Abstract
The present study examines differences in metabolic and pancreatic islet adaptative responses following streptozotocin (STZ) and hydrocortisone (HC) administration in male and female transgenic GluCreERT2/Rosa26-eYFP mice. Mice received five daily doses of STZ (50 mg/kg, i.p.) or 10 daily doses of HC (70 mg/kg, i.p.), with parameters assessed on day 11. STZ-induced hyperglycaemia was evident in both sexes, alongside impaired glucose tolerance and reduced insulin concentrations. HC also had similar metabolic effects in male and female mice resulting in classical increases of circulating insulin indicative of insulin resistance. Control male mice had larger pancreatic islets than females and displayed a greater reduction of islet and beta-cell area in response to STZ insult. In addition, female STZ mice had lower levels of beta-cell apoptosis than male counterparts. Following HC administration, female mouse islets contained a greater proportion of alpha cells when compared to males. All HC mice presented with relatively comparable increases in beta- and alpha-cell turnover rates, with female mice being slightly more susceptible to HC-induced beta-cell apoptosis. Interestingly, healthy control female mice had inherently increased alpha-to-beta-cell transdifferentiation rates, which was decreased by HC treatment. The number of glucagon-positive alpha cells altering their lineage to insulin-positive beta cells was increased in male, but not female, STZ mice. Taken together, although there was no obvious sex-specific alteration of metabolic profile in STZ or HC mice, subtle differences in pancreatic islet morphology emphasises the impact of sex hormones on islets and importance of taking care when interpreting observations between males and females.
Introduction
Epidemiological data suggest that males are at increased risk of developing type 2 diabetes (T2D) than females, but that this sex bias is absent with onset of the menopause (Saeedi et al. 2020). Clearly, there is an implication that oestrogen acts as a protective factor for pancreatic islet beta cells to help prevent development of T2D (Tiano & Mauvais-Jarvis 2012). Beyond this, males and females also show divergent responses in terms of diabetes complications, efficacy of pharmacological interventions and drug metabolism (Kautzky-Willer et al. 2023). This sexual dimorphism is poorly understood for various reasons. First, preclinical assessment of novel antidiabetic compounds generally tends to be conducted in male animals to circumvent postulated metabolic variabilities associated with the oestrus cycle (Bailey & Matty 1972, Richardson et al. 2015). Second, prior to 1993, U.S. Food and Drug Administration guidance for clinical testing of new drug entities recommended initial assessment almost exclusively in male populations (FDA 1993). Such matters were highlighted with devastating effects in the well-publicised thalidomide cases of the 1950s, resulting in the death of 2000 children and severe birth defects in thousands more (Vargesson 2015). Preclinical testing of the drug using female tissue or animals could have predicted the teratogenic nature of thalidomide (Therapontos et al. 2009). Despite efforts to promote clinical assessment in wider demographic profiles, women of child-bearing age are still under-represented in early clinical trials (Steinberg et al. 2021). As a result, approved drugs can still fail in the real-world setting due to undetected female specific side effects (Zucker & Prendergast 2020). It follows that preclinical trials should be designed to fully account for possible sex-based dimorphic impact, necessitating a greater appreciation of the underlying differences in male and female models of disease.
