As model systems, we utilized the Kimba, Akita and Akimba mouse models. The Kimba mouse model (trVEGF029) transiently overexpresses human vascular endothelial growth factor in photoreceptors and demonstrates neovascular changes associated with DR without a hyperglycemic background [22]. These DR associated changes occur at the age of 3–4 weeks postnatally [19, 22]. The Akita (Ins2Akita) mouse is a naturally occurring diabetic model that carries a dominant mutation in the insulin 2 gene. Male heterozygous Akita mice develop hyperglycaemia as early as 4 weeks of age and demonstrate some mild features of DR after 12 weeks of age [20]. By crossing Akita and Kimba mice, the Akimba (Ins2AkitaVEGF+/–) mouse model is generated, which combines the retinopathy features of the Kimba mouse with the hyperglycaemic background of the Akita mouse [21]. The Akimba mouse model displays hallmark retinal microvascular and neural abnormalities representative of human DR including microaneurysms, tortuous vessels, venous beading, vasodilation, capillary non-perfusion, increased retinal vascular leakage, edema, neovascularization, retinal layer alterations and thinning [21, 23, 24, 25]. Some of these features develop in 8 to 9-week-old young Akimba mice (microaneurysms, severe capillary nonperfusion, haemorrhages, vascular leakage and tortuous retinal vessels) [21, 24]. This model is a highly relevant model for the study of retinal vascular disease and in our study, we investigated ~7-week-old animals with the aim of evaluating the effect of DAPA in the prevention or slowing of the development of DR.

Using the Wizard Genomic DNA Purification Kit (catalogue #A1120, Promega, Madison, WI, USA) DNA was isolated from tail clippings. Genotyping of Kimba mice was carried out as described previously [22, 25]. As described by Vagaja et al. [26] Akita mice were genotyped for the Ins2 gene. Akimba mice were genotyped by using protocols for both Kimba and Akita mice.

All experimental and animal handing activities were performed at the Royal Perth Hospital animal holding facility or the Harry Perkins Institute for Medical Research animal holding facility (Perth, Western Australia, Australia) in accordance with the guidelines of institutional Animal Ethics Committee and ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Animal ethics was approved by both the Royal Perth Hospital Animal Ethics Committee (R537/17-20; approved: 15/08/17) and Harry Perkins Institute for Medical Research Animal Ethics Committee (AE141/2019; approved: 12/02/19). Specific pathogen-free ~7-week-old male C57BL6/J, Kimba, Akita and Akimba mice were obtained from the Animal Resources Centre (Perth, Western Australia, Australia).

Mice were housed under a 12-hour light/dark cycle, at 21 $\pm$ 2 °C and were given a standard chow diet (Specialty Feeds, Glen Forrest, WA, Australia) with free access to food and drinking water. Following a 7-day acclimatization period, the SGLT2 inhibitor Dapagliflozin (DAPA; catalogue #A-0840, Ark Pharma Scientific Limited, China; 25 mg/kg/day), dual SGLT1 and 2 inhibitor Sotagliflozin (SOTAG; HY-15516/CS-1069, Med Chem Express, Princeton, NJ, USA; 25 mg/kg/day) or vehicle (dimethylsulfoxide) was administered to the mice via drinking water for 8 weeks. Drinking water containing the inhibitor or vehicle was freshly prepared and replenished on a weekly basis. Urine glucose levels were measured (Keto-Diastix; catalogue #2883, Bayer, Leverkusen, Germany) before treatment to confirm the phenotype of animals and 1-week post-treatment to establish the development of glucosuria induced by treatment. On a weekly basis, mice were weighed and consumed water volumes were measured.

In a separate set of experiments, Akimba mice were treated with the SGLT2 inhibitor Empagliflozin (EMPA; catalogue #H-020496, Ark Pharma Scientific Limited, China; 25 mg/kg/day) or vehicle for a period of 8 weeks via drinking water. Drinking water containing the inhibitor or vehicle was freshly prepared and replenished on a weekly basis. At the end of week 8, mice were euthanized and eye tissue was processed for histological analysis of SGLT1 protein expression.

