AR remained mainly in the cytosolic fraction (Fig. 5G).

Figure 5. AR extranuclear location in pancreatic β-cells.

Figure 5

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(A–B) Mouse pancreas section showing an islet with AR immunofluorescent staining (green) in β-cells colocalizing with insulin (red) and DAPI (blue) merged images (the scale bar represents 10μm). (C) Human islet showing AR (green) expression (the scale bar represents 20μm). Immunofluorescent staining of AR (green) in (D) LNCaP cell and (E) INS-1 cell treated with Vehicle or DHT for 40 minutes and imaged by confocal microscopy (the scale bar represents 15μm). (F) LNCaP cells and (G) INS-1 cells treated with DHT at the indicated time points, followed by subcellular fractionation. Upper panels show representative immunoblots of AR, GAPDH (cytosolic marker), Histone H3 (nuclear marker) expression. Middle panels: AR cytosolic and nuclear localizations were quantified by dividing AR expression by the expression of the respective markers. Lower panels: AR relative nuclear translocation was calculated as the ratio of nuclear (N) over cytosolic (C) AR expression. N = 3 independent experiments.

Mechanism of AR potentiation of GSIS in pancreatic β-cells

Having established that ligand-activated AR exhibits a preferential extranuclear location in β-cells, we sought to determine whether this extranuclear location is instrumental in stimulating GSIS. To this end, we synthesized a novel androgen dendrimer conjugate (ADC) that selectively activates extranuclear AR signaling pathways, but remains outside the nucleus (Table S1 and Fig S5A–D). We successfully used a similar estrogen dendrimer conjugate to validate the function of extranuclear estrogen receptors in β-cell function and survival (Tiano and Mauvais-Jarvis, 2012a; Tiano et al., 2011; Tiano and Mauvais-Jarvis, 2012b, c; Wong et al., 2010). We confirmed that unlike DHT, ADC 1) cannot increase AR-dependent gene transcription in cells transfected with a reporter construct containing an androgen response element (ARE) (Fig. 6A), and 2) cannot increase the expression of the prostate specific antigen, an AR target gene containing an ARE (Zhu et al., 2003) (Fig. 6B). In contrast, in cultured mouse and human islets, selective extranuclear activation of AR using ADC was as efficient as DHT in enhancing GSIS (Fig. 6C and D).

Figure 6. AR amplifies GSIS in β-cells via cAMP signaling.

Figure 6

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(A) Luciferase activity measured in LNCaP cells following a 6 hours treatment by DHT (10−8 M) and ADC (10−7 M) (n=4 independent wells). (B) PSA protein expression inn 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 minutes. Results from 2 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 minutes. Results from 2 experiments (n= 6 independent wells). Human donors were a male Caucasian aged 28 with BMI 18.6 and a male Latino aged 61 with BMI 25.8. The results were the average from 2 different donors/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 3 experiments). (G) ATP levels measured on islets from male RIP-Cre and βARKO−/y islets following 30 min DHT stimulation and at 11mM glucose. In panels (C–G) 10 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.

The observation that ligand-activated extranuclear AR stimulates GSIS in β-cells led us to hypothesize that in these cells, DHT-activated AR interacts with membrane or cytosolic molecules to enhance GSIS. GSIS is triggered by glucose metabolism that leads to increased ATP/ADP ratio, the closure of ATP sensitive K+ channels that is followed by membrane depolarization, opening of Ca2+ channels, and influx of intracellular calcium [Ca2+]i (Jones et al., 1985). Using male WT islets exposed to KCl at low glucose to depolarize the cell membrane, we investigated whether AR acts through KATP channels and membrane depolarization independently of glucose metabolism. As expected, KCl-treated islets showed increased insulin secretion (Fig. 6E). However, we observed no further effect of DHT on insulin secretion, an observation demonstrating that AR does not potentiate insulin secretion triggered by depolarization alone (Fig. 6E). We next investigated whether AR activation could stimulate glucose metabolism (and therefore ATP generation) and subsequent [Ca2+]i influx. Increasing glucose concentration from 2.8 to 11 and 16.7 mM increased ATP concentration in male WT mouse islets (Fig. 6F). However, exposure to DHT did not result in a further increase in ATP concentration in these islets (Fig. 6F). Similar results were obtained when we quantified the ATP/ADP ratio (Fig S5E). Consistent with these findings, at 11mM glucose, DHT did not significantly increase ATP concentration in control or βARKO−/y islets (Fig. 6G). However, it is important to stress that βARKO−/y islets displayed decreased ATP concentration compared to control islets (Fig. 6G).

