Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Effects of antidiabetic agents on Alzheimer’s disease biomarkers in experimentally induced hyperglycemic rat model by streptozocin

  • Shatw Khalid Ali ,

    Contributed equally to this work with: Shatw Khalid Ali, Rojgar H. Ali

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing

    shatwla@yahoo.co.uk

    Affiliation Department of Pharmacology and Toxicology, Hawler Medical University, Erbil, Iraq

    ⨯
  • Rojgar H. Ali

    Contributed equally to this work with: Shatw Khalid Ali, Rojgar H. Ali

    Roles Methodology, Supervision

    Affiliation Department of Pharmacology and Toxicology, Hawler Medical University, Erbil, Iraq

    ⨯

Abstract

Background

Alzheimer’s disease is the most common cause of dementia in the elderly population. It is characterized by the accumulation of amyloid β and intraneuronal neurofibrillary tangles in the brain. Increasing evidence shows that the disturbance of insulin signalling in the brain may contribute to the pathophysiology of Alzheimer’s disease. In type 1 diabetes, these disruptions are caused by hypoinsulinemia, but in type 2 diabetes, they are caused by insulin resistance and decreased insulin secretion. Multiple studies have shown that diabetes is connected with an increased risk of acquiring Alzheimer’s disease. The aim of this study was to investigate the impact of anti-diabetic agents on Alzheimer’s disease progression and the levels of Alzheimer’s biomarkers in a hyperglycaemic rat model, which was induced by intraperitoneal injection of streptozocin to produce insulin-deficient diabetes.

Method

Thirty-six male Wistar albino rats were allocated into six groups of six rats each. Group I was the negative control group. Intraperitoneal injections of streptozocin (42mg/kg) were used once for the five experimental groups. Group II served as the positive control group. The rats in Groups III, IV, V, and VI received metformin (300mg/kg), donepezil (10mg/kg), insulin glargine (3 unit/animal), and glibenclamide (10mg/kg), respectively, for 21 days.

Results

Inducing hyperglycaemia in rats significantly increased the levels of serum glucose, haemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein, interleukin 6, tumour necrosis factor alpha, amyloid β 42, total plasma tau, and neurofilament light. A significant increase was also found in brain amyloid β 42, nitric oxide, acetylcholinesterase, malondialdehyde, β secretase, and phosphorylated microtubule-associated protein tau. The greatest statistically significant reductions in serum glucose, haemoglobin A1c, triglycerides, amyloid β 42, total plasma tau, brain amyloid β 42, acetylcholinesterase, and malondialdehyde were observed in rats treated with metformin. In contrast, rats treated with donepezil demonstrated the greatest statistically significant reduction in serum tumour necrosis factor alpha, brain nitric oxide, and β secretase. The levels of neurofilament light and phosphorylated microtubule-associated protein tau in the brains of rats treated with insulin glargine were significantly lower than the other treatment groups. The total cholesterol and low-density lipoprotein levels in rats treated with glibenclamide exhibited the most statistically significant reductions of all the treatment groups.

Conclusions

Metformin and donepezil, when administered at appropriate doses, were shown to successfully lower most plasma and brain biomarkers, including glucose, triglycerides, tumour necrosis factor alpha, amyloid β 42, nitric oxide, acetylcholinesterase, malondialdehyde, and β secretase in rats suffering from Diabetes Mellitus. As a result of this research, we suggest that metformin, either alone or in conjunction with donepezil, might be an excellent drug of choice for neuro-regeneration and risk reduction in Alzheimer’s like disease.

Citation: Ali SK, Ali RH (2022) Effects of antidiabetic agents on Alzheimer’s disease biomarkers in experimentally induced hyperglycemic rat model by streptozocin. PLoS ONE 17(7): e0271138. https://doi.org/10.1371/journal.pone.0271138

Editor: Stephen D. Ginsberg, Nathan S Kline Institute, UNITED STATES

Received: February 21, 2022; Accepted: June 23, 2022; Published: July 8, 2022

Copyright: © 2022 Ali, Ali. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly population. It is characterized by neurodegeneration affecting both the cortex and the limbic system, as well as the accumulation of amyloid β and intraneuronal neurofibrillary tangles in the brain [1]. Despite various hypotheses, the specific pathologic pathways that cause neurodegeneration in AD are still unclear. Increasing evidence shows that the disturbance of insulin signalling in the brain may contribute to the pathophysiology of AD [2]. Several studies have discovered decreased insulin levels and insulin receptor expression in the brains of AD patients [3, 4], whereas other studies have highlighted insulin resistance [5], but all the evidence points to a breakdown of the insulin-signalling system. In addition to regulating food intake and energy balance, insulin and insulin receptors in the brain play a role in cognitive performance [6].

In type 1 diabetes mellitus (T1DM), these disruptions are caused by hypoinsulinemia, but in type 2 diabetes mellitus (T2DM), they are caused by insulin resistance and decreased insulin secretion. Multiple studies have shown that DM is connected to an increased risk of acquiring AD [7–9]. Although other studies have found no clear association between DM and cognitive impairment, dementia, or AD [10, 11], they emphasize that diabetes should be considered a potential risk factor for these conditions [12].

Disorders of the insulin-signalling pathway are emerging as common hallmarks of both AD and DM; nevertheless, the emphasis thus far has been on T2DM, with hyperinsulinemia and insulin resistance serving as the key insults [12]. Little information is known about the relationship between T1DM and AD, despite the fact that hypoinsulinemia causes a comparable impairment in insulin signalling. Furthermore, cognitive deficits, such as impaired learning and memory and difficulties with problem solving and mental flexibility, are more common in individuals with T1DM than in the normal population [13, 14], suggesting that the negative effect of cerebral hyperglycaemia or hypoinsulinemia exists in hyperglycaemic individuals.

As a result of these deficiencies, degradation of the cerebral cortex [15] and neuronal loss [16] are often identified at autopsy, and these findings are more evident in individuals with T1DM than in age-matched non-diabetic patients.