Glucose tolerance testing (GTT) was performed at the end of 7 weeks of treatment after 5 hours of fasting. Blood glucose levels were measured before (0 minutes) and 15, 30, 45, 60 and 90 minutes after intraperitoneal administration of 25% glucose solution at 1 g/kg body weight. All GTT test results higher than 33.3 mmol/L could not be determined due to glucometer limitations and were plotted as 33.3 mmol/L.

At the end of the experiment, mice were fasted for 5 hours with free access to treatment water. Animals were deeply anesthetized using isoflurane inhalation. Blood samples were collected using cardiac puncture and placed on ice immediately. Fasting blood glucose was measured using the Accu-Chek Performa blood glucose monitoring system (product ID: 2581627, Roche Diagnostics, North Ryde, Australia). Blood samples were centrifuged, serum collected and stored at –80 °C. Eyes were enucleated and cleaned to remove all muscle and connective tissue. The right eye was collected into 10% buffered paraformaldehyde for histology while the left eye was fixed in ice-cold 10% buffered paraformaldehyde for 2 hours at 4 °C in preparation for retinal vascular assessment. The optic nerve tissue was collected into glutaraldehyde and was processed to obtain semi-thin transverse sections for electron microscopy.

Retinal flat mounts were prepared and analyzed with modification to a previously published method [27]. In brief, fully dissected retinas from the left eye were washed twice in 1$\times$ phosphate buffered saline (PBS) and were permeabilized for two hours at room temperature in PBS-BSA-Triton-X solution (1$\times$ PBS, 1% Bovine Serum Albumin (BSA), 1% Triton-X) followed by washing in Wash Buffer (1$\times$ PBS, 0.5% Triton-X) for five times. Retinas were treated with Isolectin GS-IB4 overnight at 4 °C (1:100 in Wash Buffer) with specificity for alpha-galactosylated glycoprotein residues on vascular endothelial cells and macrophages (Griffonia Simplicifolia Lectin I (GSL I); isolectin B4; catalog #B1205-5, Biotinylated; Vector Laboratories, Burlingame, CA, USA). Subsequently, the retinas were washed five times using 1$\times$ PBS and secondary antibody Cy3-Streptavidin (catalogue #PA43001, 1:500 in 1$\times$ PBS; GE Healthcare, Amersham, UK) was added and incubated for five hours in the dark at room temperature. Stained retinas were washed five times in 1$\times$ PBS, flat-mounted and cover-slipped with VECTASHIELD HardSet Anti-fade Mounting Medium (catalogue #H-1400, Vector Laboratories, Burlingame, CA, USA).

Enucleated eyes were fixed in 4% paraformaldehyde for 24 hours, placed in 70% ethanol overnight, followed by wax embedding and collection of 5 $\mu$m sections. A series of sections were stained with hematoxylin and eosin (H&E). Additional sections were deparaffinised in xylene (2 $\times$ 10 minutes) and rehydrated in 100% (2 $\times$ 5 minutes), 95% (1 $\times$ 1 minute) and 70% ethanol (1 $\times$ 3 minutes). Slides were then placed under running tap water for 5 minutes. Antigen retrieval was carried out by placing tissues in pre-heated EDTA (pH 8.5; Sigma–Aldrich, Sydney, Australia) and further heating with micropower for 5 minutes. Slides were washed in PBS + 0.1% Tween (2 $\times$ 5 minutes) and sections were outlined using a paraffin pen. Sections were treated with 3% H${}_2$O${}_2$ for 10 minutes, washed with PBS + 0.1% Tween (2 $\times$ 5 minutes) and blocked with 5% Fetal Bovine Serum in PBS + 0.1% Tween for 1 hour at 4 °C in a humidified chamber. Slides were incubated with a combination of Rabbit anti-SGLT1 antibodies (catalogue #ab14685, Abcam, Melbourne, Australia and Novus Biologicals, Melbourne, Australia, respectively) in 5% Fetal Bovine Serum in PBS + 0.1% (1:180 and 1:100, respectively), overnight at 4 °C in a humidified chamber and washed with PBS + 0.1% Tween (3 $\times$ 5 minutes). Retinal sections were incubated with donkey anti-rabbit horseradish peroxidase (HRP; cat #NA934V Thermo Fisher Scientific, Victoria, Australia) in PBS + 0.1% Tween (1:500) for 1 hour. Detection was performed using Ultraview Universal DAB (3, 3’-Diaminobenzidine) detection kit (Product no. 760-500, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions, followed by counterstaining of nuclei with Gill’s haematoxylin. Slides were washed, dehydrated, cleared and mounted using Dibutylphthalate Polystyrene Xylene (DPX; catalogue #06522, Sigma–Aldrich, Sydney, Australia).