In this respect, the impact that sex exerts in the response to streptozotocin (STZ) or hydrocortisone (HC) administration, frequently adopted approaches to chemically induce contrasting aetiologies of metabolic dysregulation in rodents, is not fully understood. As such, STZ is a toxic glucose analogue that is selectively taken up by beta cells via the GLUT2 transporter (Lenzen 2008). Within the beta-cell STZ alkylates proteins and fragments DNA culminating in beta-cell death and insulin-dependent diabetes (Lenzen 2008). Interestingly, female mice are considered to be less susceptible to STZ-induced diabetes than male counterparts (Kolb 1987, Kim et al. 2020), possibly due to oestrogen mediated beta-cell protective effects. However, others have questioned this opinion and highlight that female rodents are still equally susceptible to STZ-induced diabetes as male mice (Plesner et al. 2014). On the other hand, glucocorticoids, such as hydrocortisone (HC) and prednisolone, are recognised to induce insulin resistance and to some extent adversely alter insulin secretion (Hansen et al. 2010). In peripheral tissues, prolonged glucocorticoid administration increases hepatic glucose production (Dirlewanger et al. 2000) and reduces glucose uptake by decreasing GLUT4 expression (Saad et al. 1993). In addition, within pancreatic beta cells, glucocorticoids have been demonstrated to inhibit insulin synthesis and secretion, highlighting these cells as an important target for the negative metabolic actions of glucocorticoids (Delaunay et al. 1999). Despite this, the possible sex-dependent differences of HC and STZ on glucose homeostasis, and especially pancreatic islet histology and cell lineage, has not been fully investigated (Kaikaew et al. 2019).
Therefore, the present study aims to compare and contrast the metabolic responses and alterations of pancreatic islet histology in response to STZ and HC treatment in male and female transgenic GluCreERT2/Rosa26-eYFP mice. Importantly, use of GluCreERT2/Rosa26-eYFP mice permits for investigation of the possible sex-dependent effects on pancreatic alpha-cell lineage and related alpha-to-beta-cell transdifferentiation, that is now known to be important for the development and progression of diabetes (Saleh et al. 2021). In that regard, the primary focus of the work was assessment of changes in pancreatic islet morphology, as well as alpha and beta islet cell populations, in male and female mice treated with STZ or HC. This is the first study to directly compare the sex-specific impact of STZ and HC on pancreatic islet cell morphology, as well as to investigate the impact of these diabetogens on islet cell transdifferentiation events in both sexes.
Methods
Animals
Transgenic GluCreERT2/Rosa26-eYFP C57BL/6 mice were bred in house within the Biomedical and Behavioural Research Unit (BBRU) at Ulster University, Coleraine. These mice express an inducible eYFP reporter specifically in cells expressing the glucagon gene and have previously been fully characterised (Campbell et al. 2020). For experiments, mice were individually housed with ad libitum access to standard chow and water in a temperature controlled, 12-h light:12-h darkness cycle environment. The following experiments were approved by Ulster University Animal Welfare and Ethics Review Committee (AWERB) and conducted in line with the UK Animals (Scientific Procedures) Act 1986. In brief, 14-week-old male and female mice (n = 7 mice/group) received either saline control (0.9% NaCl), STZ (S0130, Sigma-Aldrich) or HC (H0888, Sigma-Aldrich) to induce insulin deficiency or insulin resistance, respectively, as detailed below. One week prior to administration of STZ or HC, all mice were administered a single i.p. dose of tamoxifen (7 mg/mouse, T2859, Sigma-Aldrich) to induce eYFP expression. To provoke insulin deficiency, STZ (50 mg/kg, freshly dissolved in citrate buffer) was administered by intraperitoneal (i.p.) injection on 5 consecutive days, with overt hyperglycaemia observed 5 days following the last injection. For insulin resistance, HC (70 mg/kg, dissolved in PBS) was administered by daily i.p. injections for 10 consecutive days. At regular intervals, body weight, non-fasting glucose and energy intake were recorded. Energy intake was assessed by weighing the food hopper of individually caged mice at regular timepoints. All experiments were terminated on day 11 with appropriate collection of blood and pancreatic tissues as described below.
Biochemical analyses
Blood glucose was measured from tail vein blood using an Ascensia Contour blood glucometer. Terminal blood samples were collected in heparin/fluoride-coated microcentrifuge tubes and centrifuged for 10 min at 1500 g. Separated plasma was then stored at −70℃ until assessment of insulin concentrations. At termination, pancreatic tissues were excised, divided longitudinally, and processed for either determination of pancreatic hormone content following acid/ethanol protein extraction or fixed in 4% paraformaldehyde for 48 h at 4℃ for histological analysis. Pancreatic and plasma insulin content were determined by radioimmunoassay (Flatt & Bailey 1981), whilst total protein content was assessed using Bradford reagent.
