The combined effects of resistance and endurance training with ursolic acid supplementation on some Alzheimer's disease-related biomarkers in a rat model of type 2 diabetes

Abstract

Background

Type 2 diabetes is associated with increased inflammation and a risk of Alzheimer's disease (AD). This study aimed to assess the impact of exercise with ursolic acid (UA) on some protein levels in the brains of aged male Wistar rats with diet-induced Type 2. We investigated the effects of exercise with UA on protein levels in rats with type 2 diabetes. The rats were divided into seven groups and underwent different exercise or UA protocols.

Results

The results showed that type 2 diabetes led to increased levels of tau, IL-1β, TNF-α, and c-Jun, and decreased levels of IRS2 protein. Endurance training improved tau, Jun, and IRS2 levels. UA reduced increased levels of tau, IL-1β, TNF-α, and c-Jun, and increased IRS2 levels. Combining the supplement with training led to further improvements.

Conclusions

These findings suggest that combining training and UA partially reversed the inflammation in the Type 2 diabetes model. However, further research is needed to understand how UA consumption with or without training protocols can reduce the risk of AD in type 2 diabetes.

Background

Most older adults suffer from insulin resistance, which causes and aggravates both type 2 diabetes and inflammation (Park et al. 2006). Type 2 diabetes and subsequent inflammation exacerbate Alzheimer's disease (AD) and are considered to be age-related diseases (Karaji et al. 2023). Thus, due to shared molecular and cellular features between type 2 diabetes and AD, and memory deficits associated with insulin resistance in AD, this disease has been called Type 3 diabetes mellitus (Shen et al. 2023). The initial and most crucial pathological occurrence that can result in insulin resistance is systemic inflammation (Vinuesa et al. 2021). It has already been established that inflammation mediates the link between type 2 diabetes and AD (Vinuesa et al. 2021). Tumor necrosis factor (TNF-α), also known as tumor necrosis factor, is a prominent inflammatory factor. Increased TNF-α affects metabolic and immune pathways (De Sousa Rodrigues et al. 2020). Conversely, TNF-α leads to the activation of c-Jun N-terminal kinases (JNK) signaling in AD models.

C-Jun proto-oncogene (c-Jun) is a protein with various cellular functions, including cell growth, death, survival, tumor formation, and tissue development. Its role as a transcription factor allows it to bind to deoxyribonucleic acid (DNA) and regulate gene transcription alone or in conjunction with other proteins. Recent studies suggest that external signals can modify c-Jun. These modifications include posttranslational modifications that occur on the protein and changes in protein levels. Moreover, c-Jun can interact with different signals to control tissue development and disease through a complex regulatory mechanism (Meng and Xia 2011).

Elevated levels of the inflammatory protein TNF-α in people with diabetes can lead to the initiation JNK. As a result, the inhibitory phosphorylation of Ser312 on insulin receptor substrate 1 (IRS1) is increased, leading to the suppression of IRS1 function (Ferreira et al. 2018). Research has indicated that inflammatory diseases can be positively affected by exercise and diet (Karaji et al. 2023). Regular physical activity can positively impact the body's anti-inflammatory pathway, leading to a reduction in inflammatory response (Karaji et al. 2023). It can also improve insulin effectiveness by activating PI3-kinase linked with IRS1 in skeletal muscles (Kirwan et al. 2000). On one hand, engaging in physical activity has the potential to decrease disability among older individuals (Rahmati et al. 2023). On the other hand, engaging in regular physical activity is beneficial in preventing and managing insulin resistance in individuals with type 2 diabetes (Kirwan et al. 2000). Studies have also demonstrated that voluntary exercise in Type 2 rat models can lead to a decrease in oxidative stress and inflammation markers, as well as a reduction in hepatic JNK activation and Ser307-phosphorylated insulin receptor substrate 1. These decreases have been associated with a lowering of insulin resistance and a decrease in the production of the primary gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (Király et al. 2010).

