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Loss of nuclear and membrane estrogen receptor-α differentially impairs insulin secretion

and action in male and female mice.

Camille Allard1, Jamie J. Morford

1,2,3, Beibei Xu

1, Benjamin Salwen

1, Weiwei Xu

1, Lucie

Desmoulins4, Andrea Zsombok

3,4, Jason K. Kim

5, Ellis R. Levin

6,7, and Franck Mauvais-

Jarvis1,2,3,8*

1Section of Endocrinology and Metabolism, Department of Medicine, Tulane University Health

Sciences Center, New Orleans, LA,

2Neuroscience Program, Tulane University, New Orleans, LA

3Brain Institute, Tulane University, New Orleans, LA,

4Department of Physiology, Tulane University Health Sciences Center, New Orleans, LA

5Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of

Massachusetts Medical School, Worcester, MA.

6Department of Medicine and Biochemistry, University of California, Irvine, CA

7Long Beach VA Medical Center, CA

8Southeast Louisiana Veterans Healthcare Medical Center, New Orleans, LA,

*Corresponding author: Franck Mauvais-Jarvis, Department of Medicine, Section of

Endocrinology and Metabolism, Tulane University Health Sciences Center, 1430 Tulane Avenue,

New Orleans, LA 70112, USA, Tel.: +1 504 988 5990; fax: +504 988 6271,

[email protected]

Short running title: ERα and sex differences in glucose homeostasis

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Keywords: Estrogen Receptor Alpha, Extra-nuclear signaling, Insulin Secretion, Insulin

Sensitivity, Sex dimorphism

Abbreviations:

E2: 17β-estradiol

EndoRa: Endogenous glucose production

ERα: Estrogen receptor alpha

ERE: Estrogen response element

GIR: Glucose infusion rate

GSIS: Glucose-Stimulated insulin secretion

Gt: Glucose turnover

GTT: Glucose tolerance test

HGP: Hepatic glucose production

ICV: intracerebroventricular

I.P.: intra-peritoneal

ITT: Insulin tolerance test

MOER: Membrane-Only ERα

NOER: Nucleus-Only ERα

PTT: Pyruvate tolerance test

CTRL: Control

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ABSTRACT

Estrogens favor glucose homeostasis primarily through the estrogen receptor alpha (ERα), but

the respective importance of nuclear and membrane ERα pools to glucose homeostasis are

unknown. We studied glucose homeostasis, insulin secretion and insulin sensitivity in male and

female mice expressing either the nuclear ERα (NOER) or the membrane ERα (MOER). Male

and female MOER mice exhibited fasting and fed hyperglycemia and glucose intolerance.

Female MOER mice displayed impaired central insulin signaling associated with

hyperinsulinemia and insulin resistance due to unrestrained hepatic gluconeogenesis, without

alterations in glucose stimulated-insulin secretion (GSIS). In contrast, male MOER mice did not

exhibit detectable insulin resistance, but showed impaired GSIS associated with reduced brain

glucose sensing. Female NOER mice exhibited milder hepatic insulin resistance and glucose

intolerance. In conclusion, nuclear ERα signaling is predominant in maintaining glucose

homeostasis in mice of both sexes. Lack of nuclear ERα alters the central control of insulin

sensitivity in females, and predominantly impairs the central regulation of insulin secretion in

males.

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Estrogens favor glucose homeostasis, and estrogen deficiency predisposes males and females to

dysglycemia (1; 2). In women, early menopause (producing prolonged 17β-estradiol -E2-

deficiency) and surgical menopause by oophorectomy (producing rapid and severe E2

deficiency) both increase the risk of type 2 diabetes compared to women with natural menopause

(2). In men, total estrogen deficiency induced by inactivating mutations of the CYP19 gene,

which codes for aromatase, produces insulin resistance, glucose intolerance and even type 2

diabetes (1). In both cases, estrogen therapy improves metabolic alterations. The metabolic

actions of estrogens are mediated via the estrogen receptor-α (ERα), ERβ and the membrane-

bound G protein-coupled estrogen receptor (GPER). ERα is believed to account for most actions

of estrogens on glucose homeostasis in vivo. Male and female mice with global knockout of ERα

(ERαKO) develop insulin resistance and glucose intolerance (3-6). Experimentally, skeletal

muscle pools of ERα are more important for systemic insulin action (4; 7). Loss of ERα in

pancreatic islets also predisposes male and female mice to pancreatic β-cell dysfunction and

failure in conditions of metabolic stress (8-10). ERα is a ligand-activated transcription factor that

regulates gene expression by binding estrogen response elements (ERE) present on the DNA or

indirect binding via tethering to other transcription factors (11). ERα is not only localized to the

nuclear compartment but is also present in extranuclear locations at the level of membranes.

Membrane targeting of ERα through palmitoylation at cysteine residues (12) represents 5 to 10%

of the total pool depending on the cell type (13). Membrane localization of ERα facilitates

membrane-initiated rapid signaling events that are important in males and females for

reproduction (14; 15), neuronal function (16) and vascular health (17). Activation of extranuclear

ERα also promotes pancreatic islet survival from apoptotic stresses (18), prevents excess islet

lipogenesis (9; 19) and stimulates insulin synthesis (20). The importance of extranuclear steroid

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receptors in metabolic homeostasis is not limited to ERs. The androgen receptor also exhibits

extranuclear/membrane location in β-cells with a critical role in insulin secretion in males (21).

Therefore, understanding the contributions of extranuclear and nuclear pools of ERα to glucose

homeostasis is a necessary step toward the development of gender-based therapies in diabetes.

Here, we studied mice of both sexes expressing either the membrane (MOER) or the nuclear

pools of ERα (NOER) (14; 22) to determine their contribution to glucose homeostasis, insulin

action and insulin secretion.

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RESEARCH DESIGN AND METHODS

Animals. The generation and genotyping of ERαKO (23), MOER (22) and NOER (14) mice was

described previously (Fig. 1A). MOER mice genotyping confirmed the absence of endogenous

expression of ERα and the presence of human E domain of ERα (Supplementary Fig. 1A-B).

Moreover, MOER and littermate controls expressed comparable amount of the human E domain

mRNA in muscle and liver, which was absent in the ERαKO and wild-type (WT)

(Supplementary Fig. 1D). NOER mice genotyping confirmed the presence of the knock-in

mutation at the position 451 of the ERα gene (Supplementary Fig. 1C). WT littermates were used

as controls; if not available, C57Bl/6N mice (Charles River) were used as controls. All

experiments were approved by the Institutional Animal Care and Use Committee of Tulane

University in accordance with NIH guidelines. Mice were studied between 5 and 12 months of

age.