For GFAP immunohistochemistry, 5 $\mu$m wax sections were mounted onto charged slides and incubated for 60 minutes at 60 °C. The immunohistochemistry was performed on a Benchmark Ultra™ staining module (material no. 05342716001, Ventana Medical Systems, Tucson, AZ, USA) using recommended reagents. In brief, the slides were deparaffinized using EZ prep (Product no. 950–102) followed by epitope retrieval (Cell conditioner no. 1, pH 8.5, Product no. 950–124) at 95 °C for 36 minutes. Sections were incubated with monoclonal mouse GFAP antibody (Clone GF2 1:3000) (catalogue #M0761, DAKO, Glostrup, Denmark) for 32 minutes at 36 °C. UltraView Universal DAB detection (Product no. 760–500) was used for visualization of the bound primary antibody using the manufacturer’s recommend protocol. The slides were then counterstained with Hematoxylin and bluing reagent prior to dehydration in graded alcohols.

Retinal whole-mounts were imaged from the vitreous side using the inverted fluorescent microscopic system Nikon Eclipse Ti (Nikon, Tokyo, Japan) with a digital camera CoolSNAP HQ2 (Photometrics, Tucson, AZ, USA) linked to a computer running the image analysis software ‘NIS-Elements Advanced Research’ V4.0 (Nikon, Tokyo, Japan). Overlapping high-resolution images of the entire retinas were captured using the 4$\times$ objective. Images were merged to construct a montage of the retina. The generated low magnification photomontages were used to count the number of retinal vascular lesions such as microaneurysms, intra retinal microvascular abnormalities (IRMA) within 2000 $\mu$m radius from the optic nerve head (NIS-Elements Advanced Research, Nikon, Tokyo, Japan). Representative areas of retinal vascular lesions were captured in the z-plane in 2 $\mu$m steps. The z-stacks were compiled into a focused image representing all superficial, intermediate and deep capillary layers.

Brightfield imaging of retinal sections stained with H&E, SGLT1 or GFAP were conducted using the microscopic system Nikon Eclipse Ti (Nikon, Tokyo, Japan) with a digital camera CoolSNAP HQ2 (Photometrics, Tucson, AZ, USA) linked to a computer running the image analysis software ‘NIS-Elements Advanced Research’ (Nikon, Tokyo, Japan).

H&E-stained retinal sections were scanned for overall gross anatomy followed by morphometric analysis. Morphometric measurements included counting the cells in the ganglion cell layer. Retinal ganglion cells were quantified by counting ganglion cells in a 100 $\mu$m linear distance in the mid retinal region. In addition, overall retinal thickness and specific nuclear layer thickness was measured.

Retinal sections were screened for GFAP expression in Müller cells across the length of each retinal section. Müller cell projections spanning beyond the ganglion cell layer were considered for Müller cell gliosis. Each retinal section was screened in the mid retinal region in a linear manner. At least two retinal sections per eye was assessed.