We then explored whether DHT could increase [Ca2+]i influx. Consistent with results for ATP concentration, raising glucose concentration increased [Ca2+]i influx in both control and βARKO−/y islets (Fig. 6H). However, consistent with results for ATP content, DHT did not further increase [Ca2+]i in control or βARKO−/y islets (Fig. 6H). Notably, at high glucose, as in the case of ATP concentration, βARKO−/y islets exhibited decreased [Ca2+]i compared to control islets (Fig. 6H). Thus, DHT increased insulin secretion without increasing glucose metabolism or [Ca2+]i influx. We interpreted the decreased ATP and [Ca2+]i in βARKO−/y islets as consequences of glucose desensitization leading to decreased glucose metabolism and ATP production. Indeed, genes involved in dedifferentiation of β-cells secondary to hyperglycemia (GK, GLUT2) were downregulated in glucose intolerant male βARKO−/y islets compared to controls. In contrast, these genes were not decreased in islets from normoglycemic female βARKO−/− mice (Table S2).

The finding that DHT acting on AR enhanced GSIS without increasing cellular ATP generation or [Ca2+]i influx suggested to us that AR activation amplifies GSIS by acting downstream and augmenting the Ca2+ signal. Incretins like glucagon-like peptide-1 (GLP-1) potentiate GSIS by increasing β-cell concentrations of cyclic adenosine monophosphate (cAMP) (Lin and Haist, 1973). We explored the effect of DHT on cAMP production in islets from male mice. In control male islets, DHT increased cAMP production by approximately two fold (Fig. 7A). In contrast, the effect of DHT in increasing cAMP levels was not observed in βARKO−/y islets (Fig. 7A) or in WT islets treated with the AR antagonist flutamide (Fig. 7B), an observation demonstrating its dependence on AR. Note that in these experiments, DHT-induced cAMP production was independent from phosphodiesterase (PDE) inhibition since DHT increased cAMP concentration over vehicle in islets preincubated with PDE inhibitors. An increase in intracellular cAMP concentration activates the cAMP-dependent protein kinase A (PKA) (Nesher et al., 2002). To determine whether DHT-mediated potentiation of GSIS was dependent on PKA activation, we used H-89, the PKA inhibitor. While DHT potentiated GSIS in cultured WT male islets, the DHT effect was ablated in the presence of H-89 (Fig. 7C). Further, activation of PKA leads to phosphorylation of the cAMP response element-binding protein (CREB). In male mouse islets, DHT exposure produced a rapid phosphorylation of CREB (Fig. 7D). Thus, AR activation by DHT potentiates GSIS in a cAMP and PKA-dependent manner.

Figure 7. Mechanism of AR stimulation of GSIS in β-cells.

Figure 7

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(A) cAMP concentrations measured in the indicated male mouse islets stimulated with DHT (10−8M) for 30 min. cAMP was measured by an enzyme-linked immunoassay. Results from 3 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 200uM IBMX in each condition. Results from 2 experiments (n = 3 independent wells) (C) GSIS measured in static incubation in islets from WT male mice treated with vehicle or DHT (10−8M) in vitro for 48 hours and H89 (10μM) 30 minutes prior to stimulation. Results from 4 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 3 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 2 experiments (n= 6 independent wells). Human donors were a male Latino aged 61 with BMI 25.8 and a male Caucasian aged 53 with BMI 33. The results were the average from 2 different donors/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 2 experiments (n= 6 independent wells). Human donor was a male Caucasian aged 53 with BMI 33. The results were a representative experiment from 2 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.