Metabolic studies and hormone measurements. Random fed blood glucose and plasma insulin

were measured in the morning (3-4 h after beginning of light cycle). Fasting blood glucose and

plasma insulin were measured after 16h fasting (overnight). Glucose tolerance test (GTT),

Glucose-stimulated insulin secretion (GSIS) and pyruvate tolerance test (PTT) were performed

after 16h fasting. Insulin tolerance test (ITT) was performed after 6h fasting. Mice were injected

ip with glucose for GTT (2 g/kg) and GSIS (3g/kg), sodium pyruvate for PTT (2 g/kg) or insulin

for ITT (0.5-0.75 U/kg for females, 0.75-1.25 U/kg for males). The brain glucose sensing

experiment was performed as described (24; 25) following right carotid catheterization and

injection of a bolus of glucose (25 mg/kg). Blood was sampled from the tail vein at indicated

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times for glucose and/or insulin assessment. Blood glucose was measured using the True Result

glucose meter (Nitro Diagnostics). Plasma insulin levels were measured by ELISA (Millipore).

At sacrifice, blood was sampled from the inferior vena cava and serum was collected for

measurement of testosterone (IBL America), luteinizing hormone (LSBio), 17β-estradiol

(Calbiotech), and IL-6 (R & D Systems) by ELISA. Non-esterified fatty acids (NEFA, Cell

Biolabs) were measured from EDTA treated-plasma following a 16h fasting.

Hyperinsulinemic, euglycemic clamp

Whole-body glucose turnover was quantified using a hyperinsulinemic, euglycemic clamp with

isotope tracer. Under isoflurane anesthesia, female MOER mice were catheterized with a silastic

catheter into the jugular, with the other end tunneled out the neck skin. Post-recovery, mice were

fasted for 6 h in a cage where they were freely moving. For some mice, a basal was done by

perfusing saline containing HPLC-purified 3-3H D-glucose (0.05 µCi/min, ARC) for 2 h before

the beginning of the clamp. A continuous infusion of insulin (4 mU/kg/min, Humulin, Lilly) was

started to induce hyperinsulinemia. The insulin solution contained glucose tracer (0.1 µCi/min,

ARC) to measure the glucose turnover (4 µl bolus in 1 min, then constant rate of 1 µl/min for

120 min). A variable glucose infusion rate (GIR) of 15% D-glucose solution was adjusted to

maintain blood glucose around 100 mg/dl. Blood glucose was measured every 20 min from the

tail vein (no restraint). Blood samples were collected at t=100, 110 and 120 min to assess glucose

specific activity. Blood was deproteinized with ZnSO4 and Ba(OH)2, supernatant was dried to

remove 3H2O, resuspended in water, and counted in scintillation fluid (Ultima Gold; Perkin

Elmer) with a beta-counter (Packard, Tulane University Shared Instrument Facility). Whole-body

glucose turnover (Gt, mg/kg/min) was determined as the ratio of the 3-3H glucose infusion rate

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(dpm/kg/min) to the specific activity of blood glucose (dpm/mg) during the final 20 minutes of

clamp. Endogenous glucose production (EndoRa) was calculated as EndoRa = Gt – GIR. Plasma

insulin was determined from blood samples at t=0 and t=120 min. At the end of the 120 min

period, mice were anesthetized and tissues were collected and snap-frozen in liquid nitrogen.

Central glucose-induced insulin secretion. In order to test in vivo the ability of the central

nervous system (CNS) to induce insulin secretion in response to glucose (glucose sensing), male

mice were injected with glucose towards the brain as previously described (24). Briefly, a

catheter was introduced into the carotid under anesthesia (Inactin, 200 mg/kg). A bolus of

glucose (30 µl, 25 mg/kg) was injected towards the brain. This dose was not sufficient to alter

peripheral blood glucose. Blood samples were collected from the tail vein 0, 1, 3 and 5 min post-

injection to measure blood glucose and plasma insulin levels.

Central insulin perfusion. The central infusion of insulin was performed as previously described

(26). Briefly, a single cannula guide (Plastic One) was inserted into the lateral ventricle. After

recovery and 16h fasting, mice were perfused intracerebroventricularly (ICV) with insulin

(Humulin, Lilly, 10 µU/µl, 1 µl in a min bolus then 1 µl/h, UltraMicroPump III, WPI) or vehicle

(artificial cerebrospinal fluid). After 3h, mice were euthanized, tissues were dissected and snap-

frozen for further analysis.

Insulin secretion in static incubation. Following islet isolation via pancreatic duct cannulation,

experiments of static incubation were performed as described (21). Insulin secretion was

expressed as percentage of total insulin content.

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Immunohistochemistry and β-cell mass measurement. Pancreases were dissected, weighed and

fixed in 10% neutral buffered formalin before paraffin-embedding. Pancreas sections (5 µm)

were prepared by the Tulane University Stem Cell Research & Regenerative Medicine Histology

Lab. Sections were dewaxed and rehydrated before antigen demasking using citrate buffer. After

permeabilization, slides were incubated with guinea-pig anti-insulin antibody (Linco or abcam)

and donkey anti-guinea pig Cy3 antibody (Jackson Laboratories). Nuclei were counterstained

using DAPI. Each islet and the entire pancreas were photographed using a Nikon Eclipse Ti and

Olympus microscope, respectively (Tulane University COBRE Molecular Core). Individual

pancreas pictures were reconstituted using the stitching function of the Metamorph software

(Nikon). Insulin-positive areas were quantified using ImageJ software. The β-cell mass (mg) was

calculated by morphometric analysis by multiplying the β-cell area (insulin-positive area/entire

pancreas area; %) by the mass of the entire pancreas.

Western Blotting. Tissues were homogenized in RIPA buffer containing anti-phosphatases and

anti-proteases cocktails (Roche) and protein concentration was determined using the BCA

method (Thermo Pierce). 30-50 µg of protein was separated by electrophoresis using 10% or

12% SDS-polyacrylamide premade gels (Bio-Rad), then transferred to nitrocellulose membranes

(Invitrogen). Membranes were incubated with the indicated antibodies (pAkt S473, pAKT T308,

Akt, pSTAT3 Y705, STAT3, Cell Signaling), followed by incubation with fluorophore coupled

anti-rabbit IgG (Li-Cor) or HRP-linked anti-rabbit IgG (SantaCruz). Fluorescent signal was

acquired using Odyssey instrument (Li-Cor) or ChemiDoc MP (Bio Rad). Band density

quantification was performed using ImageJ or Biorad’s Image Lab software.

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Real-time qPCR. RNA was extracted from the liver using a TRIzol reagent (Invitrogen Life

Technologies) following the manufacturer’s instructions. cDNA synthesis (1 µg) was performed

using iScript cDNA synthesis kit (Bio-Rad). PCR reactions were prepared using iQ SYBR Green

Supermix (Bio-Rad) and PCRs were performed in a Light-cycler 96 real-time detection system

(Roche). Quantification of a given gene (il-6, primers sequence upon request), expressed as

relative mRNA level compared with control, was calculated with the 2-ddCT comparative

method after normalization to β-actin housekeeping gene.

