Sex differences in islet stress responses support female β cell resilience

Abstract

Objective:
Pancreatic β cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in β cells, but few studies have examined male-female differences in β cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in β cell and islet gene expression and function using both human and rodent samples.

Methods:
We compared β cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased analysis of murine gene expression pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesize female islets will be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and β cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress.

Results:
Our single-cell analysis of human β cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was biologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males.

Conclusions:
Our data demonstrate sex differences in β cell gene expression in both humans and mice, and that female β cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.

Full text

Sex differences in islet stress responses support
female
b
cell resilience
George P. Brownrigg, Yi Han Xia, Chieh Min Jamie Chu, Su Wang, Charlotte Chao, Jiashuo Aaron Zhang,
Søs Skovsø, Evgeniy Panzhinskiy, Xiaoke Hu, James D. Johnson
*
, Elizabeth J. Rideout
*
ABSTRACT
Objective: Pancreatic
b
cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes
(T2D). Emerging evidence points to sex differences in
b
cells, but few studies have examined male-female differences in
b
cell stress responses
and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in
b
cell and
islet gene expression and function using both human and rodent samples.
Methods: In humans, we compared
b
cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and
females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity.
Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we
hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male
and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and
b
cell insulin production and secretion. Tran-
scriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress.
Results: Our single-cell analysis of human
b
cells revealed sex-specific changes to gene expression and function in T2D, correlating with more
robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing
revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes
linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female
mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-
stimulated insulin production and secretion during ER stress compared with males.
Conclusions: Our data demonstrate sex differences in
b
cell gene expression in both humans and mice, and that female
b
cells show a greater
ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.
Ó2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords Pancreatic islets;
b
cells; Diabetes mellitus; Endoplasmic reticulum stress; Protein synthesis; Transcriptomics
1. INTRODUCTION
Pancreatic
b
cells make and secrete insulin, an essential hormone
required to maintain whole-body glucose homeostasis. Emerging evi-
dence from multiple species points to biological sex as an important, but
often overlooked, factor that affects
b
cell biology [1e6]. Large-scale
surveys of gene expression in mice and humans show that differ-
ences exist between the sexes in the pancreas [7e9], in islets [10], and
in
b
cells specifically [4,11]. Humans also have a sex-specific
b
cell
gene expression response to aging [12], and show male-female dif-
ferences in pancreatic
b
cell number [6]. With respect to
b
cell function,
most data from rodent and human studies suggests glucose-stimulated
insulin secretion is higher in females than in males [5,10,13e16]. While
male-female differences in peripheral insulin sensitivity [15,17e27]may
contribute to these differences, sex-biased insulin secretion in humans
persists in the context of equivalent insulin sensitivity between males
and females [5]. Whether sex differences in other aspects of
b
cell gene
expression and function are similarly independentof insulin sensitivity in
rodents and humans remains unclear, as insulin sensitivity is not
routinely monitored across datasets showing sex differences in
b
cell
biology.
Biological sex also affects the risk of developing T2D. Across many
population groups, men are at a higher risk of developing T2D than
women [28e31]. Some of this differential risk is explained by lifestyle
and cultural factors [31e33]. Biological sex also plays a role; however,
as the male-biased risk of developing diabetes-like phenotypes is
conserved across multiple animal models [22,34e39]. Despite a
dominant role for
b
cell function in T2D pathogenesis [40,41], T2D-
and stress-associated changes to
b
cell gene expression and function
in each sex remain largely unexplored, as most studies on these topics
do not include biological sex as a variable in their analysis [42e49].
Collecting detailed knowledge of
b
cell gene expression and function
from each sex under physiological and pathological conditions is
therefore a key first step toward understanding whether male-female
differences in this important cell type may contribute to T2D risk.
The overall goal of our study was to provide detailed knowledge of
b
cell gene expression and function in both males and females across
multiple contexts to advance our understanding of sex differences in
Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada
*Corresponding authors. E-mails: james.d.johnson@ubc.ca (J.D. Johnson), elizabeth.rideout@ubc.ca (E.J. Rideout).
Received October 12, 2022

Revision received January 7, 2023

Accepted January 17, 2023

Available online 20 January 2023
https://doi.org/10.1016/j.molmet.2023.101678
Original Article
MOLECULAR METABOLISM 69 (2023) 101678 Ó2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
www.molecularmetabolism.com 1

this important cell type. Our data show male-female differences in
islet and
b
cell gene expression and stress responses in both
humans and mice. These differences contribute to sex differences in
b
cell resilience, where we find female
b
cells show a greater ability
to maintain glucose-stimulated insulin secretion in response to stress
and T2D in mice and humans, respectively. Given that an insulin
tolerance test indicated similar insulin sensitivity between the male
and female mice used in our study, our findings suggest biological
sex is an important variable to consider in studies on islet and
b
cell
function.
2. MATERIALS AND METHODS
2.1. Animals
Mice were bred in-house or purchased from the Jackson Laboratory.
Unless otherwise stated, islets were isolated from C57BL/6J mice aged
20e24 weeks. Animals were housed and studied in the UBC Modified
Barrier Facility using protocols approved by the UBC Animal Care
Committee and in accordance with international guidelines. Mice were
housed on a 12-hour light/dark cycle with food and drinking water ad
libitum. Mice were fed a regular chow diet (LabDiet #5053); 24.5%
energy from protein, 13.1% energy from fat, and 62.4% energy from
carbohydrates.
2.2. Islet isolation, culture, dispersion and treatment
Mouse islet isolations were performed by ductal collagenase injection
followed by filtration and hand-picking, using modifications of the
protocol described by Salvalaggio [50]. Islets recovered overnight, in
islet culture media (RPMI media with 11.1 mM
D
-glucose supple-
mented with 10% vol/vol fetal bovine serum (FBS) (Thermo:
12483020) and 1% vol/vol Penicillin-Streptomycin (P/S) (GIBCO:
15140-148)) at 37

C with 5% CO
2
. After four washes with Minimal
Essential Medium [
L
-glutamine, calcium and magnesium free] (Corn-
ing: 15-015 CV) islets were dispersed with 0.01% trypsin and
resuspended in islet culture media. Cell seedings were done as per the
experimental procedure (protein synthesis: 20,000 cells per well, live
cell imaging: 5,000 cells per well). ER stress was induced by treating
islets with the SERCA inhibitor thapsigargin. For assays less than 24 h,
we used (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S). For assays greater
than 24 h we used (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S, 10% vol/
vol FBS).
2.3. Analysis of protein synthesis
Dispersed islets were seeded into an optical 96-well plate (Perkin
Elmer) at a density of approximately 20,000 cells per well in islet
culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S, 10% vol/
vol FBS). 24 h after seeding, treatments were applied in fresh islet
culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S). After in-
cubation, fresh culture media was applied (11.1 mM
D
-glucose
RPMI, 1% vol/vol P/S), then supplemented with 20
m
MOPP(Invi-
trogen) and respective drug treatments. The assay was performed
according to manufacturer’s instructions. Cells were imaged at 10
with an ImageXpress
MICRO
high-content imager and analyzed with
MetaXpress (Molecular Devices) to quantify the integrated staining
intensity of OPP-Alexa Fluor 594 in cells identified by NuclearMask
Blue Stain.
2.4. Live cell imaging
Dispersed islets were seeded into 384-well plates (Perkin Elmer) at a
density of approximately 5,000 cells per well. Islet viability was
measured with the TC20 Automated Cell Counter (Bio-Rad: 1450102)
with Trypan Blue (Bio-Rad: 1450021). Islets were allowed to adhere for
48 h in islet culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S,
10% vol/vol FBS). Cells were stained with Hoechst 33342 (Sigmae
Aldrich) (0.05
m
g/mL) and propidium iodide (SigmaeAldrich) (0.5
m
g/
mL) for 1 h in islet culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol
P/S, 10% vol/vol FBS) prior to the addition of treatments and imaging.
384-well plates were placed into the environmentally-controlled
(37

