Many studies have reported that T2DM with sustained hyperglycemia is a serious risk factor for cognitive dysfunction and dementia (Mehta and Banerjee, 2017; Palta et al., 2014; Umegaki, 2014). To counter this condition, currently, anti-hyperglycemic medications like metformin, pioglitazone, etc., are being prescribed (Tyagi and Pugazhenthi, 2021). However, hyperglycemia is not the sole factor responsible for diabetes-associated cognitive insult (Gaspar et al., 2016b). Therefore, understanding the pathobiology of the disease and identifying the specific molecular targets with pharmacological and non-pharmacological approaches is the need of the hour. Aerobic exercise has been shown to be an excellent non-pharmacological intervention against cognitive impairment and Alzheimer’s disease (Kim et al., 2014). However, the exact mechanism is yet to be explored. The present study demonstrates the beneficial role of treadmill exercise alone and metformin coadministration on HFD + STZ-treated type 2 diabetic rats. Here, we observed that long-term treadmill exercise with metformin (70 mg/kg) treatment alleviates the neuroinflammation and also upregulates the adult hippocampal neurogenesis by upregulating the Wnt/β-catenin pathway, which is impaired in T2DM rat brain.
In the present study, we used a high-fat diet combined with a single low-dose injection of STZ to induce an experimental model of type 2 diabetes, and after fourteen weeks of diabetes induction, blood glucose levels and serum metabolic parameters like triglyceride, total cholesterol, and serum insulin levels were found to be elevated in HFD + STZ-treated rats, as previously reported (Datusalia and Sharma, 2014). Cerebral insulin resistance and glucose hypometabolism are the enigmatic factors responsible for the loss of synaptic plasticity and lead to cognitive dysfunction (Grillo et al., 2019). It has been reported that levels of IRS-1 are reduced in rats administered HFD for 12 weeks (Karvani et al., 2019; Pratchayasakul et al., 2011). In line with this, the level of IRS-1 in the hippocampal tissue decreased in HFD + STZ-treated rats compared to the control group, which signifies the loss of insulin sensitivity in the hippocampus of diabetic rats. However, treadmill exercise with metformin administration for 14 weeks partially reduced the blood glucose level, upregulated the IRS-1 level, and prevented hippocampal insulin resistance that manifests due to T2DM. Furthermore, serum metabolic parameters like triglyceride and cholesterol levels, which were elevated in diabetic animals, were also lowered after treadmill exercise and metformin treatment.
Multiple studies have reported that T2DM with chronic hyperglycemia significantly deteriorates cognitive health in human and rodent models (Datusalia and Sharma, 2014; Mehta and Banerjee, 2017). Neurobehavioural assessments like the Y-maze test, passive avoidance and novel object recognition test were performed between the 14th and 15th week before euthanasia. Fourteen-week HFD + STZ-treated rats showed significant alteration in behavioural patterns compared to the naïve control group. Long-term treadmill exercise proved beneficial for cognitive function in rodent models of Alzheimer’s disease and dementia (Hoveida et al., 2011). We also observed that 14-week treadmill exercise and exercise with metformin (70 mg/kg) treatment improved the T2DM-associated cognitive deficits in T2DM animals.
Oxidative stress is a cellular process manifested by a disbalance in producing oxidants and antioxidant defences. Chronic T2DM disrupts cerebral glucose metabolism, altering the brain's redox homeostasis and oxidative health (Muriach et al., 2014; Reis et al., 2012). MDA and GSH levels are usually considered oxidative stress markers, markedly altered in the brain during metabolic dysfunctions like obesity, diabetes, etc (Li et al., 2023). Previous reports suggest that T2DM rats with chronic hyperglycemia and dyslipidemia showed a decline in reduced GSH levels and elevated MDA levels in the hippocampal tissue (Butterfield et al., 2014; Chen et al., 2022). We also estimated the level of MDA and GSH in rats fourteen weeks after diabetes induction and observed an elevated level of MDA and a decreased level of reduced GSH in hippocampal tissue compared to control animals. However, long-term moderate treadmill exercise with metformin (70 mg/kg) partially reversed the oxidative stress markers compared to HFD + STZ-treated rats. Furthermore, we also examined the level of proinflammatory cytokines in hippocampal tissue across the groups. As previously reported, elevated levels of proinflammatory cytokines like TNF-α, IL-1β and chemokines such as MCP-1 were found in the hippocampal tissue of HFD + STZ-treated rats compared to control (Chen et al., 2022). Lang et al. reported that long-term moderate treadmill exercise ameliorates cognitive impairment in T2DM mice by alleviating neuroinflammation and increasing the BDNF level in the brain (Lang et al., 2020). In congruence with these studies, we also observed that proinflammatory cytokines were lowered upon fourteen-week treadmill exercise and metformin administration compared to the diabetic group. In addition, we also checked the level of BDNF in the hippocampal tissue, which was found to be decreased in T2DM animals, and long-term moderate treadmill exercise elevates the BDNF level in the hippocampus. Our result also showed that 14 weeks of moderate treadmill exercise and metformin administration increased the BDNF level compared to T2D rats. To further examine the astrogliosis and microglial activation, we performed GFAP and Iba-1 protein expression studies. Chronic hyperglycemia has been reported to cause astrocytic and microglial activation, ultimately leading to neuroinflammation (Nagayach et al., 2014; Paul et al., 2024). Similar to these reports, we observed elevated GFAP and Iba-1 expression levels in the hippocampal tissue of HFD + STZ-treated rats, which was downregulated upon long-term treadmill exercise and metformin coadministration.