Immunohistochemistry
Fixed tissue was embedded into paraffin wax blocks and sectioned at 5 µm slices using a microtome (Shandon Finesse 325, Thermo Scientific), and sections selected at intervals of every ten sections. Immunohistochemistry was conducted to assess islet morphology, cellular proliferation and apoptosis rates as well as alpha-cell transdifferentiation. In brief, slides were dewaxed in xylene and rehydrated in an alcohol gradient (100–50% EtOH). Antigen retrieval was carried out in citrate buffer (pH 6, 90℃), followed by blocking in 4% BSA solution. Slides were then incubated with primary antibodies (Table 1) overnight at 4℃, prior to rinsing in PBS and incubation with the appropriate fluorescently conjugated secondary antibodies (Table 1). Slides were also incubated with DAPI before mounting and imaging using a fluorescent microscope (Olympus model BX51) fitted with DAPI (350 nm), FITC (488 nm) and TRITC (594 nm) filters and a DP70 camera adapter system.
Target, host, fluorophore, dilution, supplier and combinations employed of primary and secondary antibodies employed for immunohistochemistry.
| Primary antibodies | ||||
|---|---|---|---|---|
| Target | Host | Fluorophore | Dilution | Supplier |
| Insulin | Mouse | N/A | 1:400 | Abcam (ab6995) |
| Glucagon | Rabbit | N/A | 1:1000 | Abcam (ab92517) |
| Glucagona | Guinea pig | N/A | 1:400 | Raised in-house (PCA2/4) |
| GFP | Goat | N/A | 1:500 | Abcam (ab5450) |
| Ki-67 | Rabbit | N/A | 1:500 | Abcam (ab15580) |
| Secondary antibodies | ||||
| Target | Host | Fluorophore | Dilution | Supplier |
| Mouse | Donkey | Alexa Fluor 594 | 1:400 | Invitrogen (A-21203) |
| Mouse | Donkey | Alexa Fluor 488 | 1:400 | Invitrogen (A-21202) |
| Rabbit | Donkey | Alexa Fluor 594 | 1:400 | Invitrogen (A-21207) |
| Rabbit | Donkey | Alexa Fluor 488 | 1:400 | Invitrogen (A-21206) |
| Goat | Donkey | Alexa Fluor 488 | 1:400 | Invitrogen (A-11055) |
| Guinea pig | Goat | Alexa Fluor 488 | 1:400 | Invitrogen (A-11073) |
aTo overcome species cross-reactivity when assessing alpha-cell proliferation, the guinea pig anti-glucagon antibody was employed for these experiments. For all other assessments of glucagon staining, the rabbit anti-glucagon antibody was employed.
Image analysis
ImageJ software was used to analyse islet morphology, employing the ‘closed polygon’ tool to identify regions of insulin-positive and glucagon-positive staining areas to define beta- and alpha-cell areas, respectively (Khan et al. 2016). To quantify alpha- and beta-cell apoptosis and proliferation rates, the number of insulin or glucagon positive cells expressing TUNEL or Ki-67, respectively, were quantified and expressed as a percentage of total insulin/glucagon positive cells. To assess effects on alpha-cell lineage, YFP was detected with a goat anti-GFP antibody (Table 1), which is reactive against all variants of Aequorea victoria GFP, including YFP. In this regard, cells expressing both glucagon and GFP were considered ‘mature alpha cells’, whilst cells expressing GFP but lacking in glucagon were termed ‘dedifferentiated alpha-cells’ (Lafferty et al. 2021). In addition, islet cells expressing GFP together with insulin were deemed to have undergone alpha-to-beta-cell transdifferentiation.
Statistics
Results were analysed using GraphPad PRISM (version 8.0) software, with data presented as mean ± S.E.M. Comparative analyses between groups were carried out using a one-way or two-way ANOVA, as appropriate, utilising a Bonferroni post hoc test for multiple comparisons between treatment groups and sex. In this respect, the P-values for interaction between treatment and sex fell between 0.1 and 1.0 with analysis subsequently progressing to post hoc tests to derive which groups have differences for each individual factor studied. Results were deemed significant if P < 0.05.