C, 5% CO2) ImageXpress
MICRO
high content imaging system. To
measure cell death, islet cells were imaged every 2 h for 84 h. Met-
aXpress software was used to quantify cell death, defined as the
number of Propidium Iodide-positive/Hoechst 33342-positive cells. To
measure Ins2 gene activity, dispersed islets from Ins2
GFP/WT
mice aged
21e23 weeks were used [51]. Islet cells were imaged every 30 min for
60 h. MetaXpress analysis software and custom R scripts were used to
perform single-cell tracking of Ins2
GFP/WT
b
cells as previously
described [51].
2.5. Western blot
After overnight recovery, islets were split equally per mouse into islet
culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S, 10% vol/vol
FBS) resulting in w100e150 islets per condition. Islets were treated
for 24 h with DMSO or 1
m
M Tg in islet culture media then sonicated in
RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1%
SDS, 50 mM Tris (pH 7.4), 2 mM EGTA, 2 mM Na
3
VO
4
, and 2 mM NaF
supplemented with complete mini protease inhibitor cocktail (Roche,
Laval, QC)). Equal protein amounts (8e10
m
g of protein) in equal
volumes were loaded for each experiment. Protein lysates were
incubated in Laemmli loading buffer (Thermo, J61337AC) at 95

C for
5 min and resolved by SDS-PAGE. Proteins were then transferred to
PVDF membranes (BioRad, CA) and probed with antibodies against
HSPA5 (1:1000, Cat. #3183, Cell Signalling), eIF2
a
(1:1000, Cat.
#2103, Cell Signalling), phospho-eIF2
a
(1:1000, Cat. #3398, Cell
Signalling), IRE1
a
(1:1000, Cat. #3294, Cell Signalling), phospho-
IRE1
a
(1:1000, Cat. #PA1-16927, Thermo Fisher Scientific), CHOP
(1:1000, #ab11419, Abcam),
b
-actin (1:1000, NB600-501, Novus
Biologicals). The signals were detected by secondary HRP-conjugated
antibodies (Anti-mouse, Cat. #7076; Anti-rabbit, Cat. #7074; CST) and
either Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific)
or Forte (Immobilon). Protein band intensities were quantified using
Image Studio (LI-COR).
2.6. Islet secretion and content
Glucose-stimulated insulin/proinsulin production and secretion were
assessed using size-matched islets (five islets per well, in triplicate)
seeded into 96-well V-bottom Tissue Culture Treated Microplates
(Corning: #CLS3894). Islets were allowed to adhere for 48 h in culture
media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S, 10% vol/vol FBS),
based on a published method [52], as this protocol permits analysis of
large sample numbers and treatments, and minimizes islet loss.
Adherent islets were washed with KrebseRinger Buffer (KRB; 129 mM
NaCl, 4.8 mM KCl, 1.2 mM MgSO
4
, 1.2 mM KH
2
PO
4
, 2.5 mM CaCl
2
,
5 mM NaHCO
3
, 10 mM HEPES, 0.5% bovine serum albumin) con-
taining 3 mM glucose then pre-incubated for 4 h in 3 mM glucose KRB.
1
m
M Tg was added to the 3 mM low glucose pre-incubation buffer 4 h
prior, 2 h prior, or at the start of the low glucose incubation period.
Islets were incubated in KRB with 3 mM glucose then 20 mM glucose
for 45 min each. Supernatant was collected after each stimulation. Islet
insulin and proinsulin content was extracted by freeze-thawing in
100
m
L of acid ethanol, then the plates were shaken at 1200 rpm for
10 min at 4

C to lyse the islets. Insulin was measured by Rodent
Insulin Chemiluminescent ELISA (ALPCO: 80-INSMR) and proinsulin by
Original Article
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Rat/Mouse Proinsulin ELISA (Mercodia: 10-1232-01). Measurements
were performed on a Spark plate reader (TECAN).
2.7. Blood collection and in vivo analysis of glucose homeostasis
and insulin secretion
Mice were fasted for 6 h prior to glucose and insulin tolerance tests.
During glucose and insulin tolerance tests, tail blood was collected
for blood glucose measurements using a glucometer (One Touch
Ultra 2 Glucometer, Lifescan, Canada). For intraperitoneal (i.p.)
glucose tolerance tests, the glucose dose was 2 g glucose/kg of body
mass. For insulin tolerance tests, the insulin dose was 0.75U insulin/
kg body mass. For measurements of in vivo glucose-stimulated in-
sulin secretion, femoral blood was collected after i.p. injection of 2 g
glucose/kg body mass. Blood samples were kept on ice during
collection, centrifuged at 2000 rpm for 10 min at 4

C and stored as
plasma at 20

C. Plasma samples were analysed for insulin using
Rodent Insulin Chemiluminescent ELISA (ALPCO: 80-INSMR).
Glucose-stimulated insulin secretion from an independent large
cohort of Insr
f/f
:Ins1Cre
/
:nTnG
þ/
mice reared in our facility was
replotted from a published Johnson lab study [53]. Blood glucose
was monitored using control and insulin-reduced mice. Control mice
were Ins1
/
:Ins2
f/f
:mTmG þtamoxifen and Ins1
/
:Ins2
f/f
:
Pdx1Cre
ERT
:mTmG þcorn oil [54]. Because we confirmed there were
no significant differences in blood glucose between control geno-
types, the data were combined in our analysis. Insulin-reduced mice
were generated by injecting Ins1
/
:Ins2
f/f
:Pdx1Cre
ERT
:mTmG mice
with tamoxifen (3 mg/40 g body weight, dissolved in corn oil, for 4
consecutive days) at 6e8 weeks [54].
2.8. RNA sequencing
To assess basal transcriptional differences, islets from male and fe-
male mice (n ¼9M, 8F) were snap-frozen and stored at 80

C until
RNA extraction. To assess Tg-induced transcriptional changes, islets
from each mouse were treated with DMSO or 1
m
M Tg for 6- or 12-
hours in culture media (11.1 mM
D
-glucose RPMI, 1% vol/vol P/S).
Four groups per sex (eight groups total) were analyzed: 6 h DMSO,
6 h Tg, 12 h DMSO and 12 h Tg, n ¼3e4 per group, each nrep-
resents pooled islet RNA from two mice. Islets were frozen at 80

Cin
100
m
L of RLT buffer (Qiagen) with beta mercaptoethanol (1%). RNA
was isolated using RNeasy Mini Kit (Qiagen #74106) according to
manufacturer’s instructions. RNA from 43 to 62 islets was pooled from
two mice and 19e150 ng of RNA was sequenced per pooled sample.
RNA sequencing was performed at the UBC Biomedical Research
Centre Sequencing Core. Sample quality control was performed using
the Agilent 2100 Bioanalyzer System (RNA Pico LabChip Kit). Qualifying
samples were prepped following the standard protocol for the NEBNext
Ultra II Stranded mRNA (New England Biolabs). Sequencing was per-
formed on the Illumina NextSeq 500 with Paired End 42bp 42bp
reads. Demultiplexed read sequences were then aligned to the
reference sequence (UCSC mm10) using STAR aligner (v 2.5.0b) [55].
Gene differential expression was analyzed using DESeq2 R package
[56]. Pathway enrichment analysis were performed using Reactome
[57]. Over-representation analysis was performed using NetworkA-
nalyst3.0 (www.networkanalyst.ca)[58].
2.9. Proteomics
Islets were treated with DMSO or 1
m
M Tg for 6 h in islet culture media
(11.1 mM
D
-glucose RPMI, 1% vol/vol P/S). Two groups per sex (four
groups total): 6 h DMSO and 6 h Tg, n ¼5e7 per group, each n
represents 200e240 islets pooled from two mice. Islet pellets were
frozen at 80