Statistics. Statistical analyses were performed with GraphPad Prism. Normality of the samples

was checked using the Kolmogorov-Smirnov test. When the samples assumed a Gaussian

distribution, a Student t-test or a one-way ANOVA (with Bonferroni post-hoc test) were

performed when appropriate. Otherwise, Mann-Whitney or Kruskal-Wallis tests were used.

Results are expressed as mean +/- sem. and p<0.05 was considered as significant. Significance is

expressed as follows: * p<0.05, ** p<0.01, *** p<0.001.

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RESULTS

Loss of nuclear ERα, and to a lesser extent membrane ERα, impairs glucose homeostasis in

mice of both sexes.

To determine the relative contribution of nuclear and membrane pools of ERα to glucose

homeostasis, we studied NOER and MOER male and female mice. ERαKO mice were initially

used as controls of global ERα deficiency (Fig. 1A). Female MOER mice exhibited fed and

fasted hyperglycemia (Fig. 1B and C) and hyperinsulinemia (Fig. 1E and D) to an extent similar

to ERαKO mice (Supplementary Fig. 2A). Female NOER mice exhibited fed and fasted blood

glucose levels comparable to those of littermate wild-type (WT) controls (Fig. 1B and C), even

though their fasting and fed plasma insulin levels were non-significantly increased, suggesting

insulin resistance (Fig. 1E and D). Following an intraperitoneal (I.P.) glucose tolerance test

(GTT), female MOER mice, and to a lesser extent NOER mice, exhibited glucose intolerance, as

observed in ERαKO mice (Supplementary Fig. 2B), compared to the littermate controls (Fig.

1F).

Like females, male MOER mice exhibited fed and fasted hyperglycemia (Fig. 2A and B) similar

to ERαKO mice (Supplementary Fig. 2C), whereas male NOER mice remained normoglycemic

(Fig. 2A and B). However, unlike in the case of females, plasma insulin levels of male MOER

and NOER mice were not significantly different from those of littermate controls, suggesting that

they were not insulin resistant (Fig. 2C and D). Only fasting plasma insulin levels were elevated

in male MOER mice (Fig. 2C and D) and showed a trend toward elevation in ERαKO

(Supplementary Figure 2C). Following an IP glucose challenge, male MOER and ERαKO mice,

but not NOER mice exhibited glucose intolerance (at 2h into the GTT) compared to their

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littermate controls (Fig. 2E and Supplementary Fig. 2D). Male NOER mice also responded

normally to an oral glucose challenge (Supplementary Fig. 3).

These results suggest that nuclear ERα is essential for maintaining glucose homeostasis in male

and female mice but suggests that sex-specific mechanisms are involved.

Loss of nuclear and membrane ERα produces hepatic insulin resistance in female mice.

We assessed insulin sensitivity in physiological conditions via insulin tolerance testing (ITT) in

male and female MOER and NOER mice. Female MOER mice, like ERαKO mice, exhibited

resistance to the hypoglycemic effect of insulin, as demonstrated by a smaller decrease in blood

glucose after insulin injection compared to their controls, whereas female NOER mice exhibited

normal insulin sensitivity (Fig. 3A and Supplementary Fig. 4A). In contrast, during the ITT, male

MOER mice exhibited normal insulin sensitivity that was comparable to that of male NOER and

ERαKO mice (Fig. 3B and Supplementary Fig. 4B). Both female and male MOER mice showed

a similar increase in subcutaneous inguinal, mesenteric and perigonadal adipose tissues

compared to control mice, which was not observed in NOER mice (Fig. 3C-F), eliminating a

potential role for adiposity in the insulin resistance observed in MOER females compared to

males. Note that male MOER showed an increase in fat mass without significant increase in

body weight, likely due to the deleterious effect of ERα deletion on lean mass in the male (27;

28).

Having observed that female MOER mice are hyperinsulinemic and insulin resistant during ITT,

and that female NOER mice exhibit a non-significant fasting and fed hyperinsulinemia,

suggesting insulin resistance (not detected during ITT), we explored systemic insulin sensitivity

during a euglycemic, hyperinsulinemic clamp in female MOER and NOER mice. During the

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clamp, we increased plasma insulin levels by a factor of 3 to 6 from fasted levels in MOER and

NOER mice and littermate controls (Supplementary Fig. 5A and D). Blood glucose levels were

maintained to similar levels in MOER, NOER, and their littermate controls (Supplementary Fig.

5B and E), with no difference in blood glucose levels at the end of the 2h (Supplementary Fig.

5C and F) by adjusting the glucose infusion rate (GIR). The GIR was decreased in female

MOER (Fig. 4A) and NOER mice (Fig. 4D) compared to littermate control mice demonstrating

systemic insulin resistance. Based on tracer specific activity determination at the end of the

clamp, there was no difference in insulin-stimulated whole body glucose turnover (Gt) between

female control and littermate MOER and NOER mice (Fig. 4B and E). However, the endogenous

glucose production (Endo-Ra) was more elevated in the female MOER (Fig. 4C) and to a lesser

extent NOER (Fig. 4F) mice compared to control mice, suggesting hepatic insulin resistance. We

explored the mechanism of insulin resistance in female MOER mice only. To explore liver

gluconeogenesis, we examined pyruvate incorporation into glucose via gluconeogenesis

following a pyruvate tolerance test (PTT). Following pyruvate injection, in agreement with the

Endo-Ra (Fig. 4C), female MOER mice exhibited higher blood glucose, which did not return to

basal level 2h post-injection (Fig. 4G). Together, these data suggest that female MOER mice

exhibit insulin resistance via increased hepatic glucose output from unsuppressed

gluconeogenesis. Direct insulin signaling, as assessed by insulin-stimulated Akt phosphorylation

on Ser 473 and Thr 308 in liver (Fig. 4H) and skeletal muscle (Supplementary Fig. 6) isolated

from clamped MOER mice, was unchanged compared to controls, suggesting that insulin

resistance is mediated via extra-hepatic tissues and/or other signaling pathways.

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Loss of nuclear ERα alters the central control of insulin sensitivity in female mice.

Central insulin is known to suppress hepatic glucose production via the autonomic nervous

system and a liver IL-6-STAT3 axis (26; 29). In liver isolated from hyperinsulinemic clamped

MOER mice, IL-6 mRNA expression was decreased compared to controls (Fig. 4I) without

changes in IL-6 serum concentrations (IL-6; pg/ml; Controls: 3.78 ± 1.1; MOER: 4.46 ± 0.6;

Mean ± SEM). Additionally, STAT3 phosphorylation (Fig. 4J) was also decreased compared to

controls, together suggesting a reduced ability of central insulin to lower hepatic glucose

production (HGP).