Recently, it was found that learning impairments are associated with increased tau phosphorylation, and higher amyloid β protein levels in the brain were identified in a T1DM mouse model [17]. Similar alterations have also been seen in a spontaneous T1DM rat model [18].

To further investigate the roles of insulin deficiency and hyperglycaemia in the central nervous system dysfunction and pathology, we have investigated the impact of anti-diabetic agents on AD progression and AD biomarker levels in a hyperglycaemic rat model, which was induced by streptozocin (STZ) to produce insulin-deficient diabetes. Alterations in amyloid β 42, tau, and phosphorylated microtubule-associated protein tau (pMAPT/ptau), which are the key neuropathological markers of AD, were specifically examined in both the brain and serum. Furthermore, we investigated whether T1DM effects levels of interleukin 6 (IL6), tumour necrosis factor alpha (TNFa), neurofilament light (NFL), nitric oxide (NO), acetylcholinesterase (AChE), malondialdehyde (MDA), and β secretase.

Method

Thirty-six male Wistar albino rats, weighing 200 to 300 g and aged 5 to 6 months, were evaluated in this study. One week prior to the start of the experiment, six male rats were housed per cage at 24°C and 55% ± 5% humidity with a 12-hour light-dark cycle (light on at 8:00 am and light off at 8:00 pm). Unlimited access to water and ordinary rat pellet diets were provided. The animals were acquired via the Hawler Medical University, College of Pharmacy (Iraq-Erbil). This work was authorized by the ethics committee of the Hawler Medical University, College of Pharmacy, with permission number HMUPH-EC20211129-361. For the entire duration of the research project, we adhered to the “Guide for the Care and Use of Laboratory Animals,” which was developed by the National Academy of Science and published by the National Institutes of Health.

Materials

Rat TNFa, amyloid β peptide 42, AChE, NO, β secretase, MDA, (pMAPT/ptau), tau protein, NFL polypeptide, and rat IL6 enzyme-linked assay (ELISA) kits were purchased from Shanghai Sunred Biological Technological Co., Ltd (China). STZ was bought from Glentham Life Sciences Corsham, S13 0SW, United Kingdom. Glucophage (metformin; 500mg tab), Daonil (glibenclamide; 5mg tab), Lantus (insulin glargine; 100 units/ml pen), and Aricept (donepezil; 10mg tab) were purchased in a pharmacy and were manufactured by Merck Healthcare (Darmstadt, Germany), Sanofi (Paris, France), Sanofi (Paris, France), and Pfizer (New York, United States), respectively.

Study design

In this study, the 36 male Wistar albino rats were allocated into six groups with a simple random sampling method (n = 6): Group I served as a negative control group, and Group II functioned as a positive control group.

On Day 1 of the experiment, all the rats were fasted for 12 hours prior to receiving STZ therapy and were given water as usual. A total of 21 mg of STZ was weighed into a 1.5-ml microcentrifuge tube, which was sealed with aluminium foil. One tube was used for each rat. The STZ was dissolved in 50 mM of sodium citrate buffer (pH 4.5) to a final concentration of 21 mg/ml immediately before injection. For groups II, III, IV, V, and VI, the STZ solution was injected intraperitoneally (i.p.) at 42 mg/kg (2.0 ml/kg) using a 1-ml syringe and a 23-G needle [19]. The negative control group received an equivalent amount of citrate buffer (pH 4.5) injected i.p. [20]. After injection, they were provided with normal food and maintained on 10% (w/v) sucrose water. On Day 2 of the experiment, the sucrose water was replaced with regular tap water [20].

On Day 3, all the rats were fasted for 6–8 hours in the early morning. The animals were anesthetized with 1.0 ml of xylazine (20 mg/ml), which was added to 10 ml of ketamine (100 mg/ml) and diluted by one-tenth with sterile saline [21]. Blood was collected through the medial canthus of the orbit using the end of a microhematocrit tube [22], and blood glucose concentration was measured using the Accu-Chek Instant blood glucose monitoring system to check for hyperglycaemia. More than 50% of the rats had blood glucose levels of more than 150 mg/dl (8.3 mmol/L), which was significantly higher than the control animals, suggesting that they were in the early stages of T1DM. On Day 10 of the experiment, the groups who failed the initial test for diabetes were tested again by repeating the above-mentioned procedure, and all of the rats had statistically higher blood sugar levels than the negative control group.

Most of the STZ-injected rats developed severe diabetes after 3 weeks, with blood glucose concentrations commonly ranging from 250 to 600 mg/dl (13.9–33.3 mmol/L) in most cases. According to previous studies, all of the DM complications should be apparent within 4–8 weeks [20]. Table 1 shows the variations over several days in rat blood glucose levels after STZ (42 mg/kg) injections.

thumbnail
Download:
Table 1. High blood glucose levels in rats after exposure to STZ.

Hyperglycaemia was induced in rats using a single i.p. dose of STZ (42 mg/kg). Blood glucose levels were checked before and after the STZ injection.

https://doi.org/10.1371/journal.pone.0271138.t001

Animals began treatment on the Day 61 of the experiment.

Group III: Received an oral daily dose of metformin, 300 mg/kg [23, 24].

Group IV: Received an oral daily dose of donepezil, 10 mg/kg [25].

Group V: Received a subcutaneous injection of insulin glargine, 3 units/rat [26].

Group VI: Received an oral daily dose of glibenclamide, 10 mg/kg [27, 28].

The oral medications were dissolved in normal saline, and the correct dosage was calculated. The medications were administered to the animals through oral gavage, except for insulin, which was injected subcutaneously.