Interestingly, the insulinotropic effect of DHT observed in mouse and human islets was not observed in INS-1 cells (Fig S6A) despite the fact that INS-1 cells express AR (Fig. 5), suggesting that the insulinotropic effect of DHT requires a secreted factor, produced by islet non-β-cells, that acts on β-cells in a paracrine manner. Therefore, we explored the hypothesis that AR action in β-cells amplifies GLP-1R signaling to increase cAMP production, thus potentiating the insulinotropic action of GLP-1. We reasoned that because GLP-1 is produced by cultured islet α-cells (Liu et al., 2011; Marchetti et al., 2012), DHT enhances GSIS in cultured islets (which produce GLP-1) but not in cultured INS-1 cells (which do not produce GLP-1). Consistent with this hypothesis, in mouse and human islets (Fig. 7E) and in INS-1 cells (Fig S6B), DHT amplified the insulinotropic effect of GLP-1 on GSIS. To explore the possibility that DHT amplification of GSIS requires a functional GLP1-R, we studied the effect of DHT in the presence of the selective GLP1-R antagonist exendin (9-39) and in absence of exogenous GLP-1 (Goke et al., 1993). The effect of DHT in amplifying GSIS was abolished in the presence of exendin (9-39) (Fig. 7F). Together, these studies demonstrate that DHT amplifies the insulinotropic effect of exogenous and islet-derived GLP-1 and this effect requires a functional GLP-1R.

Discussion

To study the role of testosterone in β-cells in vivo, we generated a mouse with conditional elimination of AR in these cells. Although male βARKO−/y mice also exhibit decreased AR expression in hypothalamus, the insulin-secretory defect observed in these mice results from the loss of AR in β-cells. This is supported by the following evidence: First, abnormalities observed in male βARKO−/y mice are reproduced in isolated male βARKO−/y islets and in human islets exposed to an AR antagonist, demonstrating that this defect is secondary to the loss of AR in the islets. Second, the insulin-secretory defect observed in male βARKO−/y mice is not observed in male mice that selectively lack AR in neurons. Finally, and most importantly, a second βARKOMIP mouse exhibiting selective and inducible β-cell AR elimination in adulthood recapitulates the impaired GSIS leading to glucose intolerance.

A critical finding of this study is that AR deficiency in β-cells of male mice impairs GSIS which produces glucose intolerance because activation of β-cell AR is required to enhance both first and second phase GSIS. Importantly, the insulinotropic function of the AR is present in human islets at physiological concentrations of testosterone. Together, these observations suggest that (1) testosterone is necessary for normal GSIS in men, and (2) men with androgen deficiency display a deficit in GSIS that predisposes them to T2D.

Early studies showing binding of androgen to a nuclear protein in prostate (Bruchovsky and Wilson, 1968) followed by the cloning of the AR and analysis of its structure (Chang et al., 1988; Lubahn et al., 1988; Tilley et al., 1989) led to the establishment of a paradigm in which AR acts as a nuclear ligand-activated transcription factor. Indeed, in prostate, AR is maintained in the cytosol in an inactive complex by heat-shock proteins Upon ligand binding, AR homodimerizes and translocates to the nucleus (Prescott and Coetzee, 2006). In β-cells, however, AR is mostly localized in an extranuclear compartment where it remains sequestrated following androgen stimulation. When male cells are permanently exposed to DHT in vivo, AR is localized in the nucleus of prostate cells. In contrast, under the same conditions, AR is observed in the extranuclear compartment of islet β-cells. The nongenomic actions of AR are thought to account for the rapid, transcription-independent effects of androgens (Matsumoto et al., 2013). However, to date, these nongenomic effects of androgens have been observed only in vitro and therefore await validation in vivo in animal models.

We previously described novel extranuclear actions for estrogen receptors (ERs) in β-cells (Tiano and Mauvais-Jarvis, 2012a; Tiano et al., 2011; Tiano and Mauvais-Jarvis, 2012b, c; Wong et al., 2010). Unlike the nuclear ER that acts as a ligand-activated transcription factor in breast and uterine cells, extranuclear ERs protect pancreatic islet β-cell function and survival via cytosolic interactions with kinases and transcription factors. The current study provides the first evidence of rapid androgen action via an extranuclear AR involved in the pathophysiology of insulin secretion. This novel androgen action is observed at physiological concentration of the hormone, is validated in vivo, and most importantly, it is found in human tissue.

In β-cells, GSIS is driven by glucose metabolism that generates ATP (Ashcroft, 1980) and triggers [Ca2+]i influx. Testosterone activation of the AR increases GSIS independently of increases in glucose metabolism and [Ca2+]i influx. Rather, AR activation increases GSIS from β-cells by producing cAMP and activating the cAMP-dependent PKA pathway. Consistent with AR signaling via a PKA pathway, transgenic mice with enhanced PKA catalytic activity in pancreatic islets (Kaihara et al., 2013; Song et al., 2011) exhibit increased GSIS but — like male βARKOy/− mice — show no change in β-cell mass or insulin synthesis. A previous report suggested that testosterone stimulates islet insulin mRNA and content (Morimoto et al., 2001). However, we found no evidence of AR stimulation of insulin synthesis. Because the authors used testosterone (which is converted into estrogen), the effect they described was likely due to testosterone aromatization to estrogen acting on ERs (Wong et al., 2010).