Ursolic acid (UA), also known as 3β-hydroxy-urs-12-en-28-oic acid, possesses anti-inflammatory properties that can effectively counteract both endogenous and exogenous inflammatory stimuli. Its mechanism of action involves suppressing inflammatory factor expression and inhibiting elastase activity, leading to a decrease in inflammation (Luan et al. 2022). Research suggests UA has the potential to improve cognitive function by reducing impaired hippocampal neurogenesis and alleviating deficits in cognition, hippocampal neurogenesis, and synaptic regulation in a mouse model of AD induced by beta-amyloid (1–42) (Aβ_1-42) (Mirza et al. 2021). Given these promising effects on inflammation and neurodegeneration, this study was carried out to assess the effectiveness of UA, endurance, and resistance exercise on certain inflammatory factors associated with AD in aged rats with type 2 diabetes.

Methods

Animal

Fifty-six Wistar rats, all male and between 19 and 21 months old, were acquired from the Pasteur Institute (Karaj, Iran). The study was approved by the Ethics Committee for Laboratory Animals of Shahrekord University (Shahrekord, Iran) (Approval Number IR.SKU.REC.1399.001). All methods were conducted in compliance with relevant regulations and guidelines. Additionally, the study adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. After a one-week acclimation period to the laboratory environment, the rats were randomly divided into seven groups: Healthy Control (C), Diabetic Control (CD), Diabetic + UA (U), Diabetic + Endurance (E), Diabetic + Resistance (R), Diabetic + Endurance + UA (EU), and Diabetic + Resistance + UA (RU). Each group consisted of eight animals (Fig. 1). Throughout the experiment, the rats were handled by a single person.

Fig. 1

Schematic diagram of experimental protocol

Induction of diabetes

The type 2 diabetes model was induced by a combination of low-dose streptozotocin (STZ) and a high-fat diet (HFD) containing 60% fat, 20% carbohydrates, and 20% protein, with a caloric value of 5.21 kcal/g (Liu et al. 2013). The HFD was purchased from Royan Company in Isfahan. The subjects were fed this diet for 6 weeks. In the fourth week, the group that did not develop diabetes after receiving HFD/STZ was administered a single, low-dose injection (30 mg/kg) of STZ (from Sigma-Aldrich, CAS Number: 1888366-4) intraperitoneally. The STZ was dissolved in a 0.1 M sodium-citrate buffer with a pH of 4.4. Blood glucose levels were assessed using a blood glucose meter (Roche) after the first week. Rats with a fasting blood glucose level below 16.7 mmol/L received a second STZ injection (30 mg/kg). Rats that maintained blood glucose levels above 16.7 mmol/L for the following four weeks were identified as diabetic and included in further experiments (de Bem et al. 2018). During the protocol, diabetic rats were fed a different high-fat diet containing 55% fat, 31% carbohydrates, and 14% protein.

The UA intake

For the UA group, rats received UA orally via gavage every day for 8 weeks. The dosage was 500 mg/kg body weight, which is roughly equivalent to a human dose of 250 mg/kg body weight per day. A high-fat diet containing 0.5% UA, developed by Royan Isfahan Company, was provided to the rats (Kazemi Pordanjani et al. 2023).

The test to determine VO₂max

To determine the maximum running speed, a running test was conducted on a rodent treadmill following the protocol of Leandro et al. (2007). The test consisted of ten phases, each lasting three minutes, with a starting speed of 0.3 km/h that was incrementally increased by 0.3 km/h every three minutes (no incline).

The endurance exercise protocol

The endurance exercise protocol consisted of three phases: a warm-up, a main exercise period, and a cool-down. Rats followed the endurance exercise guidelines outlined in Table 1 (Kazemi Pordanjani et al. 2023).

Table 1 The endurance exercise protocol

The test to determine maximal velocity concentric contractions (MVCCs).

The MVCC was assessed using a ladder-climbing test. A weight equal to 75% of the rat's body weight was attached to its tail. The rat was then allowed to climb a ladder with this weight. For each successful climb, an additional 30 g was added to the weight. Rats were given a 2-min rest between each attempt. If the rat failed to climb the ladder successfully on three consecutive attempts, the weight from the previous successful climb was recorded as the MVCC. MVCC measurements were taken at the beginning of the first week, the fourth week, and the end of the eighth week (Singulani et al. 2017).

Resistance training protocol

First, the animals were allowed to become accustomed to vertical climbing. No excessive weight was added to the structure. After that, resistance training consisted of climbing a special training ladder (2 cm grid, 110 cm, and 85° incline), which was performed with 80% of MVCC, 5 times a week for 8 weeks and 9–10 lifts per session (Farsani et al. 2019).