C in 100
m
L of SDS lysis buffer (4% SDS, 100 mM Tris,
pH 8) and the proteins in each sample were precipitated using acetone.
The University of Victoria proteomics service performed non-targeted
quantitative proteomic analysis using data-independent acquisition
(DIA) with LC-MS/MS on an Orbitrap mass spectrometer using 1
m
gof
protein. A mouse FASTA database was downloaded from Uniprot
(http://uniprot.org). This file was used with the 6 gas phase fraction
files from the analysis of the chromatogram library sample to create a
mouse islet-specific chromatogram library using the EncyclopeDIA (v
1.2.2) software package (Searle et al., 2018). This chromatogram li-
brary file was then used to perform identification and quantitation of
the proteins in the samples again using EncyclopeDIA with Overlapping
DIA as the acquisition type, trypsin used as the enzyme, CID/HCD as
the fragmentation, 10 ppm mass tolerances for the precursor, frag-
ment, and library mass tolerances. The Percolator version used was
3.10. The precursor FDR rate was set to 1%. Protein abundances were
log2 transformed, imputation was performed for missing values, then
proteins were normalized to median sample intensities. Differential
expression was analyzed using limma in Perseus [59].
2.10. Analysis of the transcriptome and partial proteome
Tg-induced changes to gene expression and protein levels were
compared 6 h post-treatment. Log2-transformed fold change values
were used to assess the congruence between our proteomics data and
RNAseq data. Genes and proteins that were concordantly altered by Tg
treatment at both the mRNA and protein level were searched in
PubMed for relevant literature on their role in
b
cells. The search term
used was ((“beta cell”) OR (islet) OR (“
b
cell”)) AND (Gene_Name).
Additional annotations for all mouse proteins were downloaded from
Uniprot [Dec 2022].
2.11. Data from HPAP
To compare sex differences in dynamic insulin secretion, data acquired
was from the Human Pancreas Analysis Program (HPAP-
RRID:SCR_016202) Database (https://hpap.pmacs.upenn.edu), a Hu-
man Islet Research Network (RRID:SCR_014393) consortium (UC4-
DK-112217, U01-DK-123594, UC4-DK-112232, and U01-DK-
123716).
2.12. Statistical analysis
Statistical analyses and data presentation were carried out using
GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) or R (v
4.1.1). Correlation plots were generated using the corrplot R package
(v 0.92) with default settings [60]. All R codes are published on github
(https://github.com/johnsonlabubc/ER-Stress-in-Mouse-Beta-Cells-
Data-Analysis). Student’st-tests or two-way ANOVAs were used for
parametric data. A ManneWhitney test was used for non-parametric
data. Statistical tests are indicated in the figure legends. For all sta-
tistical analyses, differences were considered significant if the p-value
was less than 0.05. *:p<0.05; **p<0.01; ***p<0.001. Data
were presented as means SEM with individual data points from
biological replicates.
3. RESULTS
3.1. Sex differences in
b
cell transcriptional and functional
responses in ND and T2D human islets
Gene expression studies on human pancreas and islets identify sig-
nificant sex differences in gene expression [7,10]. Indeed, >1500
genes expressed in the pancreas show sex-biased expression [7].
While our dataset contained fewer individuals, we also note sex dif-
ferences in mRNA levels of many genes in
b
cells (Supplementary file
MOLECULAR METABOLISM 69 (2023) 101678 Ó2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
www.molecularmetabolism.com 3

