Modulation of Oxidative Stress and Apoptosis by Alteration of Bioactive Lipids in The Pancreas, and Effect of Zinc Chelation in a Rat Model of Alzheimer's Disease

Modulation of Oxidative Stress and Apoptosis by Alteration of Bioactive Lipids in The Pancreas, and Effect of Zinc Chelation in a Rat Model of Alzheimer's Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modulation of Oxidative Stress and Apoptosis by Alteration of Bioactive Lipids in The Pancreas, and Effect of Zinc Chelation in a Rat Model of Alzheimer's Disease Alev Duygu Acun, Deniz Kantar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3993800/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jun, 2024 Read the published version in Journal of Trace Elements in Medicine and Biology → Version 1 posted You are reading this latest preprint version Abstract Increasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). It is known that peripheral insulin resistance in the early stages of AD precedes and is a precursor to amyloid-β (Aβ) deposition. Although it is known that improving the CNS insulin sensitivity of AD patients is an important therapeutic goal and that the majority of insulin in the brain comes from the periphery, there has been little attention to the changes that occur in the pancreatic tissue of AD patients. Therefore, it is crucial to elucidate the mechanisms affecting insulin resistance in pancreatic tissue in AD. It is known that zinc (Zn+2) chelation is effective in reducing peripheral insulin resistance, cell apoptosis, cell death, and oxidative stress. Aims: This study aimed to determine the bioactive lipids in the pancreas in the Aβ oligomer-induced rat model to determine the changes in amylin (AIPP), oxidative stress, and apoptosis in pancreatic cells and to reveal the therapeutic effect of the Cyclo-Z agent on them. AD and ADC rats were intracerebroventricular (i.c.v.) Aβ1-42 oligomers. Cyclo-Z gavage was applied to ADC and SHC rats for 21 days. First of all, the effects of AIPP, bioactive ceramides, apoptosis and oxidative stress on the pancreatic tissue of AD group rats were evaluated. Then, the effect of Cyclo-Z treatment on these was examined. ELISA kit was used in biochemical analyses. AIPP and ceramide (CER) levels and CER/ sphingosine-1 phosphate (S1P) ratio were increased in the pancreatic tissue of AD rats. It also increased the level of CER kinase (CERK), which is known to increase the concentration of CER 1-phosphate (C1P), which is known to be toxic to cells in the presence of excessive CER concentration. Due to the increase in CER level, it was observed that apoptosis and oxidative stress increased in the pancreatic cells of AD group rats. Cyclo-Z, which has Zn+2 chelating properties, reduced AD model rats' AIPP level and oxidative stress and could prevent pancreatic apoptosis. Similar therapeutic effects were not observed in the pancreatic tissue of Cyclo-Z administered to the SH group. For this reason, it is thought that Cyclo-Z agent may have a therapeutic effect on the peripheral hyperinsulinemia observed in the early stages of AD disease and the resulting low amount of insulin transported to the brain, by protecting pancreatic cells from apoptosis and oxidative stress by regulating their bioactive metabolites. Alzheimer's Disease Cyclo-Z Ceramide Amylin Apoptosis Oxidative Stress Figures Figure 1 Figure 2 Figure 3 1. Introduction Type-2 diabetes (T2D) increases the risk of Alzheimer's disease (AD) type dementia and the conversion from mild cognitive impairment to dementia. However, the mechanisms linking T2D to AD are largely unknown. It is well established that impaired glucose metabolism, insulin resistance, inflammation, oxidative stress; amyloid, amylin (IAPP) and advanced glycosylation end-product accumulation, and mitochondrial dysfunction are biological events that occur in both T2D and AD [ 1 ]. This is why AD has recently been called "type-3 diabetes" [ 2 ] or "diabetes of the brain" [ 3 ]. However, the existence of common molecular mechanisms underlying these events and the involvement of possible common triggers remain largely unknown. It has recently been thought that lipotoxicity may contribute to neurological dysfunction and neurodegeneration, possibly representing the missing link in the pathogenetic mechanisms linking T2D to AD. Lipotoxicity can lead to insulin resistance, oxidative stress, ceramide (CER), and amyloid accumulation, which are common biological events in the pathogenesis of T2D and AD. Interestingly, the pathways of lipotoxicity-induced damage are very similar in all tissues involved in the pathogenesis of both T2D and AD [ 1 ]. Therefore, elucidating the common molecular pathways activated by lipotoxicity in T2D and AD may lead to new therapeutic perspectives. CERs are considered potential lipotoxic molecules with the ability to modulate cellular metabolism [ 4 ]. Pancreatic β-cell lipotoxicity caused by CER accumulation was first demonstrated in 1998 [ 5 ]. CER catabolism is controlled by ceramidases, which reduce CER to free fatty acids and sphingosine that can be further phosphorylated by sphingosine kinase and produce sphingosine-1 phosphate (S1P). CER and S1P are bioactive lipids downstream of the sphingolipid pathway and play important roles in cell signaling. In physical conditions, they play opposite roles in cellular metabolism. Although CER is known to be involved in stress-related cellular responses and apoptosis; S1P