Collection of tissue

After weighing, the animals were anesthetized with a mixture of 10 mg/kg of xylazine and 90 mg/kg of ketamine. After 48 h following the final protocol, the animals were sacrificed. After that, the hippocampus was dissected and immediately frozen in liquid nitrogen. The rats were euthanized following the removal of tissue.

Collection of data

The data were collected using the Western blot method, followed by an evaluation of the differences between the groups using statistical analysis. The Western blot description and the statistical analysis are provided below.

Western blot

Hippocampal tissue analysis was performed using Western blotting. Tissue was lysed with PBS, and the supernatant was stored at − 80 °C. To quantify total protein content, the Bradford method with spectrophotometric measurements was employed. Proteins were then separated by SDS-PAGE and transferred to PVDF membranes. Following incubation with primary antibodies for TAU, TNF-α, c-JUN, and IRS2, a secondary antibody was used. β-actin served as a loading control.

Statistical analysis

The Shapiro–Wilk test was utilized to assess the normality of data. Additionally, the one-way ANOVA and Tukey's HSD test were used to compare groups with different variables. The data were defined based on the mean and standard deviation. A significance level of p ≤ 0.05 was used. SPSS 20 was used for data analysis.

Results

The impact of diabetes on the levels of the tested factors is assessed in the variance analysis shown in Table 2. Table 3 presents the variance analysis of changes in weight, glucose, MVCC, and VO₂max under the influence of ursolic acid supplementation, endurance training, and resistance training at different times. Additionally, Table 4 displays the changes in body weight, glucose, MVCC, and VO₂max of diabetic rats during an 8-week treatment with various exercise training and ursolic acid.

Table 2 Variance analysis to evaluate the effect of diabetes in changing the levels of the tested factors

Table 3 Variance analysis of changes in weight, glucose, MVCC, and VO₂max factors under the influence of ursolic acid supplementation, endurance training, and resistance training at different times

Table 4 Body weight, glucose, MVCC, and VO₂max changes of diabetic rats during 8-week treatment by different exercise training and ursolic acid

The expression of TAU protein levels

This study investigated the effects of resistance and endurance exercise, along with UA supplementation, on TAU protein levels in aged rats with type 2 diabetes. In AD, neurofibrillary tangles composed of hyperphosphorylated TAU proteins accumulate in the brain. Both AD and type 2 diabetes share the common feature of TAU accumulation. Figure 2a presents the expression of TAU protein levels in different groups of aged diabetic rats.

Fig. 2

Expression of TAU (a), IL-1β (b), TNF-α (c), c-JUN (d), and IRS2 (e) Proteins Levels in Different Groups of Aged Diabetic Rats. Different letters above bars indicate statistically significant differences (p < 0.05) determined by ANOVA and Tukey's HSD test

The type 2 diabetes model increased TAU protein levels in the hippocampus of the CD group compared to the HC group (p < 0.05) and all intervention groups (U, E, R, EU, and RU). However, there was no significant difference in TAU protein levels between the CD and R groups (p > 0.05) or between the U and E groups compared to the EU and RU groups (p > 0.05). Additionally, no difference was observed between the U and E groups (p > 0.05). Interestingly, the R group had significantly higher TAU protein levels compared to all intervention groups except the CD group (p < 0.05). Overall, these findings suggest that endurance training, UA supplementation, and the combination of endurance and UA can reduce TAU levels, while resistance training alone does not have this effect.

The expression of IL-1β protein levels

Interleukin-1β (IL-1β) is a protein implicated in both AD and diabetes mellitus. A common feature of these diseases is a sustained increase in IL-1β, which contributes to structural and metabolic damage in the body. We investigated whether the expression of IL-1β protein levels is altered by diabetes, exercise, and UA supplementation in aged rats with type 2 diabetes (Fig. 2b).