Results
Effects of STZ and HC on circulating glucose, plasma and pancreatic insulin, glucose tolerance, body weight and food intake in male and female mice
Male and female mice treated with multiple low-dose STZ developed comparable hyperglycaemia (P < 0.001, Fig. 1A and B), in conjunction with reduced plasma and pancreatic insulin concentrations (P < 0.01, Fig. 1C and D) and impaired glucose tolerance (P < 0.001, Fig. 1E and F) when compared to control mice. As expected, control male mice had elevated body weight when compared to females (26.8 ± 1.1 vs 19.6 ± 0.9 g, respectively; P < 0.01), with all STZ-treated mice presenting with reduced body weight (P < 0.05 to P > 0.001, Fig. 1G and H), and interestingly this associated with a small but significant reduction in food intake in male, but not female, mice (P < 0.05, Fig. 1I). Administration of HC reduced non-fasting blood glucose in female mice on day 11 (P < 0.01, Fig. 1B), but this effect was not apparent in male mice (Fig. 1B). Moreover, glucose tolerance was slightly improved in female, but not male, HC-treated mice (P < 0.01, Fig. 1E and F). As expected, plasma insulin levels were elevated in all HC-treated mice (P < 0.01–0.001, Fig. 1C), but significantly less so in males (P < 0.05, Fig. 1C), with no obvious changes in pancreatic insulin content (Fig. 1D). Body weights of all HC mice were reduced when compared to controls (P < 0.05–0.01, Fig. 1G and H), but this occurred in the absence of changes in food intake (Fig. 1I).
Effects of STZ or HC treatment on female and male GluCreERT2/Rosa26-eYFP mice. (A, B) blood glucose, (C) plasma insulin, (D) pancreatic insulin, (E, F) glucose tolerance, (G) body weight, (H) percentage weight change and (I) cumulative food intake. Mice received five daily doses of streptozotocin (50 mg/kg, i.p.) or 10 daily doses of hydrocortisone (70 mg/kg, i.p.) with variables measured during (A, G, I) this period or on day 11 (B–F, H). All values represent mean ± s.e.m. for seven mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared to saline controls. ΔP < 0.05 compared to respective female mice under the same treatment regimen. (B–D, F, H) Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test or (A, E, G, I) using a two-way ANOVA with a Bonferroni post hoc test.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
Effects of STZ and HC on pancreatic islet histology in male and female mice
Islet size and composition varied with sex, with healthy female mice having reduced islet area when compared to their male counterparts (P < 0.05–0.01, Fig. 2A) that were composed of fewer beta cells (P < 0.001, Fig. 2B, C, D and E). When treated with low-dose STZ, male mice presented with reduced islet (P < 0.05, Fig. 2A) and beta-cell areas (P < 0.001, Fig. 2B), an effect that was not observed in females (Fig. 2B). STZ treatment elevated alpha-cell area in female mice (P < 0.05, Fig. 2C), with alpha:beta ratio greatly increased in both sexes (P < 0.05–0.001, Fig. 2E). In addition, the number of islets with centrally distributed alpha cells was notably increased in STZ-treated mice of both sexes (P < 0.01, Fig. 2F). HC treatment had no discernible impact on pancreatic islet morphology in either male or female mice, with the exception that female mouse islets were composed of more alpha cells than their HC treated male counterparts (P < 0.01, Fig. 2D). Representative images of stained islets are shown in Fig. 2G.
