Animal procedures
All animal care protocols and experimental procedures were performed in accordance with the UK Home Office Animal Scientific Procedures Act (1986), the European Directive of the Protection of Animals used for Scientific Purposes 2010/ 63/ E, and with the approval of the University of Dundee Animal Ethics Committee. All personnel performing procedures are holders of UK Home Office personal licences. Work was performed under Home Office licence PE82c1898.
Slc2a2+/− cryopreserved mouse sperm (C57BL/6 N-Slc2a2 < tm1b(KOMP)Wtsi > /B08, Riken reference number RBRC06334) was purchased from the Riken BioResource Center (Tsukuba, Ibaraki, Japan) and used to generate pups via in vitro- fertilisation (IVF) using C57BL/6 wild-type (WT) females (C57BL/6J, JAX™, strain code 632, bred and maintained from the original JAX strain code 000,664; this strain was introduced to Charles River Laboratories France in 1981 and UK in 2004. Breeding is in accordance with The Jackson Laboratory genetic management system. Breeding pairs were purchased from Charles River, Elphinstone, Tranent, Scotland, UK, and a colony maintained within the University of Dundee Resource Unit). Resultant Slc2a2+/− animals were crossed with this same C57BL/6J WT strain to obtain Slc2a2+/+ (littermate controls) and Slc2a2+/− pups.
For experimental procedures male Slc2a2+/+ (n = 8) and Slc2a2+/− (n = 13) littermates were group housed (max 4 per cage- mixed genotype housing) and maintained at 21 °C with 12-h light/ dark cycle and had ad libitum access to water and standard chow diet (SDS Ltd, Witham, Essex, UK). Animals were weighed regularly from age 10 weeks. Behavioural tests were performed as per schematic in Additional file 1: Figure S1 in the morning of test days (before 12 pm). At week 48, animals were killed by exposure to a rising concentration of CO2 followed by cervical dislocation, following a 5 h fast. Fasted trunk blood sample was collected, after culling, into a lithium-heparin blood tube (BD, Wokingham, Basingstoke, England, UK), incubated at room temperature for 30 min and spun at 4 °C at 7500 rpm for 15 min. Collected blood serum was used to determine fasted insulin levels by enzyme-linked immunosorbent assay (ELISA; Crystal Chem, Elk Grove Village, IL, USA) to manufacturers’ instructions. Fasting insulin resistance index (FIRI) was calculated as standard ((fasting insulin × fasting glucose)/25) [13]. Behavioural data was captured using a Handycam HDR-CX405 camcorder (Sony, Minato City, Tokyo, Japan) and with Anymaze 6.4 software (Stoelting Co., Wood Dale, IL, USA). Data was analysed using Prism 8 (Graphpad) and are presented as mean +/− SEM and compared as indicated in relevant figure legends.
There are no physiological differences between Slc2a2+/+ and Slc2a2+/− animals maintained on chow diet
Animals were maintained on chow diet up to 48 weeks of age. No significant differences were noted in body mass between Slc2a2+/+ and Slc2a2+/− groups (Additional file 1: Figures S2A and B), although there was a trend for increased weight gain in Slc2a2+/− animals (Additional file 1: Fig. S2C; p = 0.0597, t = 2.003, df = 19). There were no significant differences between the mean fasting blood glucose (FBG) levels (Additional file 1: Figure 2D), circulating insulin levels (Additional file 1: Figure S2E) or FIRI (Additional file 1: Figure S2F) of the Slc2a2+/− and Slc2a2+/+ animals at 48 weeks of age. These results show that inactivating an allele of Slc2a2 does not affect body mass or glucose homeostasis in animals receiving chow diet.
Inactivating one allele of Slc2a2 does not affect spatial working memory in male mice
To examine effects on spatial working memory, animals were tested using the T-Maze spontaneous alternation task at 18 weeks (Additional file 1: Figure S1A). Animals were allowed to spontaneously explore the maze for 15 individual trials (returning to start at the end of each test of each trial by themselves). No significant differences were observed between the two groups in the percentage alternation (Fig. 1a), which both groups performed above the level of chance (50%, as indicated by blue dashed line). Both groups performed poorly in the number of Left–Right-Left or Right-Left–Right sequences performed (Fig. 1b), which were not performed above the level of chance (33%, indicated by blue dashed line). Slc2a2+/− animals took a significantly longer amount of time to complete 15 trials of the test (Fig. 1c), with longer mean trial time (Fig. 1d) but no differences in the latency to leave the start box during each of the 15 trials before they explored the rest of the maze (Fig. 1e). Overall, these results suggest that inactivating one allele of Slc2a2 has no effects on spatial working memory in mice.
Inactivating one allele of Slc2a2 does not affect anxiety behaviour in male mice
To further investigate potential behaviour differences between the two groups, animals were tested with the elevated plus maze at the age of 18 weeks (Additional file 1: Figure S1B) to assess anxiety-related behaviours. No differences were observed between the two groups in any of the elevated plus maze measurements: distance travelled (Fig. 2a), average speed (Fig. 2b), number of entries into the different areas of the maze: neutral zone (Fig. 2c), open arm (Fig. 2d) and closed arm (Fig. 2e) or the percentage time spent within each zone (Fig. 2f–h). This suggests that inactivating one allele of Slc2a2 has no effects on anxiety-related behaviours.
Inactivating one allele of Slc2a2 does not affect recognition memory in male mice
The novel object recognition (NOR) test is an established procedure to assess recognition memory [14]. Animals were assessed using both a 2 min and a 24 h NOR (as per Additional file 1: Figure S1C). No significant differences were observed during the 2 min NOR conducted at 33 weeks between Slc2a2+/+ and Slc2a2+/− groups in the discrimination index (the difference in time spent exploring novel and familiar objects; DI- Fig. 3a) or in the total time spent exploring the objects in either the encoding or retention/ retrieval phases of the tests (Fig. 3b). Both groups performed the task above the level of chance (DI of 0, meaning animals spent equal amounts of time exploring objects in the retention/ retrieval phase of the task). However, both groups performed equally poorly in discriminating between objects during the 24 h NOR conducted at 37 weeks (Fig. 3c; neither group performed above the level of chance). Slc2a2+/− animals, though, did spent less total time exploring the objects during the 10 min retention/retrieval phase of this task compared to the encoding phases (Fig. 3d). To revisit the spatial memory domain in the animals (as explored during the T-maze task), the object location test was also performed at 42 weeks (Additional file 1: Figure S1D). Again, no differences were observed between the two groups in either DI (Fig. 3e) or total exploration time measurements (Fig. 3f), however, the Slc2a2+/+ group did not perform the task above the level of chance and so it is difficult to draw clear conclusions from this task. Overall, these results show that inactivating one allele of Slc2a2 has no effect on non-spatial recognition memory in mice.
Conclusions
Most human diseases, especially chronic, age-related disease, present with minor changes in molecular pathways, which make an individual more vulnerable to environmental insults. This can be due to SNPs in key genes, that have a minor effect on the expression or function of that gene product. We have previously reported that SNPs in the GLUT2 gene (Slc2a2) influence response to the main drug for type 2 diabetes. Our results presented here show that minor alterations in Glut2 expression, such as those associated with specific SNPs within the human gene, are unlikely to influence “spontaneous memory and anxiety behaviours”. However, to establish the influence on response to challenges, such as poor nutrition, disease or infection, will require additional studies.