Consistent with previous research, the type 2 diabetes model (CD group) exhibited higher IL-1β protein levels compared to the control group (C group) (p < 0.05). This finding suggests that diabetes increases IL-1β expression. Encouragingly, UA supplementation (U group) and the combined intervention (EU group) significantly reduced IL-1β protein levels compared to the diabetic group (CD group) (p < 0.05). This suggests that UA, alone or with endurance training, may decrease IL-1β, an inflammatory marker. In contrast, the exercise groups (E, R, and RU) displayed IL-1β protein levels similar to the diabetic group (CD group) (p > 0.05). As suggested by past studies, high-intensity exercise may increase inflammatory factors, potentially explaining the lack of change in IL-1β levels in these groups. Interestingly, the UA group (U) had significantly lower IL-1β protein levels compared to the control group (C group) (p > 0.05). Further investigation is needed to elucidate this unexpected finding.

The expression of TNF-α protein Levels

Recent research suggests that TNF-α may be a promising therapeutic target to reduce the risk of developing late-onset AD in individuals with type 2 diabetes. This study investigates the effects of type 2 diabetes, exercise, and UA supplementation on the expression of TNF-α protein levels in aged rats (Fig. 2c).

As shown in Fig. 2c, TNF-α protein levels were significantly lower in the C group compared to the CD group (p < 0.05). Interestingly, the R training group displayed the highest TNF-α protein levels among all groups (p < 0.05). No significant difference in TNF-α protein levels was observed between the CD group and the exercise groups (E and RU) (p > 0.05), while the mean expression of TNF-α protein levels in the U and EU groups was significantly lower than that in the CD group (p < 0.05). These findings suggest that endurance training, UA supplementation, and the combination of endurance exercise and UA may decrease TNF-α levels, while resistance training appears to have an opposing effect.

The expression of c-JUN protein levels

Previous research suggests that diabetes can elevate TNF-α levels, which in turn can activate c-JUN. Activation of c-JUN has been associated to the development of type 2 diabetes and its complications.

As shown in Fig. 2d, the C group exhibited significantly lower c-JUN protein levels compared to the diabetic group (CD) (p < 0.05). Interestingly, the R training group displayed the highest c-JUN protein levels among all groups (p < 0.05) and no significant difference between CD and RU was observed (p > 0.05).

This research showed that no difference between the mean expression of c-JUN protein levels in the U, E, and EU groups was observed (p < 0.05), but the mean expression of c-JUN protein levels in R and RU groups was significantly higher than in the other intervention groups (p < 0.05). The intensity of resistance exercise in this study may lead to an increase in the mean expression of c-JUN protein level.

The expression of IRS2 protein levels

IRS2 is known to mediate metabolic impacts. Previous studies have shown that the IRS2 receptor is involved in diabetes as well as the impairment of learning and memory.

The study aimed to investigate the impact of Type 2 diabetes, exercise, and UA supplementation in aged rats on the levels of IRS2 protein expression (Fig. 2e). It seems that the intensity of resistance exercise in our study produced these results. Our study showed that type 2 diabetes can decrease the expression of IRS2 protein levels in aged rats while endurance exercise with or without UA and resistance with UA can improve it significantly (p < 0.05). In contrast, endurance and UA, with or without together, can improve the expression of IRS2 protein levels in Type 2 diabetes-aged rats. The levels of IRS2 protein in the RU group were significantly lower than in other intervention groups (p < 0.05). It means the competition of resistance exercise and UA didn't have a positive effect on IRS2 in aged type 2 diabetes rats. Probably, the method of resistance exercise in our study may have produced these results.

Discussion

This study shows that supplementing with UA and implementing exercise interventions can improve factors related to Alzheimer's disease in aged rats with type 2 diabetes. In a model of type 2 diabetes induced by low-dose STZ and a high-fat diet, our findings showed that diabetes increased the expression of proteins associated with Alzheimer's disease (TAU, IL-1β, TNF-α, and c-JUN) and decreased IRS2 protein levels in the hippocampus of aged rats. This study aimed to investigate the effects of UA supplementation extract, endurance training, and resistance training on these protein levels. To our knowledge, this is the first study to examine the combined effects of UA supplementation and both endurance and resistance training on IL-1β, TNF-α, c-JUN, and IRS2 protein levels in aged diabetic rats.

Our results confirm that the type 2 diabetes model (low-dose STZ and high-fat diet) increased TAU, IL-1β, TNF-α, and c-Jun protein levels, while reducing IRS2 protein levels in the hippocampus of aged rats.