Effects of STZ or HC treatment on pancreatic islet morphology in female and male GluCreERT2/Rosa26-eYFP mice. (A) Islet area, (B) beta-cell area, (C) alpha-cell area, (D) alpha:beta ratio, (E) alpha-cell distribution and (F) islet composition were assessed on day 11. (G) Representative images of islets from each group highlighting insulin and glucagon staining. All values represent mean ± s.e.m., with >50 islets analysed per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to saline controls. ΔP < 0.05, ΔΔP < 0.01 compared to respective female mice under the same treatment regimen. Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-23-0174.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
Effects of STZ and HC on pancreatic islet cell turnover in male and female mice
Multiple low-dose STZ treatment increased beta-cell apoptosis in all mice (P < 0.001, Fig. 3A and B), but this was significantly more apparent in males (P < 0.05, Fig. 3A). Likewise, alpha-cell apoptosis was elevated in male STZ-treated mice (P < 0.05, Fig. 3C and D), with STZ female mice presenting with reduced alpha-cell apoptotic rates (P < 0.05, Fig. 3C). Beta-cell proliferation was unaffected by STZ (Fig. 4A and B), whilst alpha-cell proliferation was significantly increased in all STZ mice (P < 0.05, Fig. 4C and D). Further analyses of islet cell turnover rates confirmed that STZ selectively targeted beta cells for apoptosis regardless of the sex of the mouse (Fig. 5A), with no impact on proliferation frequency of these cells (Fig. 5B). In all STZ-treated mouse islets, beta-cell apoptosis significantly outweighed proliferation (P < 0.001, Fig. 5C), but the effect was more prominent in male mice (P < 0.01, Fig. 5C). Interestingly, alpha-cell apoptosis also predominated over proliferation in STZ-treated male mice (P < 0.01 Fig. 5D), but the opposite was observed in female mice (P < 0.001 Fig. 5D).
Effects of STZ or HC treatment on islet cell apoptosis in female and male GluCreERT2/Rosa26-eYFP mice. (A) Beta-cell apoptosis and (B) alpha-cell apoptosis were assessed on day 11. Representative images of islets from each group showing (C) insulin and TUNEL or (D) glucagon and TUNEL co-staining. All values represent mean ± s.e.m., with >50 islets analysed per group. *P < 0.05, ***P < 0.001 compared to saline controls. ΔP< 0.05 compared to respective female mice under the same treatment regimen. Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-23-0174.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
Effects of STZ or HC treatment on islet cell proliferation in female and male GluCreERT2/Rosa26-eYFP mice. (A) Beta-cell proliferation and (B) alpha-cell proliferation were assessed on day 11. Representative images of islets from each group showing (C) insulin and Ki-67 or (D) glucagon and Ki-67 co-staining. All values represent mean ± s.e.m., with >50 islets analysed per group. *P < 0.05 compared to saline controls. Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-23-0174.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
Effects of STZ or HC treatment on islet cell proliferation and apoptosis ratios in female and male GluCreERT2/Rosa26-eYFP mice. (A) beta:alpha apoptosis ratio (B) alpha:beta proliferation ratio, (C) beta-cell apoptosis:proliferation ratio and (D) alpha-cell apoptosis:proliferation ratio were both determined on day 11. All values represent mean ± s.e.m., with >50 islets analysed per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to saline controls. ΔP <0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 compared to respective female mice under the same treatment regimen. Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
HC treatment elicited an increase in beta-cell apoptosis (P < 0.001, Fig. 3A and B) and proliferation (P < 0.05, Fig. 4A and B) in all mice. However, beta-cell apoptosis rates were elevated in male as compared to female HC-treated mice (P < 0.05, Fig. 3A). Alpha-cell apoptosis and proliferation were unaffected by HC treatment and not different between sexes (Fig. 3C and 4C). Accordingly, the beta:alpha cell apoptosis ratio was enhanced in female HC treated mice when compared to controls and their male counterparts (P < 0.05–0.001, Fig. 5A). In all HC treated mice, alpha:beta cell apoptosis ratio was consistently reduced when compared to control mice (P < 0.05–0.001, Fig. 5B). On specific examination of alpha and beta cells, apoptosis outweighed proliferation within beta cells of female HC mice, whilst the opposite was true in alpha cells, when compared to control mice (P < 0.05–0.01, Fig. 5C and D). In male HC mice, there was no difference in the balance of apoptosis and proliferation rates in beta cells (Fig. 5C), but apoptosis was elevated within alpha cells (P < 0.01, Fig. 5D). In addition, female HC mice had a significantly decreased apoptosis:proliferation ratio in alpha cells when compared to males (P < 0.01, Fig. 5D).