The mean SGLT1 staining intensity score of stained retinal sections was calculated in counterstained retinal tissue. The SGLT1 staining intensity for each mouse retina was scored on a scale of 0–3 (0 = no staining; 1 = low; 2 = intermediate; 3 = high).

Left and right optic nerves from Akimba mice were processed for ultrastructural studies. Tissues were initially fixed in 2.5% glutaraldehyde at room temperature for 24 hours and then rinsed in cacodylate buffer. Optic nerve preparations were post-fixed in 1% osmium tetroxide, dehydrated through graded Alcohols and Propylene Oxide and infiltrated with epoxy resin on a Lynx tissue processor. Semi-thin sections were obtained and stained with toluidine blue for screening regions of the tissue block face to obtain ultra-thin sections of optic nerve tissue. Ultra-thin sections were obtained at 100 nm and stained with uranyl acetate and Reynold’s Lead citrate. Sections were imaged using a JEOL 1200EX Transmission Electron Microscope at 120 kV (JEOL, Tokyo, Japan). A series of images were obtained from the outer edge and from the middle of the cross sectioned optic nerve tissue representing the ‘outer’ and ‘inner’ regions, respectively. The frequency of axons with structural anomalies such as interrupted axonal flow, accumulation of mitochondria, dense tubular lamellae and multi vesicular bodies was assessed in axonal profiles.

Serum was analyzed for Fibroblast Growth Factor 21 (FGF21) using the Duoset human FGF21 enzyme-linked immunosorbent assay (ELISA) kit (Cat# DY2539, R&D systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s respective instructions.

All morphometric data were analyzed using two-tailed Student’s t-test and p $\le$ 0.05 was considered as a significant threshold.

Both C57BL/6 (WT) and Kimba models lack the hyperglycemic background and hence acts as an internal control for the effectiveness of the SGLT2 inhibitor DAPA used in this study. Prior to treatment, urine glucose testing via dipstick method showed 0 mmol/L glucose in both C57Bl/6 and Kimba strains and $\ge$111 mmol/L in the diabetic Akita and Akimba mice (data not shown). As anticipated, the consumption of DAPA in drinking water at a concentration of 25 mg/kg/day, resulted in marked glucose secretion in the urine of non-diabetic C57BL/6 (Fig. 1A) and Kimba mice (Fig. 1B) while remaining at 0 mmol/L in mice receiving vehicle. Urine glucose levels of the diabetic Akita and Akimba mice were $\ge$111 mmol/L regardless of treatment. Furthermore, all C57BL/6, Kimba, Akita and Akimba mice displayed normal responses to touch, smooth and healthy body coats, bright eyes and normal posture throughout the 8 weeks of DAPA treatment.

Fig. 1.

SGLT2 inhibition with DAPA promotes glucosuria in non-diabetic wild-type C57BL/6 and Kimba mice. Representative image of glucose levels in the urine in (A) wild-type C57BL/6 and (B) Kimba mice treated with vehicle or DAPA (25 mg/kg); Blue = 0 mmol/L glucose, Dark brown $\ge$111 mmol/L glucose. SGLT2, Sodium glucose cotransporter 2; DAPA, Dapagliflozin.

Over the 8 weeks of treatment, non-diabetic C57BL/6 (WT) and Kimba mice gained weight at similar rates irrespective of treatment (Fig. 2A,C). Diabetic Akita and Akimba mice typically show poor weight gain, a hallmark of the failure to thrive phenotype in these animals. At the end of 8 weeks of treatment both Akita and Akimba mice receiving DAPA exhibited significantly increased body weight gain when compared to their counterparts receiving vehicle (Fig. 2B,D).

Fig. 2.

Effect of SGLT2 inhibition on body weight in non-diabetic and diabetic mice treated with DAPA. Graphs show the normalized body weight percentage during 8 weeks of treatment with DAPA or vehicle in (A) wild-type C57BL/6, (B) Akita, (C) Kimba and (D) Akimba mice. *p $\le$ 0.05; data represented as mean $\pm$ SEM of 5-6 mice per group. SGLT2, Sodium glucose cotransporter 2; DAPA, Dapagliflozin.