Prior studies have indicated that inflammatory mediators play a role in the elevation of Aβ production and the impairment of insulin activity in the brain. The overproduction of interleukin-1 (IL-1), interleukin-6 (IL-6), and TNF-α is associated with the presence of astrocytes and microglia surrounding beta-amyloid plaques. These cytokines can stimulate the production of Aβ precursor protein (APP) and the amyloidogenic pathway, ultimately leading to the production of Aβ_1-42 peptides (Boden 2008). APP itself also promotes an increase in Aβ levels in AD. The buildup of Aβ, accompanied by inflammation, leads to oxidative stress and a reduction in insulin signaling in the brain (Yung and Giacca 2020). Insulin enhances learning processes by increasing connections between neurons (synaptic density) and promoting the growth and flexibility of neuronal branches (dendritic plasticity) (Ajuwon and Spurlock 2005). Moreover, high levels of glucose and fat in the peripheral blood can activate immune cells, for example, macrophages, which release pro-inflammatory cytokines that bind to receptors located in neurons, leading to insulin resistance and preventing insulin's effect on nerve cells. The activation of molecules like JNK can lead to the serine phosphorylation of the IRS1 receptor (Yung and Giacca 2020). Additionally, the receptor known as IRS2 plays a role in processes related to neuroplasticity in the brain, including learning and memory (Kleinridders et al. 2014). Studies have consistently shown that insulin has a positive impact on cognitive performance (Procaccini et al. 2016). Cytokines can activate molecules like JNK in neurons, resulting in serine phosphorylation of the IRS1 receptor. This, in turn, inhibits tyrosine phosphorylation and can trigger insulin resistance (Boucher et al. 2014). As we know in the present study, diabetes caused an increase in TAU, IL-1β, TNF-α, and c-JUN protein levels. Considering the role of these factors in memory impairment and AD, it can be suggested that diabetes may lead to Alzheimer's disease in older rats.

In summary, previous research suggests that type 2 diabetes mellitus can cause metabolic changes like obesity and hyperglycemia, which contribute to the development of Aβ plaques, neuron loss, and hyperphosphorylated tau protein. Increased tau levels can trigger the production of inflammatory factors such as IL-6 and TNF-α (Nazareth 2017). These pro-inflammatory factors bind to neuronal receptors and activate molecules like JNK c-Jun, the primary cellular substrate, undergoes activation through JNK-mediated phosphorylation (Nazareth 2017), JNK activation leads to phosphorylation of c-Jun, which affects gene expression and tangle formationAdditionally, JNK activation results in serine phosphorylation of the IRS1 receptor, leading to insulin resistance and impaired insulin action in neurons (Nazareth 2017). Additionally, JNK activation results in serine phosphorylation of the IRS1 receptor, leading to insulin resistance and impaired insulin action in neurons [1]. Aβ plaques can also cause insulin resistance. Both conditions are associated with cognitive deficits, inflammation, increased oxidative stress, and insulin resistance (Nazareth 2017). Furthermore, JNK activation in adipocytes (fat cells) promotes insulin resistance and upregulates pro-inflammatory cytokines like TNF-α, IL-6, and IL-1β, creating a vicious cycle (Yung and Giacca 2020), These findings support the changes observed in the factors measured in our present study and suggest a possible underlying mechanism.

Based on Christian Riehle's research, exercise training can improve the protein content of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in hearts lacking IRS1 and IRS2. The protein content of PGC-1α was unchanged in hearts lacking IRS1 but decreased in hearts lacking IRS2. These data indicate that the roles of IRS isoforms differ in facilitating the exercise-induced hypertrophic and metabolic adaptations of the heart (Riehle et al. 2014).

In our research, we observed that UA supplementation extract, both alone and in combination with endurance training, reduced protein levels of TAU, IL-α, TNF-α, and c-JUN in the hippocampus of aged rats with type 2 diabetes. Additionally, UA supplementation increased IRS2 protein levels. However, the combination of UA and resistance exercise did not significantly reduce IL-α, TNF-α, and c-JUN protein levels, nor did it significantly increase IRS2 levels. Similarly, resistance training alone did not improve the expression of these factors. Interestingly, UA supplementation with both endurance and resistance training reduced IRS2 protein levels in the hippocampus of Type 2 diabetes-aged rats (Király et al. 2010).