Incretins, like GLP-1 and exendin 4, restore first-phase and enhance second phase insulin release in humans with T2D (Egan et al., 2002; Fehse et al., 2005). Most of these incretin effects in β-cells require activation of the cAMP-dependent PKA pathway via the G protein-coupled receptor (GPCR) of GLP-1 (Drucker and Nauck, 2006), which activates the adenylate cyclase (AC) to trigger cAMP production. We observe that in cultured mouse and human islets, the insulinotropic effect of testosterone is abolished by pharmacological inhibition of the GLP-1R in absence of exogenous GLP-1, demonstrating that the AR requires an active GLP-1R to enhance GSIS and enhances the effect of islet-produced GLP-1. Accordingly, AR activation by testosterone also amplifies the insulinotropic effect of exogenous GLP-1 in these islets. Therefore, the testosterone-AR pathway could act as an incretin sensitizer in β-cells. In the future, the effect of testosterone in amplifying the insulinotropic action of other ligands of Gs-linked GPCR, like glucagon or glucagon inhibitory polypeptide, deserves investigation. Interestingly, cultured islets from βARKO−/y mice previously exposed to a western diet secrete less insulin than controls. This suggests that AR is also necessary for islet adaption to metabolic stress. Further studies are also needed to address this issue. The mechanism through which we propose AR stimulates GSIS in male β-cells is summarized Fig.7G.

The biological basis for androgen stimulation of insulin secretion and the integration of androgenic and metabolic signals in males is likely to be anabolic since both testosterone and insulin are anabolic hormones. In contrast to males, AR deficiency in β-cells of female mice does not alter GSIS. Females have lower AR expression in β-cells compared to males, an observation that likely promotes weaker androgen signaling (Visakorpi et al., 1995). In addition, females exhibit lower serum and tissue androgen concentrations than are necessary to activate the AR (Mauvais-Jarvis, 2011). We therefore interpret the absence of phenotype of βARKO−/− female mice as a consequence of the evolution of females' lower dependence on AR activation and signaling.

This study has clinical ramifications. Selective androgen receptor modulators (SARMs) are a novel class of androgen receptor ligands. The goal of SARMs is to provide androgen therapy for age-related functional decline with customized anabolic activity on muscle and bone, but without androgenic action in the prostate (Mohler et al., 2009). Our work suggests that androgen deficiency-induced T2D is at least partially due to a loss of androgen stimulation of GSIS in β-cells. Designing SARMs with AR agonistic activity in β-cells could represent a novel strategy to prevent androgen deficiency-related glucose dysregulation in men.

In conclusion, AR action is required in males' β-cells for GSIS. This study identifies the AR as a novel β-cell receptor and enhancer of β-cell function via the cAMP-dependent pathway and has important clinical and pharmacological implications for prevention of T2D in aging men.

Materials and Methods

Generation of mutant mice

The βARKO−/y mouse was generated by crossing mice carrying the AR gene with floxed exon 2 on their X chromosome (ARlox) with transgenic mice overexpressing the Cre recombinase under control of the RIP promoter (RIP-Cre, Jackson Laboratory). Generation and characterization of ARlox−/− have been described (De Gendt et al., 2004). NARKO−/− mice were generated by crossing ARlox+/− with the Syn-Cre+/− mice (Jackson Laboratory) as described (Yu et al., 2013). To generate βARKOMIP mice we crossed ARflox mice with the Ins1-Cre/ERT (MIP-Cre+/−) transgenic mouse (Jackson Lab). We induced Tamoxifen (Tam) inactivation of AR after puberty and following an 5 day treatment with Tam (75mg/Kg). All studies were performed with the approval of Northwestern University and Tulane University Animal Care and Use Committees in accordance with the NIH Guidelines.

