Adiponectin receptor 1 could explain the sex differences in molecular basis of cognitive improvements induced by exercise training in type 2 diabetic rats

Adipokines dysregulation, the main reason for cognitive impairments (CI) induced by diabetes, shows a sex-dependent pattern inherently and in response to exercise. This study aimed to compare the attenuating effect of 8-week high intensity-interval training (HIIT) on type 2 diabetes (T2D)-induced CI between male and female rats with a special focus on adiponectin and leptin. 28 male & 28 female Wistar rats with an average age of 8 weeks were randomly assigned into four groups: control (Con), exercise (EX), Diabetes (T2D), and Type 2 diabetes + exercise (T2D + Ex). Rats in EX and T2D + EX groups performed HIIT for eight weeks (80–100% Vmax, 4–10 intervals). T2D was induced by 2 months of a high-fat diet and a single dose of STZ (35 mg/kg) administration. Leptin and adiponectin levels in serum were measured along with hippocampal expression of leptin and adiponectin receptors, AMP-activated protein kinase (AMPK), dephosphorylated glycogen synthase kinase-3 beta (Dep-GSK3β), Tau, and beta-amyloid (Aβ). Homeostasis model assessments (HOMAs) and quantitative insulin-sensitivity check index (QUICKI) indices were calculated. Our results showed that following T2D, serum levels of APN, and hippocampal levels of adiponectin receptor 1 (APNR1) were higher and HOMA-IR was lower in female than male rats (P < 0.05). However, after 8 weeks of HIIT, hippocampal levels of APNR1 and AMPK as well as QUICKI were lower and hippocampal levels of GSK, Tau, and Aβ were higher in females compared to male rats (P < 0.05). While the risk of CI following T2D was more in male than female rats HIIT showed a more ameliorating effect in male animals with APN1 as the main player.


Animals
The study was performed with the approval of the Animal Care and Ethics Committee of Kerman University of Medical Sciences (KUMS) in accordance with the institutional guidelines of KUMS (Ethics Approval Code: IR.KMU.REC.1401.033)and in compliance with the ARRIVE guidelines.Then, we purchased 56 eight-weekold Wistar rats (males and females), with an average weight of 200-250 g, from the animal farm of KUMS and kept them at 23 ± 1.4 °C and a 12:12 dark-light cycle in special polycarbonate cages.All animals had free access to water and food.After being accustomed to the laboratory environment, the animals were randomly assigned to 4 groups (each group included 14 rats, 7 females and 7 males): control (Con), type 2 diabetes (T2D), exercise (EX), and type 2 diabetes + exercise (T2D + EX).The Ex and T2D + EX groups performed eight weeks of HIIT.

Induction of type 2 diabetes
Animals in T2D and T2D + EX groups were fed a high-fat diet (HFD) for 2 months, while the animals in Con and EX groups were fed a normal and regular diet.The HFD was purchased from the Isfahan Royan Research Institute.HFD includes: 60% fat (245 g of lard and 25 g of soybean oil), 20% carbohydrate (125 g Lodex10 and 72.8 g sucrose), 19% protein (200 g of casein and 3 g of cysteine), 50 g of fiber (Solca Floc), 50 g of minerals, 3 g of vitamins, and 0.5 g of dye 6,34 (Table 1).Rats' weight and food intake were measured weekly.The criterion for obesity was a weight of 350 g 34 .After 2 months, the animals fasted for 12 h, and a single dose of 35 mg/kg of streptozotocin (STZ) was injected intraperitoneally.Animal blood glucose was measured 2 weeks after STZ injection using a glucometer.Animals with fasting blood glucose (FBG) levels above 300 mg/dl were considered diabetic and included in the study 35 .The fasting time before STZ injection and also before sampling at the end of the study was 12 h.