Effects of STZ and HC on pancreatic islet cell lineage in male and female mice
Lineage tracing of alpha cells, utilising GFP staining, revealed that STZ treatment increased the percentage of GFP-positive cells co-expressing glucagon (mature alpha cells) as well as decreasing numbers of glucagon negative, GFP-positive (dedifferentiated alpha cells) islet cells independent of mouse sex (P < 0.001, Fig. 6A and B). HC treatment produced a similar increase in cells co-expressing both glucagon and GFP and decrease in cells expressing GFP without glucagon, but interestingly only in male mice (P < 0.01, Fig. 6A and B). However, it should also be noted that saline-treated female control mice had a greater percentage of mature alpha cells than corresponding males (P < 0.05, Fig. 6A). Healthy female mice displayed higher numbers of GFP-positive cells co-expressing insulin (alpha-to-beta-cell transdifferentiation) when compared to males (P < 0.01, Fig. 6C). In female mice, only HC treatment reduced the number of GFP cells co-expressing insulin (P < 0.05, Fig. 6C), whilst STZ treatment increased the number of GFP positive cells expressing insulin only in male mice (P < 0.05, Fig. 6C). Representative images of islets from each group of mice stained for glucagon or insulin alongside GFP are depicted in Fig. 6D and E, respectively.
Effects of STZ or HC treatment on alpha-cell lineage in female and male GluCreERT2/Rosa26-eYFP mice. (A) Mature alpha cells, (B) dedifferentiated alpha cells and (C) alpha-to-beta-cell transdifferentiation were assessed on day 11. All values represent mean ± s.e.m., with >50 islets analysed per group. Representative images of islets from each group of mice stained for glucagon (D) or insulin (E) alongside GFP and DAPI are also shown. *P < 0.05, ***P < 0.001 compared to saline controls. ΔP < 0.05, ΔΔP < 0.01 compared to respective female mice under the same treatment regimen. Datasets were analysed using a one-way ANOVA with Bonferroni post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-23-0174.
Citation: Journal of Endocrinology 259, 2; 10.1530/JOE-23-0174
Discussion
The present study explored how sex impacts pancreatic islet adaptive responses to STZ and HC administration in mice. As expected, STZ and HC elicited characteristic metabolic dysregulation, which was not dependent on the sex of the animal (Tanday et al. 2020a , Daniels Gatward et al. 2021). However, subtle sex-specific differences were observed at the level of the endocrine islet that merit further exploration. This serves to emphasise the importance of understanding the implications of sex on diabetic pathophysiology, and potential related influences on drug discovery programs.
Specifically, in the current setting, male mice displayed typical STZ-induced beta-cell damage, with associated severe hyperglycaemia and weight loss, akin to previous observations (Tanday et al. 2020a ) and likely related to an increased proportion of beta cells in male mice (Yokomizo et al. 2014). There is a belief that female mice are more tolerant to the detrimental effects of STZ than males (Ariza et al. 2014, Chandramouli et al. 2018, Kim et al. 2020). In that respect, several studies have already investigated the sex-specific effects of STZ treatment in mice, with a general consensus that the diabetogen causes more detrimental effects on pancreatic islet morphology in male than female mice (Kolb 1987, Abildgaard et al. 2020, Kim et al. 2020, 2023), although this has been debated (Plesner et al. 2014). Mechanistically, increased STZ resistance in female mice has been demonstrated to be related to higher oestrogen levels, with oestrogen known to suppress inflammation (Abildgaard et al. 2020), protect against inflammatory cytokine insult (Sachs et al. 2020), promote misfolded protein degradation (Xu et al. 2018) and enhance glucose-stimulated insulin secretion (Tiano & Mauvais-Jarvis 2012). Indeed, upon ovariectomy, female mice become more susceptible to STZ with similar metabolic dysregulation as observed in male mice (Kim et al. 2023). In harmony with this, ovariectomised female mice treated with oestradiol present with an improved protection against the harmful effects of STZ (Li et al. 2018). Our data would support some inherent protection from the islet cell destructive effects of STZ in female mice, but importantly both male and female GluCreERT2/Rosa26-eYFP mice presented with blood glucose levels in excess of 15 mmol/L following STZ administration, combined with severely impaired glucose tolerance and reduced plasma and pancreatic insulin. Indeed, the only assessed metabolic parameter where STZ was more harmful to male over female mice was body weight loss, which appeared to be linked to decreased food intake. However, this observation does need to be considered in light of the initial increased body weight of male mice (de Souza et al. 2022).