To assess the ability of brain insulin to activate the hepatic IL-6-STAT3 pathway and suppress

HGP in female MOER mice, we performed intracerebroventricular (ICV) insulin injection. We

used the hippocampus as a control, insulin-sensitive brain region known to develop insulin

resistance in diabetic conditions (30). Accordingly, injection of insulin into the lateral ventricle

increased hippocampal Akt phosphorylation in control mice (Fig. 4K). However, ICV insulin

perfusion produced no increase in hippocampal Akt phosphorylation in female MOER mice,

consistent with central insulin resistance (Fig. 4K). As previously described (29), ICV perfusion

of insulin stimulated the hepatic phosphorylation of STAT3, without canonical phosphorylation

of Akt in control mice (Fig. 4L-M). However, unlike in controls, the ICV insulin perfusion did

not stimulate hepatic STAT3 phosphorylation in female MOER mice (Fig. 4L). Thus, female

MOER mice exhibit central insulin resistance associated with decreased liver IL-6-STAT3

activation and increased HGP.

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Loss of nuclear ERα alters the central control of insulin secretion in male mice.

Male MOER mice exhibited fasting and fed hyperglycemia and glucose intolerance without

alteration in insulin sensitivity during ITT, suggesting these mice might have altered insulin

secretion. We assessed glucose-stimulated insulin secretion (GSIS) in vivo following an IP

glucose challenge in male MOER mice. While control mice exhibited a 4-fold increase in first

phase-insulin release following glucose injection, the first phase-insulin release was blunted in

male MOER mice (Fig. 5A), as observed in male ERαKO mice (Supplementary Fig. 7). Male

MOER mice exhibited no alteration in β-cell mass (Fig. 5B) or pancreas insulin content (Fig.

5C), suggesting a functional defect of the β-cells in these mice. In contrast to the decreased acute

phase insulin secretion, plasma insulin levels 30 min post-glucose injection (which represents the

second phase of insulin secretion) were increased in male MOER mice (Fig. 5D). To determine

whether the defect in GSIS observed in male MOER mice was islet cell-autonomous, we

performed GSIS in static incubation from cultured isolated islets from male MOER mice. Unlike

what was observed in vivo, GSIS and islet insulin content were identical in cultured male MOER

and control islets in the presence or absence of E2 (Fig.5E-H). This suggests that the defect in

acute phase insulin release observed in MOER mice results from an extra-islet factor. Unlike

males, female MOER and NOER mice exhibited no defect in acute phase insulin release during

GSIS, and accordingly, no defect in β-cell mass and or pancreas insulin content (Fig.5K-M,

Supplementary Fig. 8A-C). However, consistent with the insulin resistance described above,

plasma insulin levels 30 min post-glucose injections were increased in female MOER and NOER

mice (Fig. 5N and Supplementary Fig. 8D). Therefore MOER mice exhibit a male-specific

defect in acute insulin release in vivo.

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To search for an extra-islet factor altering GSIS selectively in male MOER, we first studied

estrogen and androgen concentrations (31). Female MOER mice exhibited a five-fold increase in

circulating E2 concentrations and a ten-fold increase in circulating testosterone concentrations

compared to levels observed in littermate CTRL males (Table 1). The ratio T/E2 was increased

two-fold in female MOER mice. Male MOER mice did not show any differences in E2 levels but

exhibited a three-fold increase in testosterone concentrations compared to CTRL (Table 1). As a

result, male MOER mice exhibited a three-fold increase in the T/E2 ratio. To assess the potential

role of increased testosterone in the altered in vivo GSIS of males, we treated male MOER mice

with the AR antagonist flutamide. Flutamide was efficient in producing androgen resistance as

assessed by increased testosterone concentrations, but produced no improvement in GSIS in

male mice, ruling out the role of excess testosterone (Supplementary Fig. 9).

The central nervous system modulates insulin secretion in vivo, via a hypothalamo-pancreatic

axis (32). To assess the extent to which brain glucose sensing was altered in male MOER mice,

we performed a gold-standard experiment to assess central GSIS by glucose injection in the

carotid artery (24; 25). As previously reported, the carotid glucose bolus did not affect

peripheral glucose levels in control and MOER mice (Fig. 5I). In male CTRL mice, glucose

injection triggered an acute insulin secretion 1-minute post-glucose injection (Fig. 5J). In

contrast, in male MOER mice, the acute insulin secretion was blunted (Fig. 5J). Thus, the nuclear

ERα is necessary to maintain GSIS in male mice, and this is at least partially dependent on brain-

induced GSIS.

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DISCUSSION

Loss of nuclear ERα differentially impairs glucose homeostasis in males and female mice on a

normal chow diet. While insulin resistance is predominant in female mice, impaired GSIS is

central in males.

Female MOER mice, lacking nuclear ERα, are hyperinsulinemic and insulin resistant during the

physiological conditions of an insulin challenge. Under these conditions, female NOER mice,

lacking membrane ERα, are only slightly hyperinsulinemic. However both MOER and to a lesser

extent NOER mice show systemic insulin resistance during the steady-state conditions of a

euglycemic–hyperinsulinemic clamp. Thus, both nuclear and membrane ERα are involved in

insulin sensitivity in female mice, with the nuclear pool of ERα playing a predominant role.

Female mice with global (6; 33) or liver-specific knockout of ERα (LERKO) (5; 34) exhibit a

decline in insulin’s ability to suppress hepatic glucose production during euglycemic–

hyperinsulinemic clamp studies, suggesting that ERα in hepatocytes is important to whole body

and liver insulin sensitivity in female mice. Additionally, conditional elimination of ERα in

skeletal muscle promotes systemic insulin resistance and secondary failure of insulin to suppress

hepatic glucose production in female mice (7). Female MOER mice exhibit hepatic insulin

resistance (increased Endo-Ra and hepatic gluconeogenesis) without detectable abnormality in

muscle insulin action assessed by whole body glucose turnover (and insulin-stimulation of Akt

phosphorylation in muscle). Thus, the primary site of systemic insulin resistance in female

MOER mice seems to be liver. Loss of hepatocyte nuclear ERα (5; 34) or increased testosterone

action in hepatocytes (35) could promote liver insulin resistance in female MOER mice.

However, unlike female LERKO mice or female mice with testosterone excess fed a HFD, on

normal chow, female MOER mice do not exhibit insulin failure to promote Akt phosphorylation.

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Thus, the defect leading to unsuppressed HGP in female MOER mice seems to originate outside

liver.

Hypothalamic insulin action suppresses HGP in mice (36). Central insulin blunts

parasympathetic output to Kupffer cells, increasing their production of IL-6 leading to activation

of STAT3 in nearby hepatocytes which downregulates gluconeogenic genes (26; 29). Female

MOER mice exhibit central insulin resistance with failure of central insulin infusion to activate

Akt in the hippocampus. Further, female MOER mice exhibit decreased hepatic IL-6-STAT3

activation during hyperinsulinemic clamp and central insulin infusion. Taken together, these

results suggest that global nuclear ERα deficiency impairs central insulin action and the

activation of the hepatic IL-6/STAT3 pathway, leading to inappropriate suppression of HGP.