Exercise protocol
Initially, in the familiarization phase, EX + T2D and EX groups ran on a treadmill twice a day for 5 days, 10 min per day with a zero-incline and a speed of 8 m per minute.Then an incremental running test was performed to determine the rats' maximum speed (V max ).In this test, the rats ran for 2 min at a speed of 6 m per minute, and every 2 min, 2 m per minute were added to the speed until they could not maintain this speed.The last attempt of each rat was considered Vmax.The main training protocol was performed for eight weeks five days a week.The exercise protocol is shown in Table 2.At the beginning and end of each session, the rats run on a treadmill

Statistical analysis
The data are reported as mean ± standard deviation (SD).Statistical analysis was performed using GraphPad Prism version 8. Normality and homogeneity of variances were assessed using Shapiro-Wilk and Leven tests, respectively.Two-way ANOVA followed by Tukey's post-hoc were used to analyze the data.P values less than 0.05 was considered statistically significant 47,48 .

Ethics approval
All stages of keeping and scarifying the animals were performed according to the rules of the ethics committee of Kerman University of Medical Sciences (Ethic code: IR.KMU.REC.1401.033).

Results
We assessed FBG to confirm our diabetes induction method.Our results showed that blood glucose was significantly increased in both male and female rats after diabetes induction (2 months of HFD and STZ injection) (month 2) compared with the pretest (month 0) in T2D and T2D + EX group (P = 0.02), with no significant difference between these groups.In addition, HIIT reduced blood glucose significantly (P < 0.05).We saw no significant effect for gender (P = 0.09) (Table 3).Also, the animals body weight results was shown in Table 4. Animals' weight significantly increased in T2D and T2D + Ex groups after diabetes induction (month 2) in both male and female rats.In addition, the weight was decreased in T2D and T2D + EX groups, with more decrease in the T2D group (P < 0.05) in post-test (month 4).The female rats have lower body weights in all groups (P < 0.05) (Table 3).
In the first week (familiarization) there was no significant different between groups in food intake but after two months of high-fat diet, food intake in T2D and T2D + EX groups increased significantly in both sex (P < 0.0001) with no significant difference between them.After STZ injection, and started exercise training, rats' food intake decreased significantly in EX and T2D + EX groups in both sex (P < 0.0001) but there was no sex difference.www.nature.com/scientificreports/Finally, after 8-weeks HIIT rats' food intake decreased significantly in EX and T2D + EX groups in both sex (P < 0.0001) without significant difference between sexes (Fig. 1A-D).
Our results showed the same result in both genders for serum insulin levels.Serum insulin level was decreased by T2D and increased by EX (P female = 0.02, P male = 0.02).In addition, a significant interaction was seen between T2D and EX (P female = 0.02, P male = 0.02).The interaction between gender and T2D, gender and EX, and gender, T2D and EX were insignificant (P > 0.05) (Fig. 2).To confirm these data, we assessed the index of pancreatic Beta cell function (i.e., HOMAβ), which showed the same results in both genders with no significant effect for gender (P > 0.05) except that Ex did not have a significant difference with Con.The interactions were also insignificant (P > 0.05) (Fig. 2).In addition, we checked the effect of T2D and EX on IR (i.e.HOMA-IR), and insulin sensitivity (i.e.QUICKI) indices.In female rats, while T2D increases and decrease HOMA-IR and QUICKI (P < 0.05), respectively, EX failed to change these indices significantly (P > 0.05).However, there was a significant interaction between T2D and EX in both indices (P < 0.05).The same result was seen in male rats except for significant effects of EX.EX decreases HOMA-IR and increases QUICKI in male rats (P < 0.05).A gender effect was seen for HOMA-IR, with a higher level in T2D female rats.Furthermore, the interaction between T2D, EX, and gender was significant with lower levels in the male rats (P < 0.05).The gender difference was seen for QUICKI in Con, EX and EX + T2D groups (P < 0.05) (Fig. 2).
To study if T2D and EX could affect LEP and APN levels, we assessed their levels in serum.APN levels in serum decreased in T2D and increased in EX groups compared to the Con group.In addition, T2D + EX showed higher levels of APN compared to T2D in both genders (P < 0.05).We also saw gender effects, with higher levels in females (P < 0.05).In addition, the female group of T2D + EX showed higher levels of APN than the same group of male rats (P < 0.05).This difference may reflect the different levels of basal APN rather than the different effects of HIIT.
In contrast with APN, LEP did not change in EX compared with Con.This was observed in both genders.The EX, T2D interaction was also insignificant.Nevertheless, a significant gender effect was seen and the LEP level was higher in females than males (P < 0.05).The interaction between gender, T2D, and EX was not significant (Fig. 3).
We measured the LEP-R and APN-R1, and APN-R2 levels in the hippocampus.Our results showed a similar pattern for all three receptors in both genders.LEP-R, APNR1, and APNR2 showed a significant reduction in T2D compared to Con (P < 0.05).However, EX could increase them significantly (P < 0.05).A significant interaction between T2D and EX has been seen for all three receptors confirming the positive effect of HIIT in diabetic rats (P < 0.05).A significant effect of gender was seen for all three receptors in the Con group, with higher levels www.nature.com/scientificreports/  in female rats (P < 0.05).In addition, a higher level of APNR1 was seen in the male T2D + EX group compared to its female peer (P < 0.05).These results highlight the vital role of APN1 (Fig. 4).
AMPK and GSK are the points where the regulatory hippocampal cascades of APN and LEP can interact to determine the net effect.Our results showed that the hippocampal P-AMPK (active form) decreased with T2D and increased with EX significantly in both genders (P < 0.05).Compared with females, diabetic male rats had a significantly higher level of AMP-K after the training period (P < 0.05).Dep-GSK (which stimulates TAU  www.nature.com/scientificreports/hyperphosphorylation) increased by T2D and decreased after 8-week EX in both genders.A significant interaction was seen for T2D and EX in both genders, which confirms the positive effects of HIIT in female diabetic rats (P < 0.05).The gender, T2D and EX interaction was significant, with the lower levels in female rats in T2D + EX group (P < 0.05) (Fig. 5).βA and Tau, the main indices of cognitive impairments, showed the same results.Both of them increased in T2D rats and increased in EX rats (P < 0.05).A significant interaction between T2D and EX was also seen.The interaction between gender, T2D and EX were significant which showed a higher effect of HIIT in the male T2D + EX group (P < 0.05) (Fig. 5).