Following 8 weeks of treatment, there was no difference in the fasting blood glucose levels of C57BL/6 mice (WT) receiving either vehicle (14.4 $\pm$ 1.8 mmol/L) or DAPA (13.1 $\pm$ 1.4 mmol/L) (Fig. 3A). Kimba mice also had similar fasting blood glucose levels upon receiving either vehicle (10.7 $\pm$ 0.4 mmol/L) or DAPA (9.4 $\pm$ 0.6 mmol/L) for 8 weeks (Fig. 3C). The fasting blood glucose levels of both Akita (18.5 $\pm$ 2.7 mmol/L) and Akimba (15.1 $\pm$ 1.6 mmol/L) mice after 8 weeks of DAPA treatment were significantly lower than Akita (29.8 $\pm$ 1.7 mmol/L) and Akimba (27.0 $\pm$ 3.8 mmol/L) treated with vehicle (Fig. 3B,D, respectively).

Fig. 3.

Effect of SGLT2 inhibition on blood glucose in non-diabetic and diabetic mice treated with DAPA. Graphs show fasting blood glucose levels after 8 weeks of treatment with DAPA or vehicle in (A) wild-type C57BL/6, (B) Akita, (C) Kimba and (D) Akimba mice. *p $\mathrm{\le }$ 0.05, **p $\le$ 0.005; data represented as mean $\pm$ SEM of 5–6 mice per group. SGLT2, Sodium glucose cotransporter 2; DAPA, Dapagliflozin; WT, Wild-type.

As shown in Fig. 4, a GTT was performed to evaluate glucose tolerance 7 weeks after DAPA treatment. When compared to Kimba mice treated with vehicle, treatment with DAPA significantly decreased average blood glucose levels 30 minutes after glucose challenge (Fig. 4A). This significant decrease continued up to 90 minutes after glucose challenge. However, in Akimba mice treated with DAPA, the average blood glucose levels were significantly lower prior to glucose challenge (0 minutes) and continued to be lower than vehicle treated mice during the course of 90 minutes after glucose challenge (Fig. 4B).

Fig. 4.

SGLT2 inhibition with DAPA reduced glucose intolerance in diabetic Akimba mice. Blood glucose was measured at 0, 15, 45, 60, and 90 minutes after glucose challenge in (A) non-diabetic Kimba and (B) diabetic Akimba mice. *p $\mathrm{\le }$ 0.05; data represented as mean $\pm$ SEM of 5–6 mice per group. SGLT2, Sodium glucose cotransporter 2; DAPA, Dapagliflozin.

Upon fluorescent microscopic examination, there were no discernable differences in the retinal vasculature of vehicle or DAPA treated C57BL/6J (WT) and Akita mice (Supplementary Fig. 1). Furthermore, retinal vascular abnormalities such as capillary dropouts or microaneurysms indicative of diabetic retinopathy were absent in both groups of diabetic Akita mice (Supplementary Fig. 1C,D). In both models with retinopathy (Kimba) and DR (Akimba), the development of retinal vascular lesions was evident as previously published [16, 21, 22, 23, 25]. Therefore, the number of vascular lesions were counted in Kimba and Akimba mice to determine the effect of SGLT2 inhibition on the retinal vascular features. There was no appreciable difference between the number of lesions in Kimba mice receiving DAPA (32.3 $\pm$ 2.9 lesions) or vehicle (30.6 $\pm$ 2.9 lesions) (Fig. 5A,C). However, in Akimba mice treated with DAPA (53.5 $\pm$ 2.3 lesions), there were significantly less lesions observed compared to Akimba mice treated with vehicle (66.7 $\pm$ 3.8 lesions) (Fig. 5B,D). This result is highly novel.

Fig. 5.