Western Diet

Mice were weaned onto a customized diet designed to be high in saturated fat and simple sugars (sucrose and fructose) to mimic a western diet (30% AMF; 14.9% Kcal protein, 33.2% Kcal carbohydrates, 51.9% Kcal fat; Harlan Teklad) for 9 weeks.

Metabolic studies

Blood glucose was measured from tail vein blood using a One Touch Ultra glucometer (Lifescan). Insulin (Linco Research) and glucagon (ALPCO) were measured in serum by ELISA. For IP-GTT (2 g/kg) and GSIS (3 g/kg), mice were fasted overnight before glucose injection. For IP-ITT, mice were fasted for 6 hrs prior to insulin injection (0.75 U/kg). Pancreas insulin concentration was measured from acid ethanol extract as described (Tiano et al., 2011).

Immunohistochemistry and β-cell mass quantification

Insulin and glucagon staining as well as β-cell mass measurement from pancreas sections were performed as described (Tiano et al., 2011). For AR staining of human islets, and islets from βARKO−/y, βARKOMIP and their respective controls, sections were incubated with primary antibody rabbit anti-AR (PG-21, 1:100, Millipore). Secondary antibody goat biotinylated anti-rabbit (1:200; Linco) and Alexa 568 tyramide signal amplification kit (TSA, Molecular Probes) was used for signal amplification. For AR staining in the hypothalamus, tissues were fixed in 10% formalin at 4 °C and stored in 30% sucrose until sectioning in 20 μm sections. Sections were incubated with primary antibody anti-AR (N20, 1:250, Santa Cruz). Secondary goat biotinylated anti-rabbit antibody was visualized using the VECTASTAIN Elite ABC kit (Vector Laboratories). Images were captured at ×20 magnification using a fluorescent microscope (Nikon Eclipse E400). LNCaP and INS-1 cells were treated with vehicle or DHT (10−8 M) for 40 minutes, followed by fixation in 4% paraformaldehyde. LNCaP cells were incubated in the anti-AR antibody (N20, 1:200, Santa Cruz), and then in the goat anti-rabbit secondary antibody (1:400). INS-1 cells were incubated in the anti-AR antibody (N20, 1:200, Santa Cruz). The signal was amplified using TSA. The images were taken using a Nikon A1 confocal microscope.

Subcellular fractionation

The LNCaP and INS-1 cells were treated with DHT for 20 minutes, 40 minutes, 1 hour, 3 hours, and 8 hours. Subcellular fractionation was performed by first extracting the cytosolic proteins with dilution buffer, followed by extracting the nuclear protein fractions with lysis buffer. Cytosolic and nuclear protein fractions were normalized to GAPDH and Histone H3 respectively.

Islet isolation and insulin secretion in static incubation

Islet isolation was performed following pancreatic duct injection with collagenase as described (Tiano et al., 2011). For measurement of insulin secretion, islets were hand-picked under a dissection microscope, and treated with DHT (10−8 M; Steraloids), or vehicle (95% ethanol) for 48 hrs. Insulin release from islets was measured as described (Tiano et al., 2011). For experiment with inhibitors, islets were treated with flutamide (10−8 M; Sigma-Aldrich, St. Louise, MO) or H-89 (10μM;Cell Signaling).

Islet perifusion

A perifusion system (Biorep Technologies) was used to determine the insulin biphasic response. Briefly, batches of 60 mouse islets were perifused at 37 °C, at a flow of 100 μL/min. Islets were first equilibrated for 60 min with KRB solution containing 2.8 mM glucose, then stimulated for 30 min with KRB solution contained either DHT (10−8 M) or vehicle (95 % ethanol) and 16.7 mM glucose. Samples were collected in a 96-well plate and insulin concentration was determined by ELISA (Millipore).

Luciferase assay

LNCaP cells were transfected with ARR3-tk-luciferase reporter, containing three repeats of androgen response element(ARE)s in tandem, upstream of the minimal tk enhancer fused to the luciferase reporter (Snoek et al., 1996), or control plasmids containing renilla luciferase reporter gene using TurboFect transfection reagent (Thermo Scientific) and 48 hours prior to the treatment. On the experiment day, cells were lysed, and dual-luciferase reporter assay system (Promega) was used to measure firefly and renilla luciferase activity sequentially. The ratio of firefly and renilla luciferase reading was calculated to indicate the ability of DHT and ADC to activate ARE-mediated luciferase expression.