After 21 days of drug administration, all animals were anesthetized with i.p. injection of xylazine (10 mg/kg) and ketamine (125 mg/kg) [29]. Blood was collected using the cardiac puncture method [22]. After centrifuging the blood samples, the separated serums were kept in tubes and put in the freezer at -23°C for five days until the day the tests were measured. The animals were sacrificed via cervical dislocation at the end of the experiment. To confirm the animals’ deaths, the heartbeats and pupillary reactions to light were measured. The brains were removed and the brain tissues were washed with ice-cold normal saline, mixed with phosphate buffer, and homogenized by hand by using mortar and pestle. The brain tissue extracts were separated by a cold centrifuge and placed in Eppendorf tubes, which were labelled with the group name and kept in a freezer at -80°C for five days until the day the tests were measured.

Biochemical assays

Rat TNFa, amyloid β peptide 42, AChE, NO, β secretase, MDA, pMAPT/ptau, tau protein, NFL, and IL6 were measured using ELISA kits specific for rats. Serum glucose, haemoglobin A1c (HbA1c), triglycerides (TG), total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using specific reagents for each of the parameters mentioned, using the Cobas-Roche analyser.

Statistical analysis

All data were expressed as mean ± standard error mean (mean ± SEM). Differences in all the parameters between control and medication-treated rats were examined using statistical package for social science (SPSS) (version 25), and a one-way analysis of variance (ANOVA) was conducted to assess the results. A multiple comparison (Tukey) test was performed to compare the groups. A P value < 0.05 was used to determine statistical significance. The datasets used or analysed for the present investigation are available from the corresponding author without restriction.

Results

The effects of different treatment groups on serum biomarkers

The results (Table 2) showed that the rats in the positive control group (pre-treated with STZ) had significantly higher blood glucose levels (761 ± 37.95 mg/dl) than the negative control group (132.16 ± 1.66 mg/dl) (P < 0.05). A post hoc test was performed to discover the exact difference and significance level between the positive control group and the study groups. All forms of the treatments significantly reduced blood glucose levels compared to the positive control, except for donepezil (P = 0.07) and insulin glargine (P = 0.26).

thumbnail
Download:
Table 2. The effects of metformin, donepezil, insulin glargine, and glibenclamide on serum biomarkers: Glucose, HbA1c, lipid profile, IL6, TNFa, amyloid β 42, total plasma tau, and NFL.

https://doi.org/10.1371/journal.pone.0271138.t002

The results indicated that the positive control group had significantly higher blood HbA1c levels (9.68 ± 0.8%) than the negative control group (5.51 ± 0.047%) (P < 0.05). A post hoc test was performed to determine the exact difference and significance level between the groups (inter-group variation). The difference between all the groups after 21 days of treatment was not significant (P > 0.05).

The results showed that a statistically significant relationship existed between the various treatment groups’ serum total cholesterol levels. When a post hoc test was performed, it indicated that a significant reduction existed in all the treatment groups compared to the positive control group.

The findings also showed that the positive control group had significantly higher triglycerides levels (128.33 ± 26.69 mg/dl) than the negative control group (60.16 ± 3.41 mg/dl) (P < 0.05). A post hoc test was performed to determine the exact difference and significance level between the control and treatment groups, it indicated that a significant reduction existed in all the treatment groups compared to the positive control group (P < 0.05).

The results (Table 2) showed that no statistically significant association existed between the different treatment protocols and serum LDL levels. The mean serum LDL levels were similar in each group, ranging from 10–14 mg/dl. ANOVA was performed to compare the average serum LDL levels of all the groups (P = 0.392). A post hoc (Tukey) test was performed to identify the exact difference in HDL levels between the control and treatment groups. Except for glibenclamide, which differed substantially from all other treatment groups (P < 0.05), there was no significant difference across the groups.

Hyperglycaemia induction by STZ resulted in a significant increase of IL6 levels (81.6 ± 0.75 pg/ml) (P<0.05) compared to the negative control group (32.28 ± 3.3 pg/ml). A post hoc (Tukey) test was performed, and the difference between all the groups was not significant (P > 0.05), except for positive control and insulin glargine (P < 0.05), which increased IL6 levels significantly more than the positive control and all the treatment groups.

Plasma TNFa concentration in the STZ-pre-treated rats was significantly higher (44.71 ± 1.17 ng/L) than that in the negative control group (11.93 ± 0.61 ng/L) (P < 0.05). The rats treated with insulin had significantly highest plasma TNFa levels compared to the positive control group and among the other treated groups, the rats treated with metformin and donepezil had significantly lowest TNFa levels compared to the positive control group (P < 0.05).

The i.p. injection of STZ in the positive control group significantly raised the serum level of amyloid β 1–42 (700.1 ± 20.04 pg/ml) compared to the negative control group (71.91 ± 0.85 pg/ml) (P < 0.05). A post hoc test was performed, and there was a significant increase in all the treatment groups compared to the positive control group (P < 0.05), except for glibenclamide (P = 0.62).

The rats pre-treated with STZ, the serum total plasma tau protein was significantly higher (140.83 ± 8.04 ng/L) than that in the control group (22.93 ± 0.51 ng/L) (P < 0.05). A post hoc test was performed, a significant reduction existed in all the treatment groups compared to the positive control group (P < 0.05).

In the hyperglycaemia-induced rats, serum NFL levels were higher (18.41 ± 0.14 ng/L) compared to the negative control group (3.42 ± 0.15 ng/L) (P < 0.05). A post hoc test indicated that a significant reduction existed in all the treatment groups compared to the positive control group (P < 0.05).

The effects of different treatment groups on brain biomarkers

The results (Table 3) showed that the control group (pre-treated with STZ) had significantly higher brain amyloid β 42 levels (778 ± 15.85 pg/ml) than the negative control group (86 ± 1.96 pg/ml) (P < 0.05). A post hoc test was performed to determine the exact difference and significance level between the positive control group and the treatment groups, a significant reduction existed in all the treatment groups compared to the positive control group (P < 0.05).

thumbnail
Download:
Table 3. The effects of metformin, donepezil, insulin glargine, and glibenclamide on brain biomarkers: Amyloid β 42, nitric oxide, acetylcholinesterase, malondialdehyde, β secretase, and pMAPT.

https://doi.org/10.1371/journal.pone.0271138.t003

The brain nitric oxide level in STZ pre-treated rats was significantly higher (29.86 ± 0.24 μmol/L) than in the control group (7.43 ± 0.19 μmol/L) (P < 0.05). The brain nitric oxide level was significantly reduced in all the treatment groups (P < 0.05).