1). Given this differential gene expression, we wanted to define
b
cell-
specific gene expression changes in T2D in each sex. We therefore
used a recently-compiled meta-analysis of publicly available scRNAseq
datasets from male and female human islets [61]. Our goal was to use
sex-based analysis to determine whether
b
cell gene expression
changes in T2D are shared between the sexes. In line with prior reports
[12],
b
cells from non-diabetic (ND) and T2D donors showed signifi-
cant transcriptional differences. In
b
cells isolated from female T2D
donors, mRNA levels of 127 genes were significantly different from ND
female donors (77 downregulated, 50 upregulated in T2D) (Figure 1Ae
C). In
b
cells isolated from male T2D donors, 462 genes were
differentially expressed compared with male ND donors (138 down-
regulated, 324 upregulated in T2D) (Figure 1AeC). Of the 660 genes
that were differentially regulated in T2D, 71 were differentially regu-
lated in both males and females (15 downregulated, 56 upregulated in
T2D) (Figs. S1AeC); however, the fold change for these 71 shared
genes was different between males and females (Fig. S1A; Supple-
mentary file 2). This suggests that for shared genes, the magnitude of
gene expression changes in T2D was not the same between the sexes.
Beyond shared genes, we observed that the majority of differentially
expressed genes in T2D (589/660) were unique to either males or
females (Figs. S1B,C; Supplementary file 2). Indeed, the most prom-
inent gene expression changes in T2D were found in genes that were
unique to one sex (Figs. S2A,B; Supplementary file 2). While these data
do not address the reasons for the sex-biased risk of T2D, and could
reflect differences in medication [62e65], age [12,16,66e68], and
body mass index [15,68], our data suggest biological sex influences
b
cell gene expression in T2D.
To determine which biological pathways were altered in
b
cells of T2D
donors from each sex, we performed pathway enrichment analysis.
Genes that were upregulated in
b
cells isolated from T2D donors
included genes involved in Golgi-ER transport and the unfolded protein
response (UPR) pathways (Figure 1DeF; Supplementary file 2). While
these biological pathways were significantly upregulated in T2D in both
males and females, w75% of the differentially regulated genes in
these categories were unique to each sex (Table 1). Genes that were
downregulated in
b
cells from T2D donors revealed further differences
between the sexes: biological pathways downregulated in
b
cells from
female T2D donors included cellular responses to stress and to stimuli
(Figure 1E; Supplementary file 2), whereas
b
cells from male T2D
donors showed downregulation of pathways associated with respira-
tory electron transport and translation initiation (Figure 1F; Supple-
mentary file 2). Our analysis therefore suggests that sex-biased
b
cell
gene expression responses to T2D may influence different cellular
processes in males and females.
The sex-biased
b
cell transcriptional response in T2D prompted us to
compare glucose-stimulated insulin secretion in each sex from ND and
T2D human islets using data from the Human Pancreas Analysis
Program database [69]. In ND donors, islets from males and females
showed similar patterns of insulin secretion in response to various
stimulatory media (Figure 1G,H). In donors with T2D, we found that
insulin secretion was impaired to a greater degree in islets from males
than in females (Figure 1GeK). This difference cannot be fully attrib-
uted to a sex difference in disease severity, as our analysis of donor
characteristics revealed no significant correlation between sex and
HbA1c (Fig. S3A). Indeed, in male but not female islets, insulin
secretion was lower in donors with T2D following stimulation with both
high glucose and IBMX (Figure 1I,J), which potentiates insulin secre-
tion by increasing cAMP levels to a similar degree as the incretins [70].
Human islets from female donors with T2D therefore show a greater
ability to maintain glucose-stimulated insulin secretion than islets from
males with T2D (Figure 1K). Indeed, while diabetes status was the
main donor characteristic that correlated with changes in insulin
secretion (Fig. S3B), we noted that in T2D sex and age were two donor
characteristics showing trends toward an effect on insulin secretion
(Fig. S3A). Combined with our
b
cell gene expression data, these
findings suggest that
b
cell transcriptional and functional responses in
T2D are not shared between the sexes.
3.2. Sex differences in UPR-associated gene expression in mouse
islets
Our unbiased analysis of human
b
cell gene expression and function in
T2D revealed differences between male and female donors with T2D.
Because human
b
cell gene expression and function can be affected by
factors such as peripheral insulin sensitivity, disease processes, and
medication [31,33], we investigated sex differences in
b
cell gene
expression and function in another context. We generated a well-
powered islet RNAseq dataset from 20-week-old male and female
C57BL/6J mice. We used an insulin tolerance test (ITT) to show that
insulin sensitivity was similar between the sexes at this age (Fig. S4A);
however, we acknowledge that the ITT may not be as sensitive as a
hyperinsulinemic-euglycemic clamp in detecting modest sex differ-
ences in insulin sensitivity.
Principal component analysis and unsupervised clustering clearly
separated male and female islets on the basis of gene expression
(Figure 2A; Fig. S5A). We found that 17.7% (3268/18938) of genes
were differentially expressed between the sexes (1648 upregulated in
females, 1620 upregulated in males), in line with estimates of sex-
biased gene expression in other tissues [71,72]. Overrepresentation
and pathway enrichment analysis both identified UPR-associated
pathways as a biological process that differed significantly between
the sexes, where the majority of genes in this category were enriched
in female islets (Figure 2B,C; Supplementary file 3). Additional genes
that were enriched in female islets were those associated with the
gene ontology term “Cellular response to endoplasmic reticulum
stress”(GO:0034976), which included many genes involved in regu-
lating protein synthesis (Figure 2D). For example, females showed
significantly higher levels of most ribosomal protein genes (Figure 2E).
Further genes enriched in females included those associated with
protein folding, protein processing, and quality control (Figure 2D).
Given that protein synthesis, processing, and folding capacity are
intrinsically important for multiple islet cell types [73e76], including
b
cells [77,78], this suggests female islets may have a larger protein
production and folding capacity than male islets.
3.3. Female islets are more resilient to endoplasmic reticulum
stress in mice
The burden of insulin production causes endoplasmic reticulum (ER)
stress in
b
cells [79e81]. ER stress is associated with an attenuation
of mRNA translation [82], and, if ER stress is prolonged, can lead to cell
death [83e85]. Given that female islets exhibited higher expression of
genes associated with protein synthesis, processing, and folding than
males, and higher expression of genes associated with the UPR, which
is activated in response to ER stress [86], we examined global protein
synthesis rates in male and female islets under basal conditions and
during ER stress. We incubated islets with O-propargyl-puromycin
(OPP), which is incorporated into newly-translated proteins and can be
ligated to a fluorophore. Using this technique, we monitored the
accumulation of newly-synthesized islet proteins with single-cell
resolution (Fig. S6A). In basal culture conditions, male islet cells had
significantly greater protein synthesis rates compared with female islet
cells (Fig. S6B). To investigate islet protein synthesis under ER stress in
Original Article
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Figure 1: Sex differences in human islet transcriptomic and functional responses in type 2 diabetes. scRNAseq data from male and female human
b
cells. For donor
metadata see Supplementary file 8. (AeC) Venn diagrams compare the number of significantly differentially expressed genes between ND and T2D donors (p-adj<0.05). All
differentially expressed genes (A), downregulated genes (B), upregulated genes (C) in T2D human
b
cells. For complete gene lists see Supplementary file 1 and 2. (DeF) Top 10
significantly enriched Reactome pathways (ND vs T2D) from non-sex-specific (D), female (E), or male (F) significantly differentially expressed genes (p-adj<0.05). Gene ratio is
calculated as k/n, where kis the number of genes identified in each Reactome pathway, and nis the number of genes from the submitted gene list participating in any Reactome
pathway. For complete Reactome pathway lists see Supplementary file 2. (GeK) Human islet perifusion data from the Human Pancreas Analysis Program in ND and T2D donor
islets in females (F, I) and males (G, H). 3 mM glucose (3 mM G); 16.7 mM glucose (16.7 mM G); 0.1 mM isobutylmethylxanthine (0.1 mM IBMX); 30 mM potassium chloride
(30 mM KCl); 4 mM amino acid mixture (4 mM AAM; mM: 0.44 alanine, 0.19 arginine, 0.038 aspartate, 0.094 citrulline, 0.12 glutamate, 0.30 glycine, 0.077 histidine, 0.094
isoleucine, 0.16 leucine, 0.37 lysine, 0.05 methionine, 0.70 ornithine, 0.08 phenylalanine, 0.35 proline, 0.57 serine, 0.27 threonine, 0.073 tryptophan, and 0.20 valine, 2 mM
glutamine). (IeK) Quantification of area under the curve (AUC) is shown for the various stimulatory media in females (I), males (J) and donors with T2D (K). (I) In females, insulin
secretion from ND islets was not significantly higher than T2D islets under any culture condition (p ¼0.4806 [AAM þLG], p ¼0.2270 [AAM þHG], p ¼0.1384
[AAM þHG þIBMX], and p ¼0.1465 [KCl]; unpaired Student’st-test). (J) In males, insulin secretion from ND islets was significantly higher than T2D islets under 4 mM
AAM þ16.7 mM glucose (HG) þ0.1 mM IBMX stimulation (p ¼0.0442 [AAM þHG þIBMX]; unpaired Student’st-test), but not in other conditions (p ¼0.5315 [AAM þLG],
p¼0.0818 [AAM þHG], and p ¼0.2259 [KCl]; unpaired Student’st-test). (K) Total insulin secretion showed a trend toward lower secretion in T2D male islets than ND male islets
(p ¼0.1514 and p ¼0.0503 for females and males, respectively; unpaired Student’st-test). *indicates p <0.05; ns indicates not significant; error bars indicate SEM.
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each sex, we treated islets with thapsigargin (Tg), a specific inhibitor of
the sarcoplasmic/endoplasmic reticulum Ca
2þ
-ATPase (SERCA) that
induces ER stress and the UPR by lowering ER calcium levels [83,87].
At 2 h post-Tg treatment, protein synthesis was repressed as expected
in both male and female islet cells (Figure 3A,B; Fig. S6C). At 24 h
post-Tg treatment, we found that protein synthesis was restored to
higher-than basal levels in female islet cells, but not in male islet cells
(Figure 3A,B; Fig. S6C). Importantly, a two-way ANOVA showed that
recovery from protein synthesis repression was significantly different
between males and females (sex:treatment interaction p <0.0001).