Inhibitory insulin action in AgRP neurons of the hypothalamic arcuate nucleus is required to

suppress HGP via the brain-liver IL-6 pathway (36; 37). However, ERα is not expressed in

mouse AgRP neurons (38). Rather, ERα is expressed in POMC neurons, and female mice lacking

ERα specifically in POMC neurons exhibit insulin resistance on a chow diet, like female MOER

(39). Moreover, E2 enhances insulin action in POMC neurons by stimulating their neuronal

excitability and the coupling of insulin receptor to TRPC5 channels via transcriptional

mechanisms (40). Therefore, in female mice, loss of ERα transcriptional activity in POMC

neurons is likely to decrease neuronal excitability and inhibition of AgRP neurons, which could

increase HGP.

Global loss of nuclear ERα in female MOER mice promotes central insulin resistance leading to

unsuppressed HGP. In contrast, hepatocyte-specific knockout of ERα in LERKO mice promotes

peripheral insulin resistance via impairment of Akt in hepatocytes. One explanation for this

apparent discrepancy is likely related to differences in diets and models. Female MOER mice

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were fed normal chow and in these conditions the neuronal ERα seems predominant in

suppressing HGP in mice (via liver IL-6/STAT3 and without activation of hepatocytes Akt). In

contrast, female LERKO were fed a HFD. Given the importance of hepatocyte ERα in limiting

liver fat accumulation, during high fat feeding insulin resistance in hepatocytes via Akt (5; 34) is

predominant.

Male MOER mice, lacking nuclear ERα, exhibit mild fasting hyperinsulinemia but no detectable

insulin resistance. Male NOER mice, lacking membrane ERα, have no detectable abnormalities

of glucose homeostasis. Male MOER mice exhibit blunted first-phase insulin release in response

to glucose in vivo. The first-phase insulin release is central to glucose homeostasis in mice (41;

42), and impaired first-phase with exaggerated second-phase GSIS is a hallmark of the early

stages of type 2 diabetes (43). Therefore, loss of nuclear ERα in male mice produces a defect

typical of human type 2 diabetes. The GSIS defect of male MOER mice is not observed in

cultured islets, demonstrating that it is independent from the loss of nuclear ERα in β-cells and

secondary to the loss of ERα in extra-islet tissues, indirectly impairing islet function possibly via

a neural factor. To assess the role of nuclear ERα in β-cell function via the hypothalamo-

pancreatic axis, we studied acute insulin release in response to the central injection of

glucose. We observed that brain glucose sensing was impaired in male MOER mice with a

blunted acute insulin response to a bolus of glucose directed towards the brain, suggesting that

the response to central glucose and the neural control of insulin secretion are impaired by loss of

nuclear ERα in male mice. Efferent circuits that emanate from the hypothalamus innervate

pancreatic islets, and glucose sensing in the hypothalamic ARC is important to GSIS in male

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mice (32). Therefore, loss of nuclear ERα transcriptional activity in the hypothalamic ARC

neurons may impair parasympathetic outflow to the islets.

In summary, global lack of nuclear ERα and to a lesser extent membrane ERα alter the central

control of hepatic glucose production in female mice (Fig. 6). In male mice, lack of nuclear ERα

predominantly impairs the central regulation of insulin secretion (Fig. 6). Further studies are

needed to identify the neuronal population mediating these sexually dimorphic effects of ERα on

glucose homeostasis.

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ACKNOWLEDGMENTS

CA designed and performed experiments, analyzed the data and wrote the manuscript. JJM

performed experiments, analyzed the data, and revised the manuscript. BX, BS, WX, LD

performed experiments. JKK and ERL discussed the results and revised the manuscript. AZ

revised the manuscript. FMJ designed the study, analyzed the data, wrote and revised the

manuscript. FMJ is the guarantor of this work and, as such, had full access to all the data in the

study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors have no conflicts of interest to declare.

This work was supported by grants from the National Institutes of Health (R01 DK074970, and

DK107444) and a VA Merit Review Award (BX003725) to F.M-J. C.A. was supported by

American Diabetes Association Post-Doctoral Fellowship (1-16-PDF-004). A.Z. was supported

by R01 DK099598.

Part of this work has been previously presented during a Keystone Symposia.

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LH (ng/ml) E2 (pg/ml)

Testosterone

(ng/ml) Testosterone

/E2 ratio (pg/ml)

Female CTRL 2.5 ± 0.5 4.2 ± 0.4 0.2 ± 0.03 44.5 ± 6.8

Female MOER 1.2 ± 0.2 22.6 ± 5.5 * 1.9 ± 0.5 ** 84.7 ± 17.1

Male CTRL 7.4 ± 2.1 4.5 ± 0.2 1.3 ± 0.2 261.0 ± 40.2

Male MOER 4.6 ± 1.5 3.9 ± 0.2 3.5 ± 1.0 ** 892.0 ± 257.1 **

Table 1. Metabolic parameters in control and MOER mice. Serum Luteinizing Hormone

(LH), 17β-estradiol (E2), testosterone and testosterone/E2 ratio were measured in 6 month-old

MOER and control (CTRL) mice. Data correspond to the mean ± SEM (n = 4-6). * p<0.05, **

p<0.01.

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FIGURE LEGENDS

Figure 1. Female MOER mice are hyperglycemic and glucose intolerant. (A) The control

(CTRL) mice express nuclear and extranuclear (membrane-bound) ERα. Global ERαKO mice

lack ERα. NOER (Nuclear Only ERα) mice exhibit a mutation of ERα palmitoylation site,

leading to exclusive ERα nuclear location. MOER (Membrane Only ERα) mice are ERαKO

mice reexpressing the human ERα ligand-binding domain selectively at the membrane. (B)

Random fed and (C) fasting blood glucose, (D) random fed and (E) fasting plasma insulin levels,

(F) blood glucose and calculated area under the curve (AUC, inset) during a glucose tolerance

test (ipGTT, 2 g/kg) in 6 month-old female MOER and NOER mice with their respective CTRL

littermates. Data correspond to the mean ± SEM (n=6-23). * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. Male MOER mice are hyperglycemic and glucose intolerant. (A) Random fed and

(B) fasting blood glucose, (C) random fed and (D) fasting plasma insulin levels, (E) blood

glucose and calculated area under the curve (AUC, inset) during a glucose tolerance test (ipGTT,

2 g/kg) in 6 month-old male MOER and NOER mice with their respective CTRL littermates.

Data correspond to the mean ± SEM (n=6-25). * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. Female MOER mice exhibit hepatic insulin resistance. (A) Blood glucose level

during an insulin tolerance test (ipITT) in 6 month-old female (0.5-0.75 U/kg) and (B) male

(0.75-1.25 U/kg) MOER and NOER mice with their respective CTRL littermates. Data

correspond to the mean ± SEM (n=4-19). * p<0.05, ** p<0.01, *** p<0.001. Body weight of 6-

month old female (C) and male (E) NOER and MOER mice with their respective CTRL

littermates. Adipose tissue weight in the indicated fat pads of 6-month old female (D) and male

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(F) NOER and MOER mice with their respective CTRL littermates. Data correspond to the mean

± SEM (n=6-24). * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Female MOER mice exhibit central insulin resistance. Glucose infusion rate (GIR)

(left panel) and corresponding AUC (right panel) calculated during a hyperinsulinemic-

euglycemic clamp in (A) female CTRL and MOER mice and (D) female CTRL and NOER mice.