The level of AChE in hyperglycemia-induced rats was significantly higher than in the negative control group. A post hoc test was performed, and the AChE level was significantly reduced in all the treatment groups (P < 0.05).

The level of MDA was significantly higher in hyperglycaemia-induced rats than in the negative control group. According to the post hoc analysis, the brain MDA level was significantly reduced in all the treatment groups (P < 0.05).

The level of β secretase was significantly higher in hyperglycemia-induced rats than in the negative control group. A post hoc test was performed, and it revealed that the brain β secretase level was significantly reduced in all the treatment groups (P < 0.05).

The level of pMAPT in the brains of STZ pre-treated rats was significantly higher (44.55 ± 0.89 ng/L) than in the control group (16.38 ± 0.68 ng/L) (P < 0.05). Brain pMAPT levels were significantly reduced in all treatment groups (P < 0.05).

Discussion

Previously, AD and DM were seen as two distinct diseases. In contrast, a number of clinical and pre-clinical investigations have shown that AD and DM have comparable pathogenic pathways [30]. However, DM cannot be assumed to be sufficient to cause AD; rather, it may play a role as a cofactor in the progression of the illness due to abnormalities in insulin signaling that are accompanied by considerable elevation of amyloid β aggregation, tau hyperphosphorylation, inflammation, oxidative stress, and mitochondrial dysfunction [31]. Due to the same pathogenesis, it has been proposed that anti-diabetic medications may have therapeutic promise for the treatment of AD. Clinical investigations are now investigating these theories. It was previously known that some anti-diabetic drugs were helpful against a number of the hallmark AD pathologies, and it was also recognized that these treatments increased neurogenesis. Although the outcomes of these medications are promising, a thorough understanding of the shared pathomechanisms between DM and AD, the central and peripheral molecular actions of medications, and the impact of demographic changes and genetic mutations on AD development is urgently required for diagnostic and therapeutic purposes [31]. Additionally, an increase in AD knowledge, diagnosis, and possible treatments, as well as population-based research, are crucial for preventing dementia caused by diabetes in high-risk populations.

In mammals, STZ is highly toxic to the insulin-producing beta cells of the pancreas. It has the potential to selectively destroy the cells responsible for generating and releasing insulin, and it is used in medical research to establish an animal model for T1DM when administered in high dosages. STZ administration by a method such as intracerebroventricular or i.p. injection results in decreased cognition and increased cerebral aggregated amyloid β fragments, total tau protein, and amyloid β deposits [32]. Tables 2 and 3 show that i.p. administration of STZ (42 mg/kg) in rats caused significant physiological alterations that can be compared to AD.

Blood glucose level is a sensitive indication of DM, and rats with a baseline fasting blood glucose level <135 mg/dL were regarded as normal [33]. After STZ administration, blood glucose levels were considerably higher in this study. The damage to pancreatic cells that STZ caused led to hypoinsulinemia and hyperglycaemia. The fact that the elevated level persisted after 21 days of therapy indicates that the harm was irreversible.

The glycosylated haemoglobin (HbA1c) test reflects blood glucose concentrations during the previous 8–12 weeks and is often used as an indicator of average blood glucose concentration. When the HbA1c test is less than 5.7%, it is considered normal [34]. In this investigation, the HbA1c level was considerably higher than that following STZ administration. An increase in HbA1c was caused by establishing a sugar-haemoglobin connection, which indicates the presence of excessive sugar in circulation, a sign of diabetic complications [35]. This discovery is consistent with the findings of previous research in the field [34].

Lipids, as the fundamental building blocks of cell membranes, play a significant role in human health and brain function. The brain contains a high concentration of lipids, and the dysregulation of lipid homeostasis is related to aging and plays a significant role in the aetiology of AD [36]. A lipid profile is a blood test that detects problems in lipid levels and mainly measures HDL, LDL, triglycerides, and total cholesterol [37, 38]. According to the findings of this research, glibenclamide had a significant effect on lowering HDL levels in all treatment groups when compared to the positive control group and LDL levels were not significantly different amongst the groups. All the treatment groups were able to significantly lower triglycerides levels, with metformin being the most successful. This finding is in accordance with the results of past research studies [39]. All the treatments were effective in lowering total cholesterol compared to the positive control group, particularly glibenclamide, and this finding is also consistent with previous research [40].

IL6 is a pleiotropic cytokine that plays a critical role in host defence, owing to its extensive spectrum of immunological and hematologic activities and its strong capacity to elicit the acute phase response [41]. In a recent study, both mild and moderately severe AD patients had significantly higher IL6 secretion levels than healthy individuals of the same age. These elevated levels of peripheral IL6 secretion might have been responsible for the acute-phase proteins in their serum [42]. In the current study, after induction of DM, none of the treatment groups reduced the level of IL6 below that of the positive control group.

TNFa is a gliotransmitter that regulates synaptic activity in brain networks, and it has recently been shown to have an important role in the breakdown of synaptic memory pathways caused by amyloid β and amyloid-β oligomers [43]. The current research showed that DM significantly increased TNFa levels and among all the medications, metformin and donepezil could significantly reduce TNFa levels when compared to the positive control group. This conclusion is in line with earlier study findings [44, 45].