This suggests that while protein synthesis repression associated with
ER stress was transient in female islets, this phenotype persisted for
longer in male islets. Because insulin biosynthesis accounts for
approximately half the total protein production in
b
cells [88], one
potential explanation for the sex-specific recovery from protein syn-
thesis repression is a sex difference in transcriptional changes to in-
sulin. To test this, we quantified GFP levels in
b
cells isolated from
male and female mice with GFP knocked into the endogenous mouse
Ins2 locus (Ins2
GFP/WT
)[51,89]. While ER stress induced a significant
reduction in Ins2 gene activity, this response was equivalent between
the sexes. This suggests Ins2 transcriptional changes cannot fully
explain the sex difference in recovery from protein synthesis repression
during ER stress (Figs. S7AeC).
Given the prolonged protein synthesis repression in males following ER
stress, we next quantified cell death, another ER stress-associated
phenotype [86], in male and female islets. Using a kinetic cell death
assay, we observed clear sex differences in Tg-induced cell death at
0.1
m
M and 1.0
m
M Tg doses throughout the time course of the
experiment (Figure 3C,D). Notably, viability prior to the assay was not
different between males and females (Fig. S8). After 84 h of Tg
treatment, no significant increase in female islet cell apoptosis was
observed with either 0.1
m
M or 1.0
m
M Tg treatment compared with
controls (Figure 3E). In contrast, cell death was significantly higher at
both the 0.1
m
M and the 1.0
m
M doses of Tg in male islet cells
compared with vehicle-only controls (Figure 3F). Our analysis shows
the magnitude of Tg-induced cell death was larger in male islet cells
compared with female islet cells (sex:treatment interaction p ¼0.0399
[0.1
m
M], p ¼0.0007 [1.0
m
M]). While one possible explanation for
these data is that female islets are resistant to Tg-induced cell death,
we found a significant increase in apoptosis in both female and male
islet cells treated with 10
m
MTg(Figure 3G,H, sex:treatment inter-
action p ¼0.0996 [0.1
m
M]; data graphed separately due to different
DMSO control). This suggests female islets were more resilient to mild
ER stress caused by low-dose Tg than male islets.
To determine whether this increased ER stress resilience was caused
by differential UPR signaling, we monitored levels of several protein
markers of UPR activation including binding immunoglobulin protein
(BiP), phosphorylated inositol-requiring enzyme 1 (pIRE1), phosphor-
ylated eukaryotic initiation factor alpha (peIF2
a
), and C/EBP homolo-
gous protein (CHOP) [90,91] after treating male and female islets with
1
m
M Tg for 24 h. We found no sex difference in UPR protein markers
between male and female islets without Tg treatment (Figs. S9AeD)
and observed a significant increase in levels of pIRE1
a
and CHOP in
islets from both sexes and BiP in female islets after a 24-hour Tg
treatment (Figs. S9AeD). Lack of a sex difference in protein markers
suggests UPR activation by Tg treatment was similar between male
and female islets at 20 weeks of age. This finding differs from the
male-biased UPR activation reported in the KINGS mouse model of
endogenous ER stress [37]. While one potential explanation for this
discrepancy is that Tg treatment induces acute ER stress in contrast to
the chronic ER stress in KINGS mice, further experiments will be
needed to confirm this possibility. Of note, we reproduced the male-
biased induction of BiP in islets isolated from 60-week-old male and
female mice (Figs. S9EeG), suggesting that age contributes to the sex
difference in UPR activation. Together, our data indicate that despite
equivalent UPR activation in male and female islets treated with Tg,
significant sex differences exist in ER stress-associated protein syn-
thesis repression and cell death.
3.4. Female islets retain greater
b
cell function during ER stress in
mice
We next examined glucose-stimulated insulin secretion in islets
cultured under basal conditions and during Tg treatment (Figure 4A). In
all conditions tested, high glucose significantly stimulated insulin
secretion in both sexes (Fig. S10A); however, we identified sex dif-
ferences in how well islets sustained glucose-stimulated insulin
secretion during longer Tg treatments (Figure 4B,C, Fig. S10A,B).
Female islets, in both low and high glucose, maintained robust insulin
secretion during Tg treatment (Figure 4B). Specifically, we observed a
significant increase in insulin secretion after short Tg treatment (0 and
2 h post-Tg), with a return to basal secretion levels 4 h post-Tg
(Figure 4B). Because Tg is a drug that depletes ER calcium stores, it
may induce an acute rise in cytosolic calcium that could explain this
acute increase in high glucose-stimulated insulin secretion in Tg-
treated samples compared with vehicle [83]. In contrast, male islets
showed no significant increase in insulin secretion after short Tg
treatment, and there was a significant drop in insulin secretion at 4 h
post-Tg treatment (Figure 4C). This suggests female islets sustained
insulin secretion for a longer period than male islets during ER stress.
Given that insulin content measurements showed insulin content
significantly increased during the 4-hour Tg treatment in female islets,
but not male islets (Figure 4D, Fig. S10C), our data suggest one reason
female islets maintain insulin secretion during ER stress is by aug-
menting islet insulin content. Proinsulin secretion followed similar
trends to those we observed with insulin secretion (Figs. S10D,E), but
Tg treatment reduced proinsulin content to a greater degree in male
Table 1 eHuman
b
cell pathway gene numbers. The number of genes
corresponding to each T2D upregulated pathway in males, females or both
sexes.
Pathway Name Number of Pathway Genes
Unique
Male
Common Unique
Female
Asparagine N-linked glycosylation 16 9 4
Cellular responses to stimuli 49 9 6
Cellular responses to stress 47 9 6
COPI-dependent Golgi-to-ER retrograde
traffic
762
COPI-mediated anterograde transport 7 5 2
ER to Golgi Anterograde Transport 10 5 2
Golgi-to-ER retrograde transport 7 6 2
Hedgehog ligand biogenesis 12 4 1
Hh mutants abrogate ligand secretion 12 3 1
Hh mutants are degraded by ERAD 12 3 1
IRE1alpha activates chaperones 9 3 1
Metabolism of proteins 69 20 13
Signaling by Hedgehog 16 6 2
The role of GTSE1 in G2/M progression
after G2 checkpoint
14 4 1
Unfolded Protein Response (UPR) 13 3 1
XBP1(S) activates chaperone genes 8 3 1
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Figure 2: Sex-biased gene expression in mouse islet bulk RNAseq. (A) Principal component analysis (PCA) of RNAseq data from male and female mouse islets. (B) Over-
representation analysis (ORA) of all significantly differentially expressed genes (p-adj <0.01) from male and female mouse islets. Top 30 enriched KEGG pathways (large nodes;
size ¼proportional to connections, darker red color ¼greater significance) and associated genes (small nodes; green ¼male enriched, yellow ¼female enriched). (C) Top
significantly enriched Reactome pathways from the top 1000 significantly differentially expressed genes. (p-adj <0.01) for males and females. Gene ratio is calculated as k/n,
where k is the number of genes identified in each Reactome pathway, and n is the number of genes from the submitted gene list participating in any Reactome pathway. For
complete Reactome pathway lists see Supplementary file 3. (D) All transcripts of differentially expressed genes under the gene ontology term “Cellular response to ER stress”
(GO:0034976) and genes labeled by their role in transcription, translation, protein processing, protein folding, secretion and protein quality control. (E) All transcripts of differentially
expressed ribosomal genes.
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islets (Figure 4E). There was no sex difference in the ratio of
proinsulin:insulin content at any timepoint (Fig. S10G). This suggests
that in addition to a greater ability to maintain glucose-stimulated
insulin secretion during ER stress, female islets also show a larger
increase in insulin content and a smaller decrease in proinsulin
content in this context.
To determine whether female islets have improved
b
cell function under
ER stress in other contexts, we next monitored glucose-stimulated in-
sulin secretion and glucose tolerancein mice at 20 weeks, an age where
we demonstrated that insulin sensitivity was similar between the sexes
(Figure 4FeH; Fig. S4). We found that fasting plasma insulin levels were
higher in males (Figure 4F) and that the sexes showed similar glucose
Figure 3: Sex differences in mouse islet ER stress-associated phenotypes. (A, B) Protein synthesis was quantified in dispersed islet cells from 20-week-old male and female B6
mice after treatment with 1
m
M Tg for 2- or 24-hours. (A) In female islet cells, protein synthesis was significantly lower after a 2-hour Tg treatment compared to control (p <0.0001;
one-way ANOVA followed by Tukey HSD test), significantly higher after a 24-hour Tg treatment compared to a 2-hour Tg treatment (p <0.0001; one-way ANOVA followed by Tukey
HSD test) and recovered to a significantly higher level than control levels p <0.0001; one-way ANOVA followed by Tukey HSD test). (B) In male islet cells, protein synthesis was
significantly lower after a 2- and 24-hour Tg treatment compared to control (p <0.0001 for both treatments; one-way ANOVA followed by Tukey HSD test) and was not significantly
different after a 24-hour treatment compared to a 2-hour Tg treatment p ¼0.3022; one-way ANOVA followed by Tukey HSD test). The magnitude of protein synthesis repression and
recovery was significantly different in all sex:treatment interactions (p ¼0.0015 [DMSO-2hr], p <0.0001 [DMSO-24hr], p <0.0001 [2hre24hr]; two-way ANOVA followed by Tukey
HSD test). (CeH) Quantification of propidium iodide (PI) cell death assay of dispersed islets from 20-week-old male and female B6 mice treated with thapsigargin (0.1
m
M, 1
m
Mor
10
m
M Tg) or DMSO for 84 h n ¼4e6mice,>1000 cells per group. Percentage (%) of PI positive cells was quantified as the number of PI-positive/Hoechst 33342-positive cells in
female (C) and male (D) islet cells. Relative cell death at 84 h in Tg treatments compared with DMSO treatment in females (E, G) and males (F, H). The control for both 0.1 and
1.0
m
M Tg treatmentsis 0.1% DMSO (E, F). The control for 10
m
M Tg treatment is 0.2% DMSO (G, H). In female islet cells, cell death was significantly higher in 10
m
MTgcomparedto
control (p <0.0001; unpaired Student’st-test). In male islet cells, cell death was significantly higher in 0.1, 1.0 and 10
m
M Tg compared to control (p ¼0.0230 [0.1
m
M], p <0.0001
[1
m
M] and p <0.0001 [10
m
M]; unpaired Student’st-test) (D). For E-H, at 84 h the % of PI positive cells for each treatment was normalized to the DMSO control avg for each sex. *
indicates p <0.05, ** indicates p <0.01, **** indicates p <0.0001; ns indicates not significant; error bars indicate SEM.
Original Article