In our current research on older rats, we found that resistance exercise did not decrease TAU protein levels. This is despite evidence suggesting that dysfunctional insulin signaling in diabetic brains can increase TAU phosphorylation via changes in glycogen synthase kinase-3β activity (Hobday et al. 2021). Interestingly, studies show that aerobic exercise can alter TAU protein expression and phosphorylation. Joa et al. (2020) demonstrated that aerobic training following stroke modulated the expression of TAU-related proteins and prevented TAU phosphorylation and acetylation through the P-AMP-activated protein kinase (P-AMPK) and p70 ribosomal protein S6 kinase (p70-S6K) pathways. Similarly, Mankhong et al. (2020) observed that aerobic exercise in rats with middle cerebral artery occlusion (MCAO) prevented TAU modifications, particularly acetylation, while improving motor and cognitive function. On the other hand, Liu et al. (2020) reported that resistance exercise improved cognitive function in a mouse model of AD by reducing neuroinflammation. They suggest this may be due to increased presynaptic structural proteins, leading to better synaptic transmission. Their study involved older 3xTg mice participating in a 4-week resistance training program that progressively increased ladder-climbing weights from 15 to 75% of their body weight across weeks 1 to 4. Several studies have investigated the effects of exercise on TAU protein in the brain. Liu et al. (2020) showed that resistance training in a mouse model of AD lowered total TAU and hyperphosphorylated TAU levels, particularly TAU-AT180, in the brain. Interestingly, a decrease in TAU-AT180 phosphorylation was observed in the hippocampus of the exercise group, but not for p-TAU 396/404. In contrast, Gratuze et al. (2017) found no significant effects of Western diet-induced obesity on TAU phosphorylation or aggregation in mice. However, exercise was able to decrease TAU phosphorylation in obese mice expressing human TAU, even with caloric restriction. While self-initiated physical activity and calorie restriction did not significantly impact TAU in these mice, exercise seemed beneficial. These findings suggest that the impact of exercise on TAU may depend on the underlying condition. Resistance exercise might not reduce TAU in diabetic rats due to the potential increase in TAU caused by diabetes, as observed in our study. However, further research is needed to confirm this hypothesis and explore the mechanisms behind these contrasting results.

Our study found that endurance training, but not resistance training, significantly reduced IL-1β levels. Similarly, TNF-α levels remained unaffected by resistance training. These findings are consistent with previous research suggesting that acute exercise increases pro-inflammatory cytokines like IL-1β, TNF-α, IL-6, IL-10, and INF-γ (Khakroo Abkenar et al. 2019; Nikbakht et al. 2011; Salamat et al. 2016). Salamat et al. (2016) investigated the effects of different exercise modalities on pro-inflammatory cytokines in overweight men. Their results showed no significant changes in IL-1β, IL-2β, and TNF-α following resistance training. Similarly, Nikbakht et al. (2011) observed a decrease in C-reactive protein but no change in IL-1β after an 8-week resistance training program with progressively increasing intensity. In contrast, Khakroo Abkenar et al. (2019) reported that high-intensity aerobic exercise increased IL-1β, IL-8, and NLRP3, potentially leading to neuronal death. The results indicate that the intensity of exercise may influence cytokine responses. Our data aligns with this notion, as endurance training (potentially lower intensity) reduced IL-1β, whereas resistance training (potentially higher intensity) did not. Further studies are warranted to explore the specific intensity thresholds that modulate IL-1β levels during exercise. The current study found that however, from different perspectives protein levels were increased through endurance and resistance training. Male obese rats were studied by researchers to observe how the expression of hepatic IRS2 and SREBP1 proteins was affected by High-Intensity Interval Training and Coenzyme Q10. The intervention of High-Intensity Interval Training resulted in a rise in IRS2 content, but there were no significant changes detected.

CoQ10 supplementation did not significantly impact hepatic IRS2 protein levels in individuals undergoing High-Intensity Interval Training (HIIT) (Ghiasi et al. 2021). Conversely, Jorge et al. (2011) reported a significant increase in IRS1 expression following 12 weeks of resistance or combined exercise training in individuals with type 2 diabetes mellitus. The increase was 65% in the resistance group and 90% in the combined group. These findings suggest that exercise modality may differentially regulate IRS isoforms. While our study and Király et al. (2010) did not observe significant changes in IRS2 with exercise, further research is needed to definitively understand the relationship between IRS2 and exercise training.