(B) Glucose turnover (Rt, mg/kg/min) in CTRL, MOER and (E) CTRL, NOER female mice. (C)

Endogenous glucose production (Endo-Ra, mg/kg/min) in female CTRL, MOER and (F) CTRL,

NOER mice. Clamp was performed in 6 month-old mice (n=5-6). (G) Blood glucose and

calculated AUC (inset) during a pyruvate tolerance test (ipPTT, 2 g/kg) in female CTRL and

MOER mice (n=4-6). (H) Protein expression for pAkt (S473), pAkt (T308), and total Akt in liver

from female MOER mice at the end of the 2h clamp were analyzed by Western blotting.

Quantification by densitometry is shown in adjacent bar graphs (n=5-6). (I) Hepatic Il-6 mRNA

expression quantified by Q-PCR in female mice at the end of the 2h clamp (n=5-6). (J) Protein

expression for pSTAT3 (Y701) and STAT3 in liver from female MOER mice at the end of the 2h

clamp were analyzed as in (H). (K) Protein expression for pAkt (S473)/Akt in hippocampus

from female CTRL and MOER at the end of 3h ICV-perfusion of vehicle or insulin.

Quantification by densitometry is shown in adjacent bar graphs (n=2-3). Protein expression for

(L) pSTAT3/STAT3 and (M) pAkt (S473)/Akt and pAkt (T308)/Akt in liver from female CTRL

and MOER mice at the end of 3h ICV-perfusion of vehicle or insulin analyzed as in (L) (n=4-6).

Data correspond to the mean values ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001.

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Figure 5. Male MOER mice exhibit reduced first-phase insulin secretion in vivo. (A) Plasma

insulin (left panel) and calculated AUC (right panel) during an in vivo IP glucose-stimulated

insulin secretion test (GSIS, 3 g/kg) in male CTRL and MOER mice (n=14-17). (B)

Representative pictures of pancreatic islets stained for insulin (red) and calculated β-cell mass

(mg, n=6-7) in mice from (A). Scale bar 100µm. (C) Pancreatic insulin content in male CTRL

and MOER mice (n=6). (D) Plasma insulin levels 30 minutes following an I.P. injection of

glucose (2g/kg), showing the second phase insulin secretion in male CTRL and MOER mice

(n=6-16). (E) In vitro GSIS in static incubation in cultured islets isolated from MOER and CTRL

male mice without 17β-estradiol (E2) or (G) with E2 (10-8

M). (F, H) Insulin content of islets

from (E) and (G) (n = 8-19 islet batches from 3-7 different animals). (I) Blood glucose and (J)

plasma insulin levels during a brain glucose-sensing test (25 mg/kg glucose) in male CTRL and

MOER (n=10-11, 5 independent experiments). (K) Plasma insulin (left panel) and calculated

AUC (right panel) during an in vivo GSIS (3g/kg) in female CTRL and MOER mice (n=7-8). (L)

Representative pictures of female CTRL and MOER pancreatic islets stained for insulin (red)

and calculated β-cell mass (mg, n=4). Scale bar 100µm. (M) Pancreatic insulin content in female

CTRL and MOER mice (n=3-7). (N) Plasma insulin levels 30 minutes following an I.P. injection

of glucose (2g/kg), showing the second phase of insulin secretion in female CTRL and MOER

mice (n=7-18). Data were collected from 6-8 month-old mice and correspond to the mean values

± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6. Sex differences in nuclear and membrane ERα effects on glucose homeostasis. In

female mice, nuclear ERα signaling in the brain promotes the suppression of hepatic glucose

production via a brain-liver IL6-STAT3 axis. Membrane ERα also participates in control of

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hepatic glucose production. In male mice, nuclear ERα action in the brain favors glucose-

stimulated first-phase insulin secretion.

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2

3* *

CT

RL

NO

ER

of 45 Diabetes

pA

kt/

Ak

t

VE

HIN

S

VE

HIN

S

0 .0

0 .5

1 .0

1 .5

2 .0

C T R L M O E R

*

0 .0

0 .5

1 .0

1 .5

2 .0

pA

kt/

Ak

t

0

5

1 0

1 5

2 0

Glu

co

se

tu

rn

ov

er

(R

t; m

g/k

g/m

in)

-2 0

-1 5

-1 0

-5

0

5

Glu

co

se

pro

du

cti

on

(En

do

-Ra

; m

g/k

g/m

in)

* * *

C T R L

N O E R

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

1 2

1 6

2 0

2 4

2 8

3 2

C T R L

N O E R

* * * * * * * * * * * * * * * * * *

* * *

* * *

T im e (m in )

GIR

(m

g/k

g/m

in)

0

5

1 0

1 5

2 0

2 5

Glu

co

se

tu

rn

ov

er

(R

t; m

g/k

g/m

in)

0 3 0 6 0 9 0 1 2 0

0

3 0

6 0

9 0

1 2 0

1 5 0

C T R L

M O E R

* *

* * * * * ** * *


Blo

od

glu

co

se

(m

g/d

l)

pAkt S473

Akt Liv

er

A B C

G H pAkt

S473

Female MOER

I

pSTAT3

STAT3

CTRL MOER

Liv

er

J K

pAkt S473

Akt

CTRL MOER

Liv

er

VEH INS VEH INS

pAkt S473

Akt

CTRL MOER

Hip

po.

VEH INS VEH INS

L

D E F

Female NOER

Female MOER

0

3

6

9* *

AU

C

-2 0

-1 5

-1 0

-5

0

5

1 0

1 5 * * *

Glu

co

se

pro

du

cti

on

(En

do

-Ra

; m

g/k

g/m

in)

M O E R

C T R L

CT

RL

MO

ER

0 .0

0 .5

1 .0

1 .5

2 .0

*IL-6

mR

NA

ex

pre

ss

ion

M

pS

TA

T3

/ST

AT

3

CT

RL

MO

ER

0 .0

0 .5

1 .0

1 .5

2 .0

p = 0 .0 7 8

pSTAT3

STAT3

CTRL MOER

Liv

er

VEH INS VEH INS

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

1 2

1 6

2 0

2 4

2 8

3 2

C TR L

M O E R

* * ** * *

* * ** * * * * * * * *

* * *

* * *

T im e (m in )

GIR

(m

g/k

g/m

in)

GIR

AU

C (

mg

/kg

/min

2 x

10

3)

0

1

2

3

4

5

* * * *

0

1

2

3

4

GIR

AU

C (

mg

/kg

/min

2 x

10

3)