ATP and cAMP measurements

Intracellular ATP concentrations were measured in 10 islets per condition treated with either vehicle or DHT (10−8 M) for 30 minutes using EnzyLight™ ATP assay and ADP assay (BioAssay Systems) according to the manufacturer's instructions. Islets were lysed to release ATP and ADP, and luminescence was measured on a luminometer (BioTek) and quantified to ATP and ADP standards. cAMP levels were determined in mouse islets pre-treated with vehicle, DHT (10−8 M), flutamide (10−8M), or DHT plus flutamide for 30 min in the presence of 200uM 3-isobutyl-1-methylxanthine (IBMX). Islets were lysed, and the supernatant was collected to measure the intracellular cAMP level with cyclic AMP XP® Assay Kit (Cell Signaling) according to the manufacturer's instructions.

Measurement of cytoplasmic calcium and perifusion

Islet [Ca2+]i was measured with the Ca2+ sensitive dye fura-2 acetoxymethyl ester (Molecular Probes) as described (Jacobson et al., 2007). Mouse islets were plated on coverslips and dye-loaded with fura-2. Fluorescence imaging was performed using a Nikon Eclipse TE2000-U microscope equipped with an epifluorescent illuminator (Sutter instruments), a CoolSNAP HQ2 camera (Photometrics) and Nikon Elements software (Nikon). The [Ca2+]i ratios of emitted fluorescence intensities at excitation wavelengths of 340 and 380 nm (F340/F380) were determined every 5 s with background subtraction. A perifusion system (Biorep Technologies) containing DHT (10−8 M) or vehicle (95 % ethanol) 2.8 and 16.7 mM glucose was used to determine biphasic response.

Statistical analysis

Results are presented as mean ± SEM as specified in figures. All statistical analyses were performed using the unpaired Student's t test. A P value less than 0.05 was considered statistically significant. * P<0.05, ** P<0.01.

Supplementary Material

1
2

Highlights.

  • Male β-cell ARKO mice exhibit decreased glucose-stimulated insulin secretion (GSIS)

  • Testosterone enhances GSIS from cultured male mouse and human β-cells via AR.

  • The AR is extranuclear in β-cells and enhances GSIS in a cAMP-dependent manner.

  • The activated AR amplifies the insulinotropic effect of glucagon-like peptide-1.

Acknowledgments

Human islets were provided by the Integrated Islet Distribution Program (IIDP) funded by the National Institute of Diabetes and Digestive and Kidney Diseases and with support from the Juvenile Diabetes Research Foundation International. This work was supported by grants from the National Institutes of Health (DK074970, HD044405), the American Heart Association (11IRG5570010) and the American Diabetes Association (7-13-BS-101) to F.M.J. and in part from the LA CaTS Center grant 1 U54 GM104940. G.N. was supported in part by NIH Training Grant T32 DK007169. D.J. was supported by NIH grant DK097392. B.W. was supported by NIH grant DK085129. J.A.K. and S.H.K. were supported by NIH grant DK015556.

Footnotes

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Author Contribution F.M.J. Conceived the idea, designed experiments, analyzed the data, and wrote/edited the manuscript. G.N. and W.X. equally contributed in designing and performing experiments, analyzing data and writing of the manuscript. Specifically, G.N. performed experiments related to βARKO−/y and NARKO−/y mice, islet perifusion experiments and experiments of GSIS in static incubation in βARKO−/y mouse and human islets and measurements of ATP concentration and cAMP in islets. WX performed experiments in βARKOMIP mice, experiments of characterization of ADC extranuclear action in mouse and human islets, cAMP and ATP measurements in islets and experiments of characterization of AR and GLP-1 stimulation of GSIS in mouse and human islets. D.A.J. performed experiments of intracellular Ca2+ influx in isolated islets. B.W. performed western blotting experiment of CREB phosphorylation. C.A. performed experiments of βARKOMIP phenotypical characterization. G.Z. and H.Z. provided technical expertise in performing subcellular fractionation experiments and characterization of ADC extranuclear actions. K.D.G. and G.V. generated and provided the AR lox/lox mouse. S.H.K. and J.A.K. synthesized and provided ADC. H.W. provided reagents and participated in the design of in vitro experiments.

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Supplementary Materials

1
NIHMS774737-supplement-1.pdf (3MB, pdf)
2
NIHMS774737-supplement-2.docx (62.4KB, docx)

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