Treatment with DAPA reduced retinal microvascular lesions in diabetic Akimba mice. Representative areas of isolectin-B4 and Cy-3 stained vasculature from (A) Kimba and (B) Akimba mice treated with vehicle or DAPA. Quantification of retinal vascular lesions in (C) Kimba and (D) Akimba mice treated with vehicle versus DAPA. A reduced numbers of retinal microvascular lesions (yellow arrows) were observed in (D) Akimba mice treated with DAPA when compared to vehicle treated mice. *p $\le$ 0.05; data represented as mean $\pm$ SEM of 3–5 mice per group. Scale bar 100 $\mu$m; DAPA, Dapagliflozin.

The complex pathology of DR affects both vascular and neural tissue. The characteristics of neurodegeneration are well-described in animal models of DR [28]. We examined the neural retina in each group by H&E staining and observed that in vehicle treated Akimba mice (Fig. 6A,B), retinal cells appeared disorganised, intercellular space increased and retinal capillaries often penetrated the generally avascular outer retinal layers (Fig. 6A,B: yellow arrows). In particular, rosette formations (Fig. 6A,B: yellow boxes) was noted in 40% of the vehicle treated mice. In contrast, DAPA treatment preserved the well-structured, intact and tightly arranged manner of plexiform and nuclear layers and minimised abnormal blood vessel transgression which causes damage to the outer layers of the retina (Fig. 6C,D).

Fig. 6.

Treatment with DAPA improved retinal neural damage in diabetic Akimba mice. Light micrographs of H&E stained retinal sections of Akimba mice treated with vehicle (A,B) or DAPA (C,D). Akimba mice treated with vehicle showing increased number of blood vessels transgressing the normally blood vessel-free photoreceptor cell layers (A–D; yellow arrows) and rosette formation (A,B; yellow boxes). Treatment with DAPA reduced retinal neural damage (C,D). n = 3–4 mice per group. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photo receptor layer. Scale bar: 50 $\mu$m.

Neurodegeneration characterised by loss of neural cell populations accompanying thinning and alterations of retinal layers are well documented [29]. Measurements of mid-retinal thickness in non-diabetic C57B6/J (Wild-Type; WT) and diabetic Akimba mice treated with vehicle or DAPA are shown in Fig. 7. Overall, the retinal thickness in WT mice with or without SGLT2 inhibitor treatment appeared normal (Fig. 7A) and did not show any significant difference when measured (Fig. 7B). When compared to WT mice, the retinal thickness was significantly reduced in diabetic Akimba mice regardless of treatment (WT vs Akimba (Vehicle) Full retina, INL (inner nuclear layer), ONL (outer nuclear layer); *p $\mathrm{\le }$ 0.05 and WT vs Akimba (DAPA) Full retina, INL, ONL; *p $\mathrm{\le }$ 0.05). However, in DAPA treated Akimba mice (Fig. 7C; right panel and D) the retina was protected from degeneration when compared to vehicle treated Akimba mice (Fig. 7C; left panel and D). Akimba mice with DAPA treatment showed an overall significantly thicker retina, specifically the INL (Fig. 7C; right panel and D). There was no significant difference in the retinal ganglion cell number between vehicle and DAPA treated Akimba groups (Supplementary Fig. 2).

Fig. 7.

Treatment with DAPA preserved retinal nuclear layers in diabetic Akimba mice. Thickness of the total retina, outer nuclear layer and inner nuclear layer was performed in the mid retina of non-diabetic C57B6/J WT (A,B) and diabetic Akimba mice (C,D) treated with Vehicle or DAPA. *p $\mathrm{\le }$ 0.05 between Akimba vehicle (dark red bars) and Akimba DAPA (light red bars) mice. Data represented as mean $\pm$ SEM of 3–5 mice per group. INL, inner nuclear layer; ONL, outer nuclear layer; DAPA, Dapagliflozin and WT, Wild-Type. Scale bar: 50 $\mu$m.