In keeping with this, pancreatic islets from male mice were larger than females, which may represent another factor as to why male mice displayed more obvious STZ-induced reductions in islet and beta-cell areas. In some accord, female STZ mice exhibited less pronounced increases in beta-cell apoptosis, perhaps linked to protective oestrogen actions (Abildgaard et al. 2020). This may be particularly relevant given the perceived importance of modulation of programmed beta-cell death for both the onset of diabetes (Eizirik et al. 2020), as well as the effectiveness of some antidiabetic therapies including GLP-1 receptor agonists (Kapodistria et al. 2018, Díaz-Megido & Thomsen 2023). In that regard, similar investigations in ovariectomised female mice with depleted oestrogen levels would be of interest. It is perhaps also worth noting that a single low dose tamoxifen injection was employed to induce Cre–lox recombination in all our GluCreERT2/Rosa26-eYFP transgenic mice, but this is a commonly employed tool with minimal adverse effects (Lafferty et al. 2021) and only prolonged exposure at elevated doses should directly impact metabolic state (Ceasrine et al. 2019). Thus, whilst low dose tamoxifen has been suggested to alter metabolism in mice, this earlier investigation employed either repeated administration at a dose more than three times our selected 7 mg/kg tamoxifen dose, or a single tamoxifen injection some seven times greater than this (Zhao et al. 2020). That said, we are unable to completely rule out an impact of the single low-dose tamoxifen injection on metabolism and islet morphology, despite the oestrogen receptor modulator being delivered 18 days prior to pancreatic tissue extraction, since persistent effects of a single tamoxifen injection are reported under certain environments (Patel et al. 2017, Stout et al. 2021). Accordingly, GluCreERT2/Rosa26-eYFP transgenic mice require tamoxifen administration to study changes in islet cell lineage, but it would be interesting to examine the impact of STZ and HC on the other aspects of pancreatic islet architecture assessed within the current setting using wildtype male and female mice. Unfortunately, such additional studies are outside the scope of the current work, however this factor should not be totally discounted when interpreting our datasets.
Although the impact of chemical diabetogens on beta-cell health is often the primary consideration in terms of modelling the phenotype in rodents, effects on the alpha cell merit further contemplation (Gannon et al. 2018). This seems particularly relevant given recent knowledge on the plasticity of mature islet cells and ability of alpha cells to function as progenitors for insulin positive beta cells through distinct lineage alteration (Tanday et al. 2020b). Moreover, alpha cells are innately more resistant to oxidative stress and induction of apoptosis than beta cells (Eizirik et al. 2023). Indeed, there is a suggestion that alpha cells could represent a viable direct target for diabetes therapies (Klempel et al. 2022). Notably, in the current study female mouse islets were composed of more alpha cells than males, with no obvious difference in turnover rates. Thus, our observations of increased alpha-to-beta-cell transdifferentiation in female mice may be related to this phenomenon as an inherent adaptation to increased alpha-cell area, but this still needs to be confirmed. A small study in 52 non-diabetic subjects reports that alpha-cell mass is not different between men and women (Henquin & Rahier 2011), whereas others suggest females have slightly more beta cells than males (Marchese et al. 2015), highlighting some uncertainty in this regard. However, such observations also need to be considered with respect to the apparent differences between human and rodent alpha-cell signalling and function (Moede et al. 2020).