Tau is a protein that controls the formation of microtubules and their structural integrity under physiological conditions [46]. Additionally, amyloid β is well known for forming amyloid plaque on nerve cells in the brains of AD patients [47]. According to studies, tau phosphorylation and amyloid β buildup both have a role in the development and pathophysiology of AD [47]. There is also a relationship between tau and DM, since both insulin and insulin growth factor 1 are involved in tau phosphorylation, which is linked to the formation of neurofibrillary tangles and synaptic loss. When insulin communication in the brain is disrupted, tau phosphorylation begins, resulting in reduced Akt kinase activity (also known as protein kinase B) and increased glycogen synthase kinase 3 beta (GSK-3) activity [48]. GSK-3 regulates glucose levels in the blood by contributing to glycogen production [49]. Increased levels of total tau and phosphorylated tau were identified in the brains of both T1DM and T2DM patients compared to healthy controls [50, 51]. In addition, T1DM patients have a greater concentration of amyloid β peptide 42 in their brains than healthy controls [51]. Finally, advanced glycation end (AGE) products are formed as a result of dysregulated glucose metabolism in DM. Increased AGE levels in the brain have been observed to enhance amyloid β 42 aggregation by preventing its clearance. The density of AGE receptors is enhanced in AD and is engaged in amyloid β related inflammatory processes [52].

Several clinical trials have shown that diabetic people who use metformin for a long time have greater cognitive function than those who take other anti-diabetic medicines. Metformin has been shown to reduce tau phosphorylation and amyloid β formation by inhibiting AChE activity and therefore raising acetylcholine content in the brain, which is important for learning and memory [53]. Overall, it seems that the commonly used AD biomarkers (tau protein hyperphosphorylation and amyloid β accumulation) are linked to memory impairment in people with DM, suggesting that these biomarkers may be useful in DM patients.

In this study, the level of amyloid β in serum and brain tissue was significantly increased after the induction of DM. Among all the treatment groups, particularly metformin, had significantly reduced amyloid β levels relative to the positive control group. This result is in line with previous studies that suggest that metformin may be an ideal choice for neuro-regeneration and risk reduction of AD [54]. The results of this study on plasma tau show that after the induction of DM, a significant rise in plasma tau levels occurred. All the treatment groups were associated with significantly lower tau levels than the positive control group, with metformin and insulin glargine being the most effective. This finding is similar to that reported in a previous study on metformin [55].

It has recently been proposed that the NFL chain is a neuron-specific structural protein that can be measured in cerebrospinal fluid and plasma [56, 57] to detect axonal injury and neurodegeneration in a wide variety of neurological disorders, including AD [58]. Higher NFL levels have been associated with increased mortality in AD and other neurodegenerative disorders [59]. The current results indicate that all the treatment groups were able to significantly reduce NFL levels, with insulin glargine and glibenclamide being the most effective in returning the levels to normal.

NO is an enzymatic product of NO synthase that has major physiological activities. An accumulating body of data shows that NO pathways are linked to a range of neurological illnesses, including AD and other neurodegenerative dementias. Aging with a vascular risk factor reduces cerebral blood flow, resulting in microvasculopathy with reduced NO release and localized metabolic dysfunction [60]. This study found that across all the treatment groups, only metformin and donepezil were able to significantly reduce NO levels to normal, which is consistent with results from other studies [61, 62].

AChE is a cholinergic enzyme present in postsynaptic neuromuscular junctions, especially in muscles and nerves. Several studies have revealed that AChE activity and its molecular forms differ in AD tissues. Many of these studies examined whether differences in AChE isoform distribution can be used as a biochemical Alzheimer’s diagnosis [63, 64]. In this study, the level of AChE in the brain increased significantly after the induction of DM, and all treatments significantly lowered AChE levels compared to the positive control group, with metformin, donepezil, and insulin glargine being the most effective at returning the levels to near-normal, which is consistent with results from other studies [53, 65, 66].

MDA is a regularly used biomarker for lipid peroxidation and oxidative stress. Lipid peroxidation is a series of reactions that produce active molecules that cause cellular damage [67], and oxidative stress has been related to several diseases, including AD [68]. After induction of DM, MDA levels increased significantly, and only metformin returned the levels to near-normal. This result is in line with earlier research findings [69, 70].

β secretase (BACE1, or APP-cleaving enzyme 1) is an aspartic proteinase involved in cell differentiation, immunoregulation, and cell death. BACE1 is the major β secretase in neurons that produces amyloid-β peptides [71], and growing evidence links BACE1 activity changes to diseases like AD [72]. Following DM induction, the level of β secretase increased significantly, and among all the treatment groups, donepezil and metformin were the most successful in decreasing the β secretase level. This discovery also aligns with prior research findings [53, 73].

Tau proteins are crucial for microtubule stability in the body. These proteins are abundant in nerve cells but far less so in oligodendrocytes and astrocytes [74]. Pathologies of the nervous system, such as AD, may occur when tau proteins become deficient and fail to appropriately maintain microtubules [75]. The level of pMAPT significantly increased following the induction of DM, and insulin glargine was the only therapy that could return it to a normal level. This finding is in keeping with earlier results [76], although the other treatments only slightly reduced the pMAPT level compared to the positive control group.

Conclusion

Metformin and donepezil, when administered at 300mg/kg and 10mg/kg, respectively, were shown lower most plasma and brain biomarkers, including glucose, triglycerides, tumour necrosis factor a, amyloid β 42, nitric oxide, acetylcholinesterase, malondialdehyde, and β secretase in rats suffering from diabetes mellitus. As a result of this research, we suggest that metformin, either alone or in conjunction with donepezil, might be an excellent drug of choice for neuro-regeneration and risk reduction in Alzheimer’s like disease.

Acknowledgments

The authors express their gratitude to Muhaeman Yusif, Rafel Abdulrazak, and Govand Tawfeeq for their efforts in providing the required facilities for the study to be carried out.