Exercise has been shown to activate the c-JUN N-terminal kinase pathway in skeletal muscle, potentially linking contractile activity to gene expression changes (Aronson et al. 1998). Cui et al. (2022) demonstrated that swimming exercise reversed the negative effects of a high-fat diet in mice, including hyperlipidemia, liver steatosis, and cell apoptosis. This reversal was associated with decreased downstream MAPK kinase 4 phosphorylation and JNK activation, suggesting exercise can modulate the JNK pathway and potentially mitigate liver lipotoxicity. Interestingly, our study found that endurance training reduced c-JUN levels, while resistance training had the opposite effect. These findings highlight the complex interplay between exercise modality and JNK signaling.

Previous research suggests that inflammatory factors can phosphorylate the IRS1 receptor, leading to insulin resistance and impaired insulin action in neurons (Kirwan et al. 2000). Endurance exercise, however, may counteract these effects by preventing the rise of inflammatory factors and subsequently reducing JUN levels. Since JUN and its downstream effector JNK contribute to cell death, their downregulation could potentially delay the onset of diabetes. Our findings, along with prior studies, indicate that exercise may improve brain function and alleviate insulin resistance by modulating interleukin and TNF-α levels (Király et al. 2010). This suggests that exercise may lower the risk of AD in diabetic individuals, although further research is warranted to confirm this hypothesis. Interestingly, resistance training in this study did not decrease IL-1β and TNF-α levels, consequently failing to positively impact JUN protein levels. This could be attributed to the high intensity of the exercise protocol employed and the potential influence of inflammatory factors on exercise tolerance. Future studies comparing different resistance training intensities and their effects on these key factors are needed to validate this notion.

Previous research by Chen et al. (2022) demonstrated that UA supplementation can decrease IL-1β and fasting blood glucose levels, slow pancreatic β-cell deterioration, and regulate gut microbiota and immune function in rats with streptozotocin-induced type 1 diabetes (T1DM). Similar anti-inflammatory properties of UA have been observed in cases of cerebral ischemia and reperfusion injury. Wang et al.'s study (citing Wang et al. here) suggests that UA reduces the production of pro-inflammatory cytokines, protecting the brain from this type of injury (Schwaiger et al. 2011). UA is known for its neuroprotective, antioxidant, anti-inflammatory, and blood sugar regulation effects (Kazemi Pordanjani et al. 2023), potentially making it beneficial in managing chronic inflammatory conditions (Schwaiger et al. 2011).

Our study investigated the effects of UA supplementation on protein levels like IL-1β, TNF-α, JUN, and IRS2 in aged rats with type 2 diabetes. These proteins play a significant role in the development of AD. The observed changes in protein levels suggest that UA supplementation may be a valuable tool for preventing or treating AD in diabetic individuals. However, further research is necessary to confirm these findings.

The study results represent an initial step in gaining a better understanding of the role of exercise and nutritional supplements in preventing and treating Alzheimer's disease in individuals with diabetes. However, larger clinical studies are required to validate these findings in human subjects.

The limitations of the research

The rats' life experiences, such as their living conditions and handling, can impact their well-being, which can then affect the reliability of our research findings. By having a control group, having one person handle the rats, and regularly monitoring blood sugar levels, we hoped to minimize the potential impact of these limitations on the study results.

Conclusions

This study investigated the effects of resistance and endurance training, with or without UA supplementation, on inflammatory markers in aged rats with type 2 diabetes. We confirmed the independent effect of UA on some inflammatory factors. However, only endurance training significantly impacted TAU, TNF-α, c-JUN, and IRS2 levels. Resistance training only affected c-JUN and IRS2. Further research is needed to explore the interaction between UA concentration and exercise intensity. Our findings suggest that UA supplementation combined with specific exercise modalities, such as endurance training, may be a promising therapeutic strategy for preventing AD in older diabetic patients. Endurance training appears to be more effective than resistance training in modulating key inflammatory factors associated with AD development. This approach warrants further investigation to determine its efficacy in improving health outcomes for type 2 diabetes in older adults.