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Figure 4: Sex differences in ex vivo and in vivo insulin secretion. (A) Experimental workflow of static glucose-stimulated insulin secretion. (B, C) Relative high glucose (20 mM;
high glucose, HG) in treatments compared with DMSO in female (B) and male (C) islets. Female islet HG secretion was significantly higher compared with control after 0- and 2-
hour Tg pre-treatments (p ¼0.0083 [0-hour] and p ¼0.0371 [2-hour]; Mann Whitney test). Male islet HG secretion was significantly lower compared with control after a 4-hour Tg
pre-treatment (p ¼0.0013; Mann Whitney test). (D) Insulin content. Female islet insulin content was significantly higher compared with control after a 4-hour Tg pre-treatment
(p ¼0.0269; Mann Whitney test). (E) Proinsulin content. Female islet proinsulin content was significantly lower compared with control after a 2-hour Tg pre-treatment (p ¼0.0437;
Mann Whitney test). Male islet proinsulin content was significantly lower compared with control after 2- and 4-hour Tg pre-treatments (p ¼0.0014 [2-hour] and p ¼0.0005 [4-
hour]; Mann Whitney test). (FeH) Physiology measurements after a 6-hour fast in 20-week-old male and female B6 mice. (F, G) Insulin levels from glucose-stimulated insulin
secretion tests (F: nM, G: % basal insulin) following a single glucose injection (2 g glucose/kg body weight, i.p). Area under the curve (AUC) calculations (n ¼13 females, n ¼18
males). (F) Insulin levels were significantly higher in male mice at 0 min and 30 min post injection (p ¼0.0063 [0 min] and p ¼0.0009 [30 min]; Student’st-test). AUC was
significantly higher in males (p ¼0.0159; Student’st-test). (G) Insulin levels (% baseline). Glucose-stimulated insulin secretion was significantly higher in female mice 15 min post
injection (p ¼0.0279; Student’st-test). (H) Glucose levels from glucose tolerance tests following a single glucose injection (2 g glucose/kg body weight). AUC calculations (n ¼11
females, n ¼11 males). For B-E, grey triangles indicate the concentration of insulin or proinsulin from five islets, black circles indicate the average values per mouse. *indicates
p<0.05, ** indicates p <0.01, *** indicates p <0.001; ns indicates not significant; error bars indicate SEM.
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tolerance (Figure 4H). To determine the magnitude of the acute glucose-
stimulated insulin secretion response in each sex, we normalized
glucose-stimulated insulin secretion to basal insulin secretion levels. We
found that females showed a trend toward higher acute glucose-
stimulated insulin secretion response in C57BL/6J mice (Figure 4G),
and significantly higher glucose-stimulated insulin secretion in 10- and
22-week-old Insr
f/f
:Ins1Cre
/
:nTnG
þ/
mice (Figs. S11AeD;data
replotted from a prior Johnson lab study [53]). These findings align with
data showing higher glucose-stimulated insulin secretion from prior
studies in humans and rodents [5,10,16,92,93]. We also found that
fasting blood glucose levels in female mice were more resilient to the
near-total insulin gene knockout in Ins1
/
;Ins2
fl/fl
;Pdx1CreER mice
given tamoxifen (Fig. S11E; replotted from published [54] and unpub-
lished Johnson lab data). We cannot rule out all potential factors that
may contribute to the sex differences in blood glucose levels following
near-total loss of insulin gene function (e.g. RNA stability, translation
efficiency); however, given that the burden of insulin production [54]
leads to ER stress even in normal physiological conditions, our data add
further support to a model in which
b
cells in female mice show a
greater ability to maintain glucose-stimulated insulin production and
secretion during ER stress.
3.5. Sex differences in islet transcriptional and proteomic
responses to ER stress in mice
To gain insight into the differential ER stress-associated phenotypes in
male and female islets, we investigated global transcriptional changes
after either a 6- or 12-hour Tg treatment in each sex. Principal
component analysis and unsupervised clustering shows that islets
clustered by sex, treatment, and treatment time (Figure 5A; Fig. S12A).
The majority of the variance was explained by treatment (Figure 5B),
and pathway enrichment analysis confirms the UPR as the top upre-
gulated pathway in Tg-treated male and female islets at both 6- and
12-hours after treatment (Figs. S13A,B; Supplementary file 4). While
some UPR-associated genes differentially regulated by Tg treatment
were shared between the sexes (6-hour: 29/36, 12-hour: 25/31),
biological sex explained a large proportion of variance in the gene
expression response to ER stress. This suggests the transcriptional
response to ER stress was not fully shared between the sexes. Indeed,
after a 6-hour Tg treatment, 32.6% (2247/4655) of genes that were
differentially expressed between DMSO and Tg were unique to one sex
(881 to females, 1376 to males). After a 12-hour Tg treatment, 29%
(2259/7785) were unique to one sex (1017 to males, 1242 to females).
To describe the transcriptional response of each sex to Tg treatment in
more detail, we used a two-way ANOVA to identify genes that were
upregulated, downregulated, or unchanged in male and female islets
between 6- and 12-hours post-Tg (Supplementary file 5). By per-
forming pathway enrichment analysis, we were able to determine
which processes were shared, and which processes differed, between
the sexes during Tg treatment. For example, we observed a significant
increase in mRNA levels of genes corresponding to pathways such as
cellular responses to stimuli, stress, and starvation in both male and
female islets between 6- and 12-hour Tg treatments (Figure 5C;
Supplementary file 4), suggesting Tg has similar effects on genes
related to these pathways in both sexes. Similarly, the Tg-induced
changes in mRNA levels of genes related to apoptosis were largely
shared between the sexes (Figs. S14A,B). While this suggests that the
sex-specific regulation of genes related to apoptosis does not fully
account for the susceptibility of male islets to low-dose Tg-induced cell
death, in-depth studies of
b
cell apoptosis will be needed to confirm
this point.
In contrast to these non-sex-specific changes in gene expression,
there was a male-specific increase in mRNA levels of genes associated
with translation during Tg treatment (Figure 5C; Supplementary file 4).
In females, there was a decrease in mRNA levels of genes associated
with
b
cell identity, such as Pklr,Rfx6,Hnf4a,Slc2a2,Pdx1, and MafA
(Fig. S15A), and in genes linked with regulation of gene expression in
b
cells (Figure 5C). Neither of these categories were altered between 6-
and 12-hour Tg treatments in males (Figure 5C; Fig. S15B). While our
data suggests some aspects of the gene expression response to ER
stress were shared between the sexes, we found that many genes
corresponding to important cellular processes were differentially
regulated during Tg treatment in only one sex.
Beyond sex-specific transcriptional changes following Tg treatment,
ER stress also had a sex-specific effect on the islet proteome. Although
the majority of proteins were downregulated by Tg treatment due to the
generalized repression of protein synthesis under ER stress
(Figure 5D), we identified 47 proteins (35 downregulated, 12 upre-
gulated in Tg) that were differentially expressed in female islets and 82
proteins (72 downregulated, 10 upregulated in Tg) that were differ-
entially expressed after Tg treatment in male islets (Supplementary
Table 1). Proteins downregulated only in females include proteins
associated with the GO term ‘endoplasmic reticulum to Golgi vesicle-
mediated transport’(GO:0006888) (BCAP31, COG5, COG3, GOSR1),
whereas proteins downregulated only in males include proteins
associated with GO terms ‘insulin secretion’(GO:0030073) (PTPRN2,
CLTRN, PTPRN) and ‘lysosome pathway’(KEGG) (NPC2, CTSZ, LAMP2,
PSAP, CLTA). Importantly, only seven differentially expressed proteins
were in common between the sexes (Figure 5D). This suggests that as
with our phenotypic and transcriptomic data, the proteomic response
to Tg treatment was largely not shared between the sexes.
To integrate our islet transcriptome and partial islet proteome data, we
assessed the direction of changes to mRNA and protein levels
following Tg treatment. In females, the number of differentially
expressed islet proteins with concordant mRNA changes was 43% (20/
47), whereas the number of differentially expressed islet proteins in
males with concordant mRNA changes was 49% (40/82) (Fig. S16;
Supplementary file 6). This data suggests that many genes with dif-
ferential mRNA expression during Tg treatment show congruent
changes in protein abundance. When we next asked whether the islet
genes with concordant changes in mRNA and protein levels during Tg
treatment were shared between the sexes, we found that only 7% (4/
56) of these genes were differentially expressed in both sexes during
Tg treatment (Tmem27,Emb,Fkbp9,Pdcd4). The remaining 93% (51/
55) of islet genes with concordant changes in mRNA and protein levels
during Tg treatment were differentially expressed in only one sex.
Thus, when taken together, our islet transcriptome and partial prote-
ome data suggest that male and female islets show distinct responses
to ER stress.
Of the islet genes with concordant changes in mRNA and protein levels,
several have been linked with
b
cell and/or islet function. For example,
Tmem27 plays a role in enhancing GSIS [94], and Pdcd4 expression is
associated with
b
cell death under stressed conditions [95,96]. Atp6ap2
and Lamp2 have important roles in autophagy [97e99], Ptprn2 is
required for the accumulation of insulin granules [100], and both Ptprn2
and Chga are involved in glucose-stimulated insulin secretion
[101,102]. Chga is also important for maintaining islet volume and islet
cell composition [103], and studies show that Tbc1d1 influences insulin
secretion and
b
cell mass in rodents [104,105]. Because the Tbc1d1
study [104,105], and others [94e103], used single- or mixed-sex
models, or cell lines, future studies will need to address whether
these effects on
b
cell and/or islet function are shared between the
Original Article
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Figure 5: Sex-specific transcriptomic and proteomic profiles following ER stress in mouse islets. (A) Principal component analysis (PCA) of RNAseq data from male and
female mouse islets treated with DMSO or 1
m
M Tg for 6- or 12-hours. (B) Spearman correlation depicting the variance for the first 5 principal components. (C) Top significantly
enriched Reactome pathways from the top 1000 significantly differentially expressed genes (p-adj<0.01) for females and males that were upregulated or downregulated between
6 and 12 h of Tg treatment. Gene ratio is calculated as k/n, where k is the number of genes identified in each Reactome pathway, and n is the number of genes from the submitted
gene list participating in any Reactome pathway. (D) Protein abundance from proteomics data of female and male mouse islets treated with DMSO or 1
m
M Tg for 6 h. Top 45
differentially expressed proteins are shown (p <0.05).
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www.molecularmetabolism.com 11