Moreover, acetylcholinesterase (AchE) serves as an important marker for cognitive impairment, which is found to be elevated in chronic diabetic rodent brains (Rajput and Sarkar, 2017). Mehta et al. observed a decreased AchE level in type 2 diabetic rats upon long-term aerobic exercise (Mehta et al., 2019). As per the reports, we also observed a rise in AchE levels in hippocampus tissue, which lowered significantly after 14 weeks of treadmill exercise and metformin co-administration in T2DM rats. Furthermore, type 2 diabetes significantly affects the survivability of pyramidal neurons and decreases the neuronal density in the hippocampal tissue (Tian et al., 2022). Zhou et al. reported that eight weeks of treadmill exercise partially reverses the hippocampal neuronal viability in db/db mice (Zhou et al., 2022). In line with this, we performed the nissl staining to check the neuronal viability. We further carried IHC for NeuN (a marker for mature neurons) in hippocampal tissue and found a significant decrease in viable neurons in HFD + STZ-treated animals compared to the control. Moreover, treadmill exercise for 14 weeks with metformin co-administration ameliorated the loss of neurons in the hippocampal region.
Hippocampal neurogenesis is severely affected by chronic hyperglycemia (Lang et al., 2009). Treadmill exercise increases hippocampal neurogenesis by various mechanisms (Lou et al., 2008; Nokia et al., 2016; Zhao et al., 2017). However, the effect of treadmill exercise on hippocampal neurogenesis in type 2 diabetes has been poorly understood. Tanokashira et al. reported that long-term metformin (250 mg/kg, p.o.) administration ameliorates the diabetes-associated decline in hippocampal neurogenesis by phosphorylation of IRS-1 (Tanokashira et al., 2018). In the present study, we report that 14 weeks of chronic hyperglycemia significantly impaired the hippocampal neurogenesis, characterised by altered protein expression of various neurogenesis markers like Ki-67, DCX, and NeuN. We found a significant downregulation of these proteins in the hippocampal region, and long-term treadmill exercise with metformin administration upregulates these markers. Next, we explored the possible pathways associated with adult hippocampal neurogenesis targeting the canonical Wnt/β-catenin signalling pathway, which plays an important role in hippocampal neurogenesis (Mishra et al., 2019; Varela-Nallar and Inestrosa, 2013). Immunoblotting analysis revealed that HFD + STZ treatment for fourteen weeks significantly altered the canonical Wnt/β-catenin pathway by modulating the upstream and downstream signalling molecules. The protein expression of Wnt-3a, β-catenin, and p-GSK-3β was downregulated in the hippocampus of T2DM rats compared to the control group and the protein expression of DKK-1, an endogenous antagonist of the Wnt ligand (Ren et al., 2019), was upregulated in the diabetic group. Moreover, fourteen-week treadmill exercise and metformin (70 mg/kg) co-treatment ameliorate the dysregulation of the canonical Wnt/β-catenin pathway. We also performed the immunofluorescence assessments and obtained the altered protein expression of the signalling proteins like Wnt-3a, β-catenin and DKK-1 in line with the immunoblotting experiment. The proposed mechanism for treadmill exercise and metformin-associated neuroprotection against diabetes-associated cognitive decline has been summarized in Fig. 10.