References

  1. 1. Terry RD. Alzheimer’s disease and the aging brain. Journal of geriatric psychiatry and neurology. 2006;19(3):125–8. pmid:16880353
  2. 2. Gasparini L, Netzer WJ, Greengard P, Xu H. Does insulin dysfunction play a role in Alzheimer’s disease? Trends in pharmacological sciences. 2002;23(6):288–93. pmid:12084635
  3. 3. Frölich L, Blum-Degen D, Bernstein H-G, Engelsberger S, Humrich J, Laufer S, et al. Brain insulin and insulin receptors in aging and sporadic Alzheimer’s disease. Journal of neural transmission. 1998;105(4):423–38.
  4. 4. Steen E, Terry BM, J Rivera E, Cannon JL, Neely TR, Tavares R, et al. Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease–is this type 3 diabetes? Journal of Alzheimer’s disease. 2005;7(1):63–80. pmid:15750215
  5. 5. Craft S. Insulin resistance and Alzheimer’s disease pathogenesis: potential mechanisms and implications for treatment. Current Alzheimer Research. 2007;4(2):147–52. pmid:17430239
  6. 6. Zhao W-Q, Chen H, Quon MJ, Alkon DL. Insulin and the insulin receptor in experimental models of learning and memory. European journal of pharmacology. 2004;490(1–3):71–81. pmid:15094074
  7. 7. Leibson CL, Rocca WA, Hanson V, Cha R, Kokmen E, O’brien P, et al. Risk of dementia among persons with diabetes mellitus: a population-based cohort study. American journal of epidemiology. 1997;145(4):301–8. pmid:9054233
  8. 8. Ott A, Stolk R, Hofman A, van Harskamp F, Grobbee D, Breteler M. Association of diabetes mellitus and dementia: the Rotterdam Study. Diabetologia. 1996;39(11):1392–7. pmid:8933010
  9. 9. Xu W, Qiu C, Winblad B, Fratiglioni L. The effect of borderline diabetes on the risk of dementia and Alzheimer’s disease. Diabetes. 2007;56(1):211–6. pmid:17192484
  10. 10. Curb J, Rodriguez B, Abbott R, Petrovitch H, Ross G, Masaki K, et al. Longitudinal association of vascular and Alzheimer’s dementias, diabetes, and glucose tolerance. Neurology. 1999;52(5):971–. pmid:10102414
  11. 11. MacKnight C, Rockwood K, Awalt E, McDowell I. Diabetes mellitus and the risk of dementia, Alzheimer’s disease and vascular cognitive impairment in the Canadian Study of Health and Aging. Dementia and geriatric cognitive disorders. 2002;14(2):77–83. pmid:12145454
  12. 12. Akomolafe A, Beiser A, Meigs JB, Au R, Green RC, Farrer LA, et al. Diabetes mellitus and risk of developing Alzheimer disease: results from the Framingham Study. Archives of neurology. 2006;63(11):1551–5. pmid:17101823
  13. 13. Biessels GJ, Deary IJ, Ryan CM. Cognition and diabetes: a lifespan perspective. The Lancet Neurology. 2008;7(2):184–90. pmid:18207116
  14. 14. Ryan C, Vega A, Drash A. Cognitive deficits in adolescents who developed diabetes early in life. Pediatrics. 1985;75(5):921–7. pmid:3991281
  15. 15. RESKE‐NIELSEN E, LUNDBæK K. Diabetic encephalopathy: diffuse and focal lesions of the brain in long‐term diabetes. Acta Neurologica Scandinavica. 1963;39(S4):273–90. pmid:14057514
  16. 16. DeJong RN. CNS manifestations of diabetes mellitus. Postgraduate Medicine. 1977;61(1):101–7. pmid:834675
  17. 17. Jolivalt C, Lee C, Beiswenger K, Smith J, Orlov M, Torrance M, et al. Defective insulin signaling pathway and increased glycogen synthase kinase‐3 activity in the brain of diabetic mice: parallels with Alzheimer’s disease and correction by insulin. Journal of neuroscience research. 2008;86(15):3265–74. pmid:18627032
  18. 18. Li Z-g, Zhang W, Sima AA. Alzheimer-like changes in rat models of spontaneous diabetes. Diabetes. 2007;56(7):1817–24. pmid:17456849
  19. 19. Donovan J, Brown P. Parenteral injections. Curr Protoc Immunol. 2006;Chapter 1:Unit 1.6. pmid:18432964
  20. 20. Furman BL. Streptozotocin‐induced diabetic models in mice and rats. Current Protocols. 2021;1(4):e78. pmid:33905609
  21. 21. Donovan J, Brown P. Current Protocols in Immunology. Current protocols in immunology / edited by John E Coligan [et al]. 2001;Chapter 1:Unit 1.4.
  22. 22. Donovan J, Brown P. Blood collection. Current Protocols in Immunology. 2006;73(1):1.7. 1–.7. 9. pmid:18432965
  23. 23. Shin AC, Balasubramanian P, Suryadevara P, Zyskowski J, Herdt TH, MohanKumar SMJ, et al. Metformin effectively restores the HPA axis function in diet-induced obese rats. Int J Obes (Lond). 2021;45(2):383–95. pmid:32951009
  24. 24. Mostafa DK, Ismail CA, Ghareeb DA. Differential metformin dose-dependent effects on cognition in rats: role of Akt. Psychopharmacology (Berl). 2016;233(13):2513–24. pmid:27113224
  25. 25. Kim SH, Chung DK, Lee YJ, Song CH, Ku SK. Neuroprotective effects of Danggui-Jakyak-San on rat stroke model through antioxidant/antiapoptotic pathway. J Ethnopharmacol. 2016;188:123–33. pmid:27163672
  26. 26. Luippold G, Bedenik J, Voigt A, Grempler R. Short- and Longterm Glycemic Control of Streptozotocin-Induced Diabetic Rats Using Different Insulin Preparations. PLoS One. 2016;11(6):e0156346. pmid:27253523
  27. 27. Behl T, Velpandian T, Kotwani A. Terminalia catappa Fruit Extract Reverses Streptozotocin-Induced Diabetic Retinopathy in Rats. Endocr Metab Immune Disord Drug Targets. 2021;21(6):1053–65. pmid:33121423