sexes. Additional studies will also be needed on genes identified in our
analysis that were not previously linked with
b
cell and/or islet function
(Supplementary file 6). This will elucidate whether the sex-specific
regulation of mRNA and protein levels during Tg treatment affects
b
cell and/or islet function in either sex.
4. DISCUSSION
Emerging evidence shows biological sex affects many aspects of
b
cell
gene expression and function. Yet, many studies on
b
cells do not
include both sexes, or fail to analyze male and female data separately.
To address this gap in knowledge, the goal of our study was to provide
detailed information on sex differences in islet and
b
cell gene
expression and function in multiple contexts. In humans, we used a
large scRNAseq dataset from ND and T2D donors to reveal significant
male-female differences in the magnitude of gene expression changes,
and in the identity of genes that were differentially regulated, between
ND and T2D donors. While these data do not address the reasons for the
sex-biased risk of T2D, our findings suggest
b
cell gene expression
changes in T2D are not fully shared between the sexes. These data
provide a useful resource to support future studies on how medication,
disease progression, age, or body mass index contribute to the sex
difference in
b
cell gene expression in T2D. Sex-based analysis of
human
b
cell gene expression data will also clarify the mechanisms
underlying our finding that
b
cells from female donors with T2D
maintain higher insulin production than male donors with T2D.
In mice, we generated a large RNAseq dataset using islets isolated from
20-week-old males and females. We used an ITT to show that male
and female mice have similar insulin sensitivity at this age. We
acknowledge that the ITT may not be sensitive enough to pick up small
differences, so future studies will need to use a hyperinsulinemic-
euglycemic clamp to further compare insulin sensitivity between the
sexes. Despite this potential limitation, our unbiased analysis of gene
expression in islets from males and females revealed sex differences in
genes associated with the UPR under normal physiological conditions.
This differential gene expression was significant, as female islets were
more resilient to phenotypes caused by ER stress and UPR activation
than male islets, showed sex-specific transcriptional and proteomic
changes in this context, and had a greater ability to maintain glucose-
stimulated insulin production and secretion during ER stress. Collec-
tively, these data suggest that in rodents,
b
cells from females are more
resilient to ER stress. Considering the well-established links between
ER stress and T2D [90,106e108], our data suggests a model in which
female
b
cells have a greater ability to maintain glucose-stimulated
insulin secretion in T2D because they are more resilient to ER stress
and UPR activation. While future studies are needed to test this working
model, and to assess the relative contribution of sex differences in
b
cells to the sex-biased risk of T2D, our findings highlight the impor-
tance of including both sexes in islet and
b
cell studies.
Including both sexes in our analysis of
b
cell gene expression in human
ND and T2D allowed us to uncover genes that were differentially
regulated in T2D in each sex. Because many of these genes may have
been missed if the scRNAseq data was not analyzed by sex, our findings
advance knowledge of
b
cell changes in T2D by identifying additional
genes that are differentially regulated in this context. This knowledge
adds to a growing number of studies that identify sex differences in
b
cell gene expression during aging in humans [12], and in mice fed either
anormal[4,11] or a high fat diet [11]. Further, given that our RNAseq on
islets from male and female mice with similar insulin sensitivity identifies
genes and biological pathways that align with previous studies on sex
differences in murine
b
cell gene expression [4,11], our data suggests
that sex differences in islet and
b
cell gene expression cannot be
explained solely by a male-female difference in peripheral insulin
resistance. Instead, there is likely a basal sex difference in
b
cell gene
expression that forms the foundation for sex-specific transcriptional
responses to perturbations such as ER stress and T2D. By generating
large islet gene expression datasets from male and female mice with
similar peripheral insulin sensitivity and from islets subjected to phar-
macological induction of ER stress, our studies provide a foundation of
knowledge for future studies aimed at studying the causes and con-
sequences of sex differences in islet ER stress responses and
b
cell
function following UPR activation. This will provide deeper mechanistic
insight into the sex-specific phenotypic effects reported in animal
models of
b
cell dysfunction [35e39,109e112] and the sex-biased risk
of diseases such as T2D that are associated with
b
cell dysfunction
[12,22,113,114].
Beyond gene expression,our sex-based analysis of mouse islets allowed
us to uncover male-female differences in ER stress-associated pheno-
types (e.g. protein synthesis repression, cell death). While previous
studies identify a sex difference in
b
cell loss in diabetic mouse models
[37,39,115], and show that estrogen plays a protective role via estrogen
receptor
a
(ER
a
) against ER stress to preserve
b
cell mass and prevent
apoptosis in cell lines, mouse models, and human islets [39,115,116],
we extend prior findings by showing that differences in ER stress-
induced cell death were present in the context of similar insulin sensi-
tivity between the sexes. This suggests sex differences in ER stress-
associated phenotypes do not solely depend on male-female differ-
ences in peripheral insulin sensitivity. Indeed, islets isolated from males
and females with similar insulin sensitivity also showa sex difference in
protein synthesis repression, a classical ER stress-associated phenotype
[86]. While estrogen affects insulin biosynthesis via ER
a
[117], future
studies will need to determine whetherestrogen also allows female islets
to restore protein synthesis to basal levels faster than male islets
following ER stress. We currentlylack this knowledge, as most studies on
UPR-mediated recovery from protein translation repression use single-
and mixed-sex animal groups, or cultured cells [118e123].
Assessing whether the recovery of protein synthesis contributes to
reduced cell death in female islets following ER stress will also be an
important task for future studies, as prior work suggests the inability to
recover from protein synthesis repression increases ER-stress induced
apoptosis [118]. Ideally, this type of study would also monitor the
activity of pathways known to regulate protein synthesis repression
during ER stress. For example, while we did not detect any changes in
levels of phosphorylated eIF2
a
(also known as Eif2s1), which is known
to mediate UPR-induced protein synthesis repression [86], our chosen
timepoints did not overlap with the rapid changes in phospho-eIF2
a
following ER stress published in other studies [124,125]. A more
detailed time course will therefore be necessary to assess p-eIF2
a
levels during ER stress in both sexes, and to test a role for phospho-
eIF2
a
in mediating differences in protein synthesis repression. More
work will also be needed to determine whether males and females
differ in
b
cell replication [126], another ER stress-related phenotype.
Ultimately, a better understanding of sex differences in ER stress-
associated phenotypes in
b
cells will provide a mechanistic explana-
tion for the strongly male-biased onset of diabetes-like phenotypes in
mouse models of
b
cell ER stress (e.g. Akita, KINGS, Munich mice)
[37,38,109]. Given the known relationship between ER stress,
b
cell
death, and T2D, studies on the male-female difference in
b
cell ER
stress-associated phenotypes may also advance our understanding of
the male-biased risk of developing T2D in some population groups.
Original Article
12 MOLECULAR METABOLISM 69 (2023) 101678 Ó2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
www.molecularmetabolism.com