* * *

CTRL MOER

pAkt T308

Akt

CTRL MOER

Liv

er

CTRLMOER

pAkt

T308

pAkt T308

Akt

CTRL MOER

Liv

er

VEH INS VEH INS

pS

TA

T3

/ST

AT

3

VE

HIN

S

VE

HIN

S

0 .0

0 .5

1 .0

1 .5

2 .0

C T R L M O E R

*

pA

kt/

Ak

t

VE

HIN

S

VE

HIN

S

0

1

2

3

C T R L M O E R

pA

kt/

Ak

t

VE

HIN

S

VE

HIN

S

0 .0

0 .5

1 .0

1 .5

2 .0

C T R L M O E R

of 45Diabetes

of 45 Diabetes5

Nuclear ERα signaling

♀ ♂

Liver

HGP

GSIS

Pancreas

Brain

nucleus

ERαSignaling

Estrogens

ERα

Insulin signaling

IL-6

pSTAT3

Glucose sensing

Estrogens

Signaling

HGP

Liver

♀

MembraneERα signaling

?

of 45Diabetes

C: NOER genotyping L iv e r

CT

RL

MO

ER

CT

RL

ERK

O

0 .0 0

0 .0 1

0 .4

0 .8

1 .2

1 .6

2 .0

2 .4

l itte rm a tes litte rm a tes

hE

do

ma

in m

RN

A

ex

pre

ss

ion

M u sc le

CT

RL

MO

ER

CT

RL

ERK

O

0 .0 0 0

0 .0 0 5

1

2

3

4

5

6

l itte rm a tes litte rm a tes

hE

do

ma

in m

RN

A

ex

pre

ss

ion

Supplementary Fig. 1. Representative gel showing amplicons from genomic DNA extracted from ear

punches of MOER and NOER mice. (A) For the MOER, PCR products using primers to detect estrogen receptor

α (forward: GGCATTACCACTTCTCCTGGGAGTCT, reverse: TCGCTTTCCTGAAGACCTTTCATAT). WT mice

show an 815 bp band, KO mice show a 255 bp band and heterozygous mice show the two bands. Mice #1 and 4

are MOER. Mice #2 and 3 are CTRL. Mouse #5 is heterozygous and will not be used for experiments. (B) For

the MOER, PCR products using primers to detect human E domain (forward: CGTGTACGGTGGGAGGTCTA,

reverse: ATGTGAACCAGCTCCCTGTC), 226 bp band. (C) For the NOER, PCR products using primers to detect

estrogen receptor α knock-in mutation (forward: CTAAACAAGCTTCAGTGGCTCCTAG, reverse:

ACCTGCAGGGAGAAGAGTTTGTGGC). CTRL mice show a 600 bp band, KO mice show a 400 bp band,

heterozygous show the two bands. Mice #1 and 5 are NOER. Mice #2 and 4 are CTRL. Mouse #3 is

heterozygous and will not be used for experiments. (D) Human E domain expression quantified with RT-qPCR

from liver and muscle in CTRL and MOER littermates (normalized to beta-actin expression) compared to CTRL

and ERαKO littermates. Primers are: forward, AGCACCCTGAAGTCTCTGGA; reverse,

GATGTGGGAGAGGATGAGGA. Data correspond to the mean ± SEM (n=1-6).

A: MOER genotyping

ERα Edomain

1 2 3 4 5 1 2 3 4 5

WT: 815 bp

KO: 255 bp Positive: 226 bp

B: MOER genotyping

1 2 3 4 5

100 100

100

WT: 600 bp KI: 400 bp

D

of 45 Diabetes

0 3 0 6 0 9 0 1 2 0

0

5 0

1 0 0

1 5 0

2 0 0

C TR L

ER K O

* * *

* *


Blo

od

glu

co

se

(m

g/d

l)

CTR

L

ERKO

0

40

80

120

160

*

Fe

d b

loo

d

glu

co

se (

mg

/dl)

CTR

L

ERKO

0

20

40

60

80*

Fa

ste

d b

loo

d

glu

co

se (

mg

/dl)

CTR

L

ERKO

0

2

4

6

8

***

Fed

in

su

lin

(n

g/m

l)

CT

RL

ERK

O

0 .0

0 .5

1 .0

1 .5

* * *

Fa

ste

d i

ns

uli

n (

ng

/ml)

Female

Male

CTR

L

ERKO

0

5

10

15

20 *

AU

C

CTR

L

ERKO

0

20

40

60

80

100

120

140

Fe

d b

loo

d

glu

co

se (

mg

/dl)

CTR

L

ERKO

0

20

40

60

80

100 ***

Fa

ste

d b

loo

d

glu

co

se (

mg

/dl)

CTR

L

ERKO

0

2

4

6

8

10

Fed

in

su

lin

(n

g/m

l)

CTR

L

ERKO

0

1

2

3

4

Faste

d in

su

lin

(n

g/m

l)

Supplementary Fig. 2. Random fed and overnight-fasted blood glucose and random fed and overnight-

fasted plasma insulin levels in female (A) and male (C) in 6-9 month-old ERαKO mice and their control

littermates (CTRL). Blood glucose and calculated area under the curve (AUC, insert) during a glucose tolerance

test (ipGTT, 2 g/kg) in female (B) and male (D) 6-9 ERαKO mice and their CTRL littermates. Data correspond to

the mean ± SEM (n=7-13). Significance was evaluated using one-way ANOVA analysis. * p<0.05, ** p<0.01, ***

p<0.001.

0 30 60 90 1200

50

100

150

200

250

CTRL

ERKO

Time after injection (min)

Blo

od

glu

co

se

(m

g/d

l)

CT

RL

ERK

O

0

5

1 0

1 5

2 0

AU

C

A B

C D

of 45Diabetes

TIme (min)

Blo

od g

luco

se

(m

g/d

l)

0 30 60 90 1200

50

100

150

200

CTRL

NOER

AU

C

CTR

L

NOER

0

5

10

Supplementary Fig. 3. Blood glucose and calculated area under the curve (AUC, insert) during an oral

glucose tolerance test (oGTT, 2 g/kg) in male 9-12 month-old NOER mice and their control littermates (CTRL). Data correspond to the mean values ± SEM (n=7-8).

of 45 Diabetes

0 3 0 6 0 9 0 1 2 0

4 0

6 0

8 0

1 0 0

C TR L

ER K O


Hy

po

gly

ce

mic

eff

ec

t o

f in

su

lin

(% f

ro

m b

as

al

blo

od

glu

co

se

)

0 3 0 6 0 9 0 1 2 0

4 0

6 0

8 0

1 0 0

C TR L

ER K O


Hy

po

gly

ce

mic

eff

ec

t o

f in

su

lin

(% f

ro

m b

as

al

blo

od

glu

co

se

)

-8-6-4-20

*

AU

C

ab

ove b

aselin

e CTR

L

ERK

O

-8-6-4-20

AU

C

ab

ove b

aselin

e CTR

L

ERK

O

Female Male

Supplementary Fig. 4. Blood glucose level during an insulin tolerance test (ipITT) in 6-month old female

(0.5-0.75 U/kg) and (B) 6-9 month old male (0.75-1.25 U/kg) ERαKO mice and their control littermates (CTRL).