  28. 28. Ortiz MI. Blockade of the antinociception induced by diclofenac, but not of indomethacin, by sulfonylureas and biguanides. Pharmacol Biochem Behav. 2011;99(1):1–6. pmid:21397627
  29. 29. Veilleux-Lemieux D, Castel A, Carrier D, Beaudry F, Vachon P. Pharmacokinetics of ketamine and xylazine in young and old Sprague-Dawley rats. J Am Assoc Lab Anim Sci. 2013;52(5):567–70. pmid:24041212
  30. 30. Gasecka A, Siwik D, Gajewska M, Jaguszewski MJ, Mazurek T, Filipiak KJ, et al. Early Biomarkers of Neurodegenerative and Neurovascular Disorders in Diabetes. J Clin Med. 2020;9(9). pmid:32872672
  31. 31. Jayaraj RL, Azimullah S, Beiram R. Diabetes as a risk factor for Alzheimer’s disease in the Middle East and its shared pathological mediators. Saudi J Biol Sci. 2020;27(2):736–50. pmid:32210695
  32. 32. Kamat PK. Streptozotocin induced Alzheimer’s disease like changes and the underlying neural degeneration and regeneration mechanism. Neural regeneration research. 2015;10(7):1050. pmid:26330820
  33. 33. Santoso HB, Rahmi RA, Kartikasari D, editors. Blood glucose level of white rats (Rattus norvegicus) after giving catfish biscuit (Pangasius hypothalmus). BIO Web of Conferences; 2020: EDP Sciences.
  34. 34. Qinna NA, Badwan AA. Impact of streptozotocin on altering normal glucose homeostasis during insulin testing in diabetic rats compared to normoglycemic rats. Drug design, development and therapy. 2015;9:2515. pmid:26005328
  35. 35. Miedema K. Standardization of HbA1c and optimal range of monitoring. Scandinavian journal of clinical and laboratory investigation. 2005;65(sup240):61–72. pmid:16112961
  36. 36. Kivipelto M, Solomon A. Cholesterol as a risk factor for Alzheimer’s disease–epidemiological evidence. Acta Neurologica Scandinavica. 2006;114:50–7. pmid:16866911
  37. 37. Kao Y-C, Ho P-C, Tu Y-K, Jou I, Tsai K-J. Lipids and Alzheimer’s disease. International journal of molecular sciences. 2020;21(4):1505. pmid:32098382
  38. 38. Coelho VG, Caetano LF, Liberatore Júnior RDR, Cordeiro JA, Souza DRS. Lipid profile and risk factors for cardiovascular diseases in medicine students. Arquivos Brasileiros de Cardiologia. 2005;85(1):57–62. pmid:16041456
  39. 39. Sin HY, Kim JY, Jung KH. Total cholesterol, high density lipoprotein and triglyceride for cardiovascular disease in elderly patients treated with metformin. Archives of pharmacal research. 2011;34(1):99–107. pmid:21468921
  40. 40. Al-neaimy KSA. Comparative effect of Metformin and Glibenclamide on Lipid profile in type 2 Diabetic Patients. Tikrit J Pharm Sci. 2011;7(1).
  41. 41. Hüll M, Fiebich BL, Lieb K, Strauss S, Berger SS, Volk B, et al. Interleukin-6-associated inflammatory processes in Alzheimer’s disease: new therapeutic options. Neurobiol Aging. 1996;17(5):795–800. pmid:8892354
  42. 42. COJOCARU IM, COJOCARU M, MIU G, ŞAPIRA V. Study of Interleukin-6 Production in Alzheimer’s Disease. ROMANIAN JOURNAL OF INTERNAL MEDICINE. 2011;49(1):255. pmid:22026253
  43. 43. Tobinick EL, Gross H. Rapid cognitive improvement in Alzheimer’s disease following perispinal etanercept administration. Journal of neuroinflammation. 2008;5(1):1–10. pmid:18184433
  44. 44. Tang X, Di X, Liu Y. Protective effects of Donepezil against endothelial permeability. European journal of pharmacology. 2017;811:60–5. pmid:28576405
  45. 45. Huang N-L, Chiang S-H, Hsueh C-H, Liang Y-J, Chen Y-J, Lai L-P. Metformin inhibits TNF-α-induced IκB kinase phosphorylation, IκB-α degradation and IL-6 production in endothelial cells through PI3K-dependent AMPK phosphorylation. International journal of cardiology. 2009;134(2):169–75. pmid:18597869
  46. 46. Medeiros R, Baglietto-Vargas D, LaFerla FM. The role of tau in Alzheimer’s disease and related disorders. CNS Neurosci Ther. 2011;17(5):514–24. pmid:20553310
  47. 47. Chen GF, Xu TH, Yan Y, Zhou YR, Jiang Y, Melcher K, et al. Amyloid beta: structure, biology and structure-based therapeutic development. Acta Pharmacol Sin. 2017;38(9):1205–35. pmid:28713158
  48. 48. Kubis-Kubiak A, Dyba A, Piwowar A. The Interplay between Diabetes and Alzheimer’s Disease-In the Hunt for Biomarkers. Int J Mol Sci. 2020;21(8).
  49. 49. Gratuze M, Joly-Amado A, Buee L, Vieau D, Blum D. Tau, Diabetes and Insulin. Adv Exp Med Biol. 2019;1184:259–87. pmid:32096044
  50. 50. Lu Y, Jiang X, Liu S, Li M. Changes in cerebrospinal fluid tau and β-amyloid levels in diabetic and prediabetic patients: a meta-analysis. Frontiers in Aging Neuroscience. 2018;10:271. pmid:30364261
  51. 51. Zhao X, Han Q, Lv Y, Sun L, Gang X, Wang G. Biomarkers for cognitive decline in patients with diabetes mellitus: evidence from clinical studies. Oncotarget. 2018;9(7):7710–26. pmid:29484146
  52. 52. Chatterjee S, Mudher A. Alzheimer’s disease and type 2 diabetes: A critical assessment of the shared pathological traits. Frontiers in neuroscience. 2018;12:383. pmid:29950970