Gliosis of Müller cells can be present in various retinal pathologies and is a distinct feature of DR [30]. Reactive Müller cells are characterised by the expression of GFAP in the fine projections of Müller cells spanning across the neural retina indicating the gliosis process [31]. In vehicle treated Akimba mice, radial Müller cell projections indicating gliosis of Müller cells were noted (Fig. 8A,B; black closed-arrows), while the GFAP expression in DAPA treated mice were limited to astrocytes and the endfeet of Müller cells (Fig. 8C and D; black open-arrows) located in the NFL and GCL.

Fig. 8.

Müller cell gliosis in the mid-retinal region was reduced with DAPA treatment in Akimba mice. Paraffin sections were stained for GFAP expression and visualised in the Müller cell projections spanning across retinal layers in the mid-retinal region. Vehicle treated Akimba mice (A,B) showed Müller cell gliosis (black closed-arrows) while DAPA treated mice (C,D) showed GFAP expression confined to astrocytes and Müller cell end-feet (black open-arrows). Data represented from n = 5 mice per group. DAPA, Dapagliflozin; NFL, Nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer and ONL, outer nuclear layer. Scale bar: 100 $\mu$m.

Transmission electron microscopic analysis of Akimba optic nerves showed myelinated fibers forming axonal bundles. The axoplasms of the fibers were light and contained mitochondria, microtubules, and neurofilaments. In both inner and outer optic nerve regions, DAPA treated mice (Fig. 9B,D) showed more compact bundles of myelinated fibres when compared to vehicle treated mice (Fig. 9A,C). Furthermore, thinly myelinated fibers, nude axons and collapsed myelin sheaths were observed more frequently in Akimba mice receiving the vehicle compared to Akimba mice receiving DAPA. Overall, DAPA treatment resulted in a more stable architecture of the optic nerve.

Fig. 9.

Treatment with DAPA improved optic neuropathy in diabetic Akimba mice. Transmission electron microscopy of the inner (A,B) and outer (C,D) regions of the optic nerve of Akimba mice treated with vehicle (A,C) or DAPA (B,D) for 8 weeks. Key features; thinly myelinated fibers (blue arrows) and nude axons (red arrows). Scale bar 1 $\mu$m; DAPA, Dapagliflozin.

Diabetic Akita mice (Fig. 10A) treated with DAPA (59.8 $\pm$ 27.0 pg/mL) had a higher serum FGF21 concentration than Akita mice treated with vehicle (8.2 $\pm$ 4.0 pg/mL). Furthermore, DAPA treatment significantly elevated FGF21 serum levels in Akimba mice (55.8 $\pm$ 14.6 pg/mL) in comparison to Akimba mice receiving vehicle (17.6 $\pm$ 5.5 pg/mL) (Fig. 10B).

Fig. 10.

Treatment with DAPA increased the beneficial growth factor FGF21 in diabetic Akita and Akimba mice. Graphs show FGF21 levels after 8 weeks of treatment with DAPA or vehicle in (A) Akita and (B) Akimba mice. *p $\mathrm{\le }$ 0.05; data represented as mean $\pm$ SEM of 4–7 mice per group. FGF21, Fibroblast Growth Factor 21; DAPA, Dapagliflozin.

Our recent studies show that diabetic Akimba mice have increased SGLT2 expression in the eye compared to non-diabetic Kimba mice [16]. The SGLT family member, SGLT1, is an integral membrane protein responsible for the mechanism of active glucose transport and it may be expressed in the eye/retina [17, 32]. We then asked the question whether SGLT2 inhibition may result in SGLT1 upregulation to act as a compensatory mechanism. Interestingly, when compared to vehicle treated mice (Fig. 11A,B), inhibition of SGLT2 with either EMPA (Fig. 11C,E) or DAPA (Fig. 11D,F) increased SGLT1 in the retinas of diabetic Akimba mice. The overall SGLT1 expression was confined to the plexiform layers of the retina. It was consistently observed that the increased intensity of SGLT1 in the EMPA (Fig. 11C) and DAPA (Fig. 11D) treated groups were mainly limited to the inner and outer plexiform layers where the neuronal synaptic contacts occur in the retina.