Data correspond to the mean values ± SEM (n=6-11). Significance was evaluated using one-way ANOVA

analysis* p<0.05, ** p<0.01, *** p<0.001.

A B

of 45Diabetes

0 1 2 0 0 1 2 0

0

5

1 0

1 5

C T R L M O E R

*

*

* * *P

las

ma

in

su

lin

(n

g/m

l)

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

0

5 0

1 0 0

1 5 0

2 0 0

C T R L

M O E R

T im e (m in )B

loo

d g

luc

os

e (

mg

/dl)

CTR

L

MOER

0

50

100

150

Blo

od

glu

co

se (

mg

/dl)

Supplementary Fig. 5. Plasma insulin before (0 min) and at the end (120

min) of a hyperinsulinemic-euglycemic clamp (4 mU/kg/min) with the

perfusion of 3-3H-D-glucose tracer (0.09µCi/min) in female MOER (A),

NOER (D) mice and their littermates controls. Blood glucose during the

clamp in female MOER (B), NOER (E) mice and their littermates controls.

Blood glucose at the end of the 120min clamp in female MOER (C), NOER

(F) and their littermates controls. Data correspond to the mean ± SEM (n=5-

6). Significance was evaluated using one-way ANOVA analysis or paired t-

test * p<0.05, *** p<0.001.

A B C

D E F

CTR

L

NOER

60

80

100

120

140

Blo

od

glu

co

se (

mg

/dl)

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

C T R L

N O E R

T im e (m in )

Blo

od

glu

co

se

(m

g/d

l)

0 1 2 0 0 1 2 0

0

2

4

6

8

1 0

C T R L N O E R

* * *

Pla

sm

a i

ns

uli

n (

ng

/ml) p = 0 .1

Female MOER

Female NOER

of 45 Diabetes

Supplementary Fig. 6.

Phosphorylation of Akt at S473 is not

altered in MOER female muscle. (A)

pAkt S473/Akt protein expression in

muscle studied by western-blotting in

female mice at the end of a

hyperinsulinemic-euglycemic clamp,

representative image (left panel) and

quantification (right panel, Data

correspond to the mean ± SEM )

(n=5-6).

WT

MO

ER

0 .0

0 .5

1 .0

1 .5

pA

kt/

Ak

t

Muscle

Akt

CTRL MOER

Muscle

pAkt

pAkt S473

of 45Diabetes

Supplementary Fig. 7. Plasma insulin

(left panel) and calculated area under the

curve (AUC, right panel) during an in vivo

glucose-stimulated insulin secretion test

(ipGSIS, 3 g/kg) in 6-8 month old male

CTRL and ERαKO mice (n=5-6). This test

shows the first phase of insulin secretion.

Significance was calculated using one-

way ANOVA in each group. * p<0.05

between T=0min and T=2min in WT mice.

Time after injection (min)P

lasm

a in

su

lin

(n

g/m

l)0 1 2 3 4 5

0

1

2

3 CTRLERKO

*

AU

C (

mg

/dl x m

in)

CTR

L

ERKO

0

5

10

15

of 45 Diabetes

Supplementary Fig. 8. (A) Plasma insulin (left panel) and

calculated AUC (right panel) during an in vivo glucose-

stimulated insulin secretion test (I.P. GSIS, 3 g/kg) in female

CTRL and NOER mice (n=4-5). (B) In vitro GSIS in cultured

islets isolated from MOER and CTRL male mice in static

incubation under different glucose concentration. (C) Insulin

content from 10 islets isolated from NOER and CTRL female

mice after the GSIS (n = 8-9 islet batches from 4 different

animals).(D) Plasma insulin levels 30 minutes following an I.P.

injection of glucose (2g/kg), showing the second phase insulin

secretion in female NOER and littermate control (CTRL) mice

(n=5-7). Data correspond to the mean ± SEM. Significance was

evaluated using unpaired, paired t-test or Wilcoxon test

accordingly ** p<0.01.

CT

RL

NO

ER

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Pla

sm

a i

ns

uli

n 3

0 m

in

po

st-

inje

cti

on

(n

g/m

l)

* *

0 1 2 3 4 5

0

1

2

3

4C T R L

N O E R


Pla

sm

a i

ns

ulin

(n

g/m

l)

Ins

uli

n s

ec

reti

on

(% i

ns

ulin

co

nte

nt)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

C T R L N O E R

2 .8 m M 1 6 .7 m M

* *

* *

CT

RL

NO

ER

0

1

2

3

4

5

Ins

ulin

co

nte

nt

in 1

0 i

sle

ts (

g)

CT

RL

NO

ER

0

5

1 0

1 5

2 0

AU

C (

mg

/dl

x m

in)

A

D B C

of 45Diabetes

Supplementary Fig. 9. (A) Plasma insulin (left panel) and calculated area under the curve (AUC,

right panel) during an in vivo glucose-stimulated insulin secretion test (ipGSIS, 3 g/kg) in 6-8 month

old male control (CTRL) and MOER mice. This test shows the first phase of insulin secretion,

before any treatment. (B) Plasma testosterone in male CTRL and MOER after 7 days of treatment

with an androgen receptor antagonist, Flutamide (Flut, 50 mg/kg, SC) or Vehicle (V). (C) Plasma

insulin (left panel) and calculated area under the curve (AUC, right panel) during an in vivo ipGSIS

(3 g/kg) in 6-8 month old male CTRL and MOER mice (same animals as in A) following one week

of treatment with Flutamide or Vehicle. Data correspond to the mean ± SEM (n=4-9). Significance

was evaluated using unpaired Student t-test or one-way ANOVA * p<0.05, ** p<0.01.

CT

RL

MO

ER

0

2

4

6

8

1 0

*A

UC

(m

g/d

l x

min

)

CT

RL+V

MO

ER

+V

MO

ER

+F

lut

0

5

1 0

1 5

2 0

Pla

sm

a t

es

tos

tero

ne

(ng

/ml)

CT

RL+V

MO

ER

+V

MO

ER

+F

lut

0

2

4

6

8 **

AU

C (

mg

/dl

x m

in)

A f te r F lu ta m id e

0 1 2 3 4 5

0 .0

0 .5

1 .0

1 .5

2 .0C T R L + V

M O E R + V

M O E R + F lu t

T im e a fte r

in je c tio n (m in )

De

lta

in

su

lin

(n

g/m

l)

Before Flutamide

0 1 2 3 4 5

0.0

0.5

1.0

1.5

2.0CTRL

MOER

Time afterinjection (min)

Delt

a in

su

lin

(n

g/m

l)

A B C

of 45 Diabetes

Page 1 of 45 Diabetes...2018/10/03 · (Humulin, Lilly, 10 µU/µl, 1 µl in a min bolus then 1...

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