A further benefit of additional studies on the sex difference in
b
cell ER
stress responses will be to identify mechanisms that support
b
cell
insulin production. In rodents, we found that female islets maintained
high glucose-stimulated insulin secretion and increased insulin content
following ER stress, whereas male islets showed significant repression
of high glucose-stimulated insulin secretion under the same condi-
tions. In humans, while a study using a mixed-sex group of T2D donors
shows
b
cells experience ER stress associated with
b
cell dysfunction
[63], we found that changes to
b
cell insulin secretion in T2D were not
the same between the sexes. Specifically, the magnitude of the
reduction in insulin release by
b
cells from female donors with T2D
was smaller than in
b
cells from male donors with T2D. Together with
our data from rodents, this suggests female
b
cells maintain enhanced
insulin production and/or secretion in multiple contexts, and the
increased
b
cell function cannot be solely attributed to a sex difference
in peripheral insulin sensitivity.
Clues into potential ways that female
b
cells maintain improved insulin
production and secretion emerge from our examination of the tran-
scriptional response to ER stress in mice of each sex. Our data shows
that Tg treatment induces gene expression changes characteristic of
ER stress [127], and revealed similar biological pathways that were
upregulated in T2D donors. Furthermore, we identified significant
differences between male and female islets in the transcriptional
response to ER stress over time. One notable finding was that a greater
number of
b
cell identity genes were downregulated between 6- and
12-hour Tg treatments in females, but not in males. Because most
studies on the relationship between
b
cell identity and function used a
mixed-sex pool of islets and
b
cells [79,128,129], more studies will be
needed to test whether there are sex-specific changes to
b
cell identity
during ER stress, and to determine the functional consequences of this
sex-specific effect.
Overall, our data demonstrates sex differences in
b
cell function in
multiple contexts. One potential explanation for these differences is the
sex-specific regulation of
b
cell ER stress responses and function.
Indeed, sex differences in ER stress and protein markers of apoptosis
were observed in mouse kidney cells [130], suggesting that studying
b
cell ER stress may provide insight into this difference in other cell
types. Alternatively, it is possible that there are sex differences in
b
cell
number that account for the male-female differences in
b
cell function
that we observe. For example, considering two published studies
indicate males have fewer
b
cells [6,37], the burden of maintaining
glucose homeostasis may fall on a smaller number of cells in males,
leading to higher susceptibility to ER stress. Future studies will need to
address sex differences in
b
cell number relative to pancreas size and
body size to test this possibility. Ultimately, a better understanding of
changes to
b
cell gene expression and function in males and females
will suggest effective ways to reverse disease-associated changes to
this important cell type in each sex, improving equity in health out-
comes [131].
4.1. Conclusions
Our study reports significant sex differences in islet and
b
cell gene
expression and stress responses in both humans and mice. These
differences likely contribute to sex differences in
b
cell resilience,
allowing female
b
cells to show a greater ability to maintain glucose-
stimulated insulin production and secretion across multiple contexts.
This knowledge forms a foundation for future studies aimed at un-
derstanding how sex differences in
b
cell function affect physiology
and the pathophysiology of diseases such as T2D.
AUTHOR CONTRIBUTIONS
G. P. B. conceived studies, conducted experiments, interpreted ex-
periments, wrote the manuscript
Y. X. performed bioinformatic analysis and data visualization
J. C. created custom R scripts (single-cell GFP tracking)
S. W. analyzed data (human RNAseq)
C. C. created custom R scripts (mouse RNAseq analysis)
J. A. Z. analyzed data (HPAP perifusions)
S. S. conducted experiments (in vivo physiology)
E. P. conducted experiments (islet western blots)
X. H. conducted experiments (dissections)
J. D. J. conceived studies, interpreted experiments, edited the
manuscript
E. J. R. conceived studies, interpreted experiments, edited the
manuscript, and is the guarantor of this work
FUNDING
This study was supported by operating grants to E.J.R. from the
Michael Smith Foundation for Health Research (16876), Canadian In-
stitutes of Health Research (GS4-171365), the Canadian Foundation
for Innovation (JELF-34879), and Diabetes Canada (OG-3-22-5646-
ER), and to J.D.J. (PJT-152999) from the Canadian Institutes of Health
Research, and core support from the JDRF Centre of Excellence at UBC
(3-COE-2022-1103-M-B). J.D.J. was funded by a Diabetes Investi-
gator Award from Diabetes Canada.
DATA AVAILABILITY
Details of all statistical tests and p-values as well as the raw data are
provided in Supplementary files. Supplementary files are available as
Brownrigg, George (2023), “Sex differences in islet stress responses
support female
b
cell resilience”, Mendeley Data, V1, doi: https://doi.
org/10.17632/ftcs6xj9ft.1. RNAseq data is available at PRJNA842443
and PRJNA842371.
ACKNOWLEDGEMENTS
We thank members of the Rideout and Johnson lab for valuable feedback. We thank
our animal care staff for supporting our animal husbandry, UBC Biomedical Research
Center for performing RNA sequencing, and UVic Genome BC Proteomics Center for
performing proteomics. We acknowledge that our research takes place on the
traditional, ancestral, and unceded territory of the Musqueam people; a privilege for
which we are grateful.
CONFLICT OF INTEREST
The authors declare no competing interest.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data to this article can be found online at https://doi.org/10.1016/j.
molmet.2023.101678.
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