  53. 53. Markowicz-Piasecka M, Sikora J, Szydłowska A, Skupień A, Mikiciuk-Olasik E, Huttunen KM. Metformin–a future therapy for neurodegenerative diseases. Pharmaceutical research. 2017;34(12):2614–27. pmid:28589443
  54. 54. Ou Z, Kong X, Sun X, He X, Zhang L, Gong Z, et al. Metformin treatment prevents amyloid plaque deposition and memory impairment in APP/PS1 mice. Brain, behavior, and immunity. 2018;69:351–63. pmid:29253574
  55. 55. Oliveira WH, Braga CF, Lós DB, Araújo SMR, França MR, Duarte-Silva E, et al. Metformin prevents p-tau and amyloid plaque deposition and memory impairment in diabetic mice. Experimental Brain Research. 2021;239(9):2821–39. pmid:34283253
  56. 56. Khalil M, Teunissen CE, Otto M, Piehl F, Sormani MP, Gattringer T, et al. Neurofilaments as biomarkers in neurological disorders. Nature Reviews Neurology. 2018;14(10):577–89. pmid:30171200
  57. 57. Thompson AG, Mead SH. Fluid biomarkers in the human prion diseases. Molecular and Cellular Neuroscience. 2019;97:81–92. pmid:30529227
  58. 58. Zhou W, Zhang J, Ye F, Xu G, Su H, Su Y, et al. Plasma neurofilament light chain levels in Alzheimer’s disease. Neuroscience letters. 2017;650:60–4. pmid:28428015
  59. 59. Kaeser SA, Lehallier B, Thinggaard M, Häsler LM, Apel A, Bergmann C, et al. A neuronal blood marker is associated with mortality in old age. Nature Aging. 2021;1(2):218–25.
  60. 60. Togo T, Katsuse O, Iseki E. Nitric oxide pathways in Alzheimer’s disease and other neurodegenerative dementias. Neurol Res. 2004;26(5):563–6. pmid:15265275
  61. 61. Obafemi TO, Owolabi OV, Omiyale BO, Afolabi BA, Ojo OA, Onasanya A, et al. Combination of donepezil and gallic acid improves antioxidant status and cholinesterases activity in aluminum chloride-induced neurotoxicity in Wistar rats. Metabolic Brain Disease. 2021;36(8):2511–9. pmid:33978901
  62. 62. Garg G, Singh S, Singh AK, Rizvi SI. Antiaging effect of metformin on brain in naturally aged and accelerated senescence model of rat. Rejuvenation research. 2017;20(3):173–82. pmid:27897089
  63. 63. McHardy SF, Wang H-YL, McCowen SV, Valdez MC. Recent advances in acetylcholinesterase inhibitors and reactivators: an update on the patent literature (2012–2015). Expert opinion on therapeutic patents. 2017;27(4):455–76. pmid:27967267
  64. 64. Talesa VN. Acetylcholinesterase in Alzheimer’s disease. Mech Ageing Dev. 2001;122(16):1961–9. pmid:11589914
  65. 65. Obafemi TO, Olasehinde OR, Olaoye OA, Jaiyesimi KF, Adewumi FD, Adewale OB, et al. Metformin/Donepezil combination modulates brain antioxidant status and hippocampal endoplasmic reticulum stress in type 2 diabetic rats. Journal of Diabetes & Metabolic Disorders. 2020;19(1):499–510. pmid:32550202
  66. 66. Sugimoto H. Donepezil hydrochloride: a treatment drug for Alzheimer’s disease. The Chemical Record. 2001;1(1):63–73. pmid:11893059
  67. 67. Singh Z, Karthigesu IP, Singh P, Rupinder K. Use of malondialdehyde as a biomarker for assessing oxidative stress in different disease pathologies: a review. Iranian Journal of Public Health. 2014;43(Supple 3):7–16.
  68. 68. Collin F, Cheignon C, Hureau C. Oxidative stress as a biomarker for Alzheimer’s disease. Future Medicine; 2018. p. 201–3. pmid:29436240
  69. 69. Ohira M, Yamaguchi T, Saiki A, Ban N, Kawana H, Nagayama D, et al. Metformin reduces circulating malondialdehyde-modified low-density lipoprotein in type 2 diabetes mellitus. Clinical and Investigative Medicine. 2014:E243–E51. pmid:25090264
  70. 70. Ma J, Yu H, Liu J, Chen Y, Wang Q, Xiang L. Metformin attenuates hyperalgesia and allodynia in rats with painful diabetic neuropathy induced by streptozotocin. European journal of pharmacology. 2015;764:599–606. pmid:26054810
  71. 71. Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, et al. BACE1 is the major β-secretase for generation of Aβ peptides by neurons. Nature neuroscience. 2001;4(3):233–4. pmid:11224536
  72. 72. Wang H, Li R, Shen Y. β-Secretase: its biology as a therapeutic target in diseases. Trends in pharmacological sciences. 2013;34(4):215–25. pmid:23452816
  73. 73. Jacobson SA, Sabbagh MN. Donepezil: potential neuroprotective and disease-modifying effects. Expert opinion on drug metabolism & toxicology. 2008;4(10):1363–9. pmid:18798705
  74. 74. Shin R, Iwaki T, Kitamoto T, Tateishi J. Hydrated autoclave pretreatment enhances tau immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissues. Laboratory investigation; a journal of technical methods and pathology. 1991;64(5):693–702. pmid:1903170
  75. 75. Lei P, Ayton S, Finkelstein DI, Adlard PA, Masters CL, Bush AI. Tau protein: relevance to Parkinson’s disease. The international journal of biochemistry & cell biology. 2010;42(11):1775–8. pmid:20678581
  76. 76. Lv H, Tang L, Guo C, Jiang Y, Gao C, Wang Y, et al. Intranasal insulin administration may be highly effective in improving cognitive function in mice with cognitive dysfunction by reversing brain insulin resistance. Cognitive Neurodynamics. 2020;14(3):323–38. pmid:32399074