Discussion
This study was designed to compare the attenuating effects of HIIT on APN and LEP signaling pathways in the hippocampus of male and female rats with T2D.
Our findings showed that T2D had destructive effects on all signaling molecules while HIIT could reverse these changes partly.Our findings revealed that APNR1, AMPK, GSK, TAU and Aβ expression were different between male and female rats after 8 weeks of HIITT.In addition, diabetes-induced IR was higher in male compared to female rats.While diabetes was induced in both gender the lack of a protective effect of estrogen may contribute to higher IR in male rats 49,50 .Added to this is the association between APN and IR in both genders.APN could reduce IR by decreasing triglyceride content in muscle and liver in obese and diabetic animals.This effect results from increased expression of molecules involved in fatty-acid metabolism such as PPAR-α 51,52 .Circulating APN was higher in female than male rats.The biological basis for this sex difference has been described in introduction.The sex difference in adiponectin could also be explained by an inhibitory effect of testosterone.Treatment of both sham-operated and castrated male mice with testosterone was accompanied by a reduction in serum APN 53 .In addition, our results showed that while APNR1 expression was higher in Con female but this was reversed after 8 weeks of HIIT.Kaminska et al. 54 reported that the expression of APNR1 is sex dependent and it is higher in male than female animals which is in consistent with our results.Animal breed and age may explain inconsistent results.It has been shown that the level of APNR1 decreased in male rats following T2D 55 .Furthermore, The stimulatory effect of exercise on APNR1 showed a dose dependent pattern and HIIT is considered as the most power full stimulant 56 .This stimulatory effect is shown to be mediated by serum testosterone in T2D rats 57 .This could be the reason for higher APNR1 expression in diabetic male than female rats after 8 weeks of HIIT.APNR1 inhibits Dep-GSK3β expression and due to higher level of APNR1 in male than female rats after HIIT, Dep-GSK3β expression showed the opposite pattern which is in line with our previous study 6 .It has been shown that APNR1 suppression exacerbates AD-like pathologies (i.e.increased Tau accumulation).One study showed that in ovariectomized female animals, the expression of APNR1 decreased and hippocampal accumulation of Tau increased compared to the healthy animals.In line with this result, our data showed lower accumulation of Tau in hippocampus of diabetic male compare to female rats after HIIT.APNR1 can also stimulate AMPK expression and this could be the reason for higher AMPK expression in male than female rats after HIIT.Brown et al. showed that AMPK expression was higher in male compared to female mice in response to 12 weeks of Endurance exercise 58 .On other hand, estrogen can increase AMPK expression in brain 59,60 and this can explain the high AMPK in healthy, not exercised female than male rats.
In general, previous studies have described the importance of some types of exercise in regulated adiponectin pathway in peripheric tissues as well as in the brain.For example, it has been shown that HIIT induced PGC-1 and APNR1 gene expressions and improved insulin sensitivity in obese individuals 61 .Also, resistance training improved insulin signaling in skeletal muscle of T2D rats 62 .Cho et al. revealed that exercise training improves whole body insulin resistance via APNR1 63 .Furthermore, some researches showed that acute physical exercise increases adiponectin signaling in the hypothalamus of lean mice 64,65 .
Another important factor in cognitive impairment induced by T2D is accumulation of Aβ which is under direct control of AMPK and therefore decreased more in male than female rats after HIIT.To the best of our knowledge, this is the first study to compare sex difference in response to cognitive improvements induced by exercise training in T2D rats.However, some studies showed that exercise can reduced Aβ deposition in hippocampus [66][67][68][69] .It should be noted that testosterone can increase Aβ clearance 70 and as exercise is an increase testosterone stimulator 71,72 , some studies considered testosterone a mediator of exercise induced Aβ clearance 32 .