Fig. 11.

SGLT2 inhibition promoted over expression of SGLT1 protein levels in the retinas of diabetic Akimba mice. Light micrographs of SGLT1 stained retinal sections of (A,B) Akimba vehicle, (C) Akimba EMPA and (D) Akimba DAPA treated mice. Graphs show SGLT1 levels after 8 weeks of treatment in Akimba mice with vehicle versus (E) EMPA or (F) DAPA mice. *p $\mathrm{\le }$ 0.01; data represented as mean $\pm$ SEM of 3–5 mice per group. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photo receptor layer. Scale bar 100 $\mu$m; SGLT1, Sodium glucose cotransporter 1; SGLT2, Sodium glucose cotransporter 2; EMPA, Empagliflozin; DAPA, Dapagliflozin.

Using a novel immunohistochemistry method, we have demonstrated for the first time that SGLT1 protein levels are significantly increased in the retinas of diabetic Akimba mice (Fig. 12B,C) when compared to non-diabetic Kimba mice (Fig. 12A,C). When compared to non-diabetic Kimba mice, the diabetic Akimba mice displayed increased SGLT1 expression which was confined to the nerve fiber, inner plexiform, outer plexiform and photoreceptor layers (Fig. 12B). This phenomenon appears to not only be confined to the retina. When compared to C57BL/6J (WT) mice, type 1 diabetic Akita mice also showed increased SGLT1 expression in pancreatic islet cells (Fig. 13).

Fig. 12.

Diabetic retinopathy promotes retinal SGLT1 protein expression in diabetic Akimba mice. Light micrographs of SGLT1 stained retinal sections of (A) non-diabetic Kimba and (B) diabetic Akimba mice treated with vehicle. (C) Graph shows SGLT1 levels after 8 weeks of vehicle treatment in Kimba vs Akimba mice. ***p $\le$ 0.001; data represented as mean $\pm$ SEM of 8–9 mice per group. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photo receptor layer. Scale bar: 100 $\mu$m; DAPA, Dapagliflozin; SGLT1, Sodium glucose cotransporter 1.

Fig. 13.

Increased SGLT1 expression in the pancreata of diabetic Akita mice. (A) Quantitation of SGLT1 positive islet cells; intensity scale (0 = no staining; 1 = low staining; 2 = intermediate staining; 3 = high intensity). Representative image of (B) SGLT1immunohistochemistry in the islet cells of diabetic Akita mice (red arrows: brown SGLT1 positive cells). ***p $\le$ 0.0001; data represented as mean $\pm$ SEM of 4–5 mice per group. Scale bar: 100 $\mu$m; SGLT1, Sodium glucose cotransporter 1; WT, Wild-type.

Given that SGLT2 inhibition results in SGLT1 upregulation, we sought to assess the effect of dual SGLT1 and 2 inhibition on SGLT1 expression with Sotgliflozin (SOTAG) in diabetic Akimba mice. Interestingly, when compared to vehicle treated Akimba mice (Fig. 14A), SOTAG treated mice showed a significant increase in the SGLT1 protein level (Fig. 14B,C). The increased SGLT1 protein expression in SOTAG treated Akimba mice was predominantly observed in the inner plexiform layer (Fig. 14B).

Fig. 14.

Dual inhibition of SGLT1 and 2 with SOTAG upregulates SGLT1 protein levels in the retinas of diabetic Akimba mice. Light micrographs of SGLT1 stained retinal sections of (A) Akimba Vehicle and (B) Akimba SOTAG treated mice. (C) Graph shows SGLT1 levels after 8 weeks of treatment in Akimba mice with vehicle versus SOTAG. *p $\le$ 0.05; data represented as mean $\pm$ SEM of 3 mice per group. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; and PRL, photo receptor layer. Scale bar: 100 $\mu$m; SGLT1, Sodium glucose cotransporter 1; SOTAG, Sotagliflozin.