Interestingly, in response to multiple low-dose STZ treatment, male and female mice had opposing effects on alpha-cell apoptotic rates, but similar increases in proliferation, resulting in elevated alpha-cell area especially in female mice. Alpha-to-beta-cell transdifferentiation was increased in male STZ mice, likely as an adaptive response to help preserve beta-cell mass. Thus, in the absence of oestrogen signalling that possesses independent beta-cell protective actions (Kim et al. 2023), islet cell lineage alteration in the face of a specific beta-cell insult could be paramount. Whether oestrogen could be exploited as an antidiabetic therapy is debatable, and detrimental off-target side effects may ultimately limit overall effectiveness (Sjögren et al. 2016). However, approaches utilising GLP-1/oestrogen conjugates to allow for more targeted oestrogen delivery are encouraging (Schwenk et al. 2014, Sachs et al. 2020).
Insulin resistance is a major risk factor for T2D and can be modelled preclinically by sustained administration of glucocorticoids. Whilst females appear to have inherent protection against developing insulin resistance (Aldhoon-Hainerová et al. 2014), this is absent upon menopause (Janssen et al. 2008). In keeping with this, the risk of insulin resistance increases in females when the menopause is surgically induced (Christakis et al. 2020) and decreases with hormone replacement therapy (Salpeter et al. 2006). Intriguingly, in our model, HC treatment resulted in mild insulin resistance typified by elevated plasma insulin levels, especially in female mice, with no real alteration of glucose homeostasis. Morphologically, pancreatic islets were largely unaltered in HC treated mice, that perhaps contrasts with other reports (Vasu et al. 2014, Mohan et al. 2022) but agrees with our observations of equivalent increases in beta-cell proliferation and apoptosis rates that was unrelated to sex. Interestingly, despite a putative protective effect of oestrogen on beta-cell survival (Sachs et al. 2020), female HC mice displayed particularly high rates of beta-cell apoptosis. Androgens have been shown to sensitise mice to glucocorticoid-induced insulin resistance, and it may be interesting to assess the impact of orchidectomy in our male HC mice (Gasparini et al. 2019). Perhaps most fascinating was the decrease in alpha-to-beta-cell lineage switch in female HC mice, suggesting that at least in female mice this pathway is less important for regulating beta-cell mass in the face of insulin resistance. That said, male HC mice had increased numbers of mature, and decreased numbers of dedifferentiated alpha cells, suggesting specific islet cell lineage changes that merit more in-depth assessment. Collectively, alpha-to-beta-cell transdifferentiation was elevated in male STZ mice, but decreased by HC administration in female mice, possibly reflecting both sex-specific actions and the contrasting aetiologies of these rodent models (Daniels Gatward et al. 2021).
In conclusion, STZ- and HC-induced insulin deficiency or resistance, respectively, in GluCreERT2/Rosa26-eYFP transgenic mice results in characteristic metabolic outcomes that are similar in male and female mice. However, there are notable sex-specific differences in associated adaptive responses within the pancreatic islets of these mice. We have described alterations in both islet morphology as well as cellular turnover and transdifferentiation rates between sexes, that merit further consideration and should be remembered when extrapolating observations from males to females.
Declaration of interest
All authors declare no conflict of interest.
Funding
These studies were supported by Ulster University Research Funding support and award of an Ulster University Vice Chancellors Research Studentship to VD. RCM is supported by an RD Lawrence Fellowship awarded by Diabetes UK.
Data availability
The authors declare that the data supporting the findings of this study are available within the article. Any additional raw data supporting the conclusions of this article will be made available by the lead author, without undue reservation.
Author contribution statement
NT, RCM, PRF and NI conceived/designed the study. NI and NT drafted the manuscript. NT, AC-P, KS and VD participated in the conduct/data collection and analysis and interpretation of data. All authors revised the manuscript critically for intellectual content and approved the final version of the manuscript.
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