Conclusion
In summary, we concluded that the risk of CI following T2D was more in female than male rats due to high deposition of Tau and Aβ in hippocampus but HIIT had more ameliorating effect in male animals than female.We believed that the difference in APNR1 expression and its downstream may be a main mechanism of this sex difference (Fig. 6).

Figure 3 .
Figure 3. Serum level of APN (A) and Lep (B).(mean ± SD) in all groups.*Significant difference with Con female.# Significant difference with Con male.& Significant difference with a female peer.Con control, T2D Type 2 diabetic, Ex exercise, and T2D + Ex Type 2 diabetic + exercise.

Figure 4 .
Figure 4. APNR1 (A), APNR2 (B) and LepR (C).(mean ± SD) in all groups.*Significant difference with Con female.# Significant difference with Con male.& Significant difference with a female peer.Con control, T2D Type 2 diabetic, Ex exercise, and T2D + Ex Type 2 diabetic + exercise.Original blots/gels are presented in Supplementary Fig. 1 which were cropped from different gels.

Figure 5 .
Figure 5. P-AMPK (A), Dep-GSK3B (B), AB (C) and Tau (D).(mean ± SD) in all groups.*Significant difference with Con female.# Significant difference with Con male.& Significant difference with a female peer.Con control, T2D Type 2 diabetic, Ex exercise, and T2D + Ex Type 2 diabetic + exercise.Original blots/gels are presented in Supplementary Fig. 1 which were cropped from different gels.

Figure 6 .
Figure 6.Adiponectin receptor 1 could explain the sex differences in molecular basis of cognitive improvements induced by exercise training in type 2 diabetic rats (HFD: High fat diet, LEP: Leptin, APN: Adiponectin).

Table 1 .
High-fat and regular diet ingredients.

Table 3 .
FBG (mean ± SD) before starting the intervention (month 0), after diabetes induction (2 months of high-fat diet and STZ injection) (month 2), and 48 h after the last training session (month 4) in all groups.FBG Fasting blood glucose, Con control, T2D Type 2 diabetic (STZ injected), Ex exercise only, and T2D + Ex Type 2 diabetic + exercise.*Significant difference with pretest.# Significant difference with month 2.