stimulates cell survival, proliferation, and tissue regeneration. Therefore, cells need to maintain the balance between CER and S1P [ 6 ]. At the same time; phosphorylation of CER by CER kinase (CERK) produces CER 1-phosphate (C1P), which has the opposite effects of CER and is a key regulator of cell homeostasis. To date, the only enzyme identified to induce C1P biosynthesis is CERK [ 7 ]. However, it has been noticed that there are changes in CER levels in pathological conditions such as AD and T2D [ 6 ]. Changes in SL metabolism enzymes observed at different stages of AD lead to an increase in CER levels (pro-apoptotic) and a decrease in S1P and C1P (anti-apoptotic). In this case, it may determine or promote cell fate in the pathophysiology of AD [ 7 ]. CER deposition-induced lipotoxicity is a mechanism also involved in the pathogenesis of AD. In the early stages of Alzheimer's disease, increased CER levels have been observed in the brains of AD patients [ 8 ], and high serum CER levels may increase the risk of developing AD [ 9 ]. Moreover, insulin resistance, mainly caused by lipotoxicity, is known to be actively involved in the pathophysiology of AD. Peripherally synthesized CERs can cross the blood-brain barrier (BBB), alter insulin signaling, and induce insulin resistance in the brain. [ 1 ]. It has been shown that the increase in CERs in rat pancreatic islets leads to impaired insulin secretion as well as a decrease in the proliferative capacity of β-cells [ 10 ]. Pancreatic beta cells are characterized by low expression of antioxidant enzymes, so they are more vulnerable to oxidative stress. At the same time, many studies show that lipotoxicity induces β-cell oxidative stress, thereby impairing β-cell function and survival [ 1 ]. Therefore, reducing CER levels in the pancreas can be considered an important therapeutic target in the pathogenesis of AD disease. At the same time, the increase in insulin secretion demand due to insulin resistance leads to the overproduction of pancreatic islet amyloid polypeptide (IAPP), also known as amylin, which accumulates and tends to form aggregates [ 11 ]. IAPP, a pancreatic β-cell hormone co-secreted with insulin, is expressed mainly in the pancreas. Although not expressed in the brain, people with AD have increased concentrations of IAPP in the blood, cerebrospinal fluid (CSF), and brain parenchyma. It has been found that intravenously injected IAPP enters the mouse brain and is taken up by neurons and microglia, contributing to AD pathology [ 12 ]. İnsulin resistance and the resulting increase in demand for insulin secretion leads to overproduction of IAPP and tends to accumulate and form aggregates [ 1 ]. Remarkably, IAPP aggregates can induce oxidative stress and apoptosis, thus being cytotoxic to beta-cells [ 11 , 13 , 14 ]. Increased apoptotic rate of β-cells is also associated with increased CER levels in pancreatic islets [ 15 ]. Therefore, we can conclude that AIPP accumulation, which develops due to CER accumulation in the pancreas, causes pancreatic β-cells and further exacerbates insulin resistance. It has been shown that Zn + 2 chelation can reverse Zn + 2 -induced expression level and accumulation and is also effective in reducing cell apoptosis cell death and oxidative stress [ 16 ]. Additionally, it has been shown that the mitogenic effects of S1P can be enhanced by a Zn + 2 -dependent mechanism [ 17 ]. Cyclo (His-Pro) (CHP) has a chelating effect on Zn + 2 and assists Zn + 2 transport via an intestinal CHP transport mechanism independent of the normal elemental Zn + 2 transport system [ 18 ]. CHP is a metabolite of thyrotropin-releasing hormone (TSH) containing the amide chemical formula PGlu-His-Pro [ 19 ]. CHP is ubiquitous in the CNS, blood, and gastrointestinal tract as well as several body fluids [ 20 ], and is a cyclic form of two amino acids, L-histidine and proline [ 21 ]. This cyclic form is also required for active transport through the gut [ 22 ] and the BBB [ 23 – 25 ]. While the therapeutic application of Zn + 2 or CHP alone in the control of blood glucose levels is minimally effective; Cyclo-Z, the form in which CHP and Zn + 2 are applied together, has been very effective [ 18 ]. Cyclo-Z treatment given to amyloid-beta (Aβ) oligomer (AβO)-induced Alzheimer rats has been shown to have a positive effect on peripheral insulin resistance and brain insulin pathway [ 26 ]. The effect of Cyclo-Z treatment on pancreatic lipotoxicity, defined as the common molecular mechanism of T2D and AD disease, is unknown. Therefore, it is crucial to elucidate the effect of Cyclo-Z treatment on bioactive lipids in the pancreas and the resulting insulin resistance and oxidative stress mechanisms. 2. Material and Methods 2.1. Animal preparation 3 months male albino Wistar rats weighing 250–300 g were obtained from Akdeniz University Animal Care Unit. Groups of 4 rats per cage were housed in stainless steel cages and given food and water ad libitum. Animals were kept at a constant temperature of 23 ± 1°C and subjected to a 12-hour light-dark cycle. The animals were divided into four groups with 8 animals in each group. 1) sham surgery (1.25 µl DMSO + 8.75 µl PBS/10µl) intracerebroventricular (i.c.v) injection plus water gavage (SH); (2) sham surgery (1,25 µl DMSO + 8,75 µl PBS/10µl) i.c.v. injection plus Cyclo-Z gavage (10 mg Zn/kg and 0,2 mg SHP/kg) (SHC); (3) Aβ42 oligomer (2,5 nmol/10 µl) i.c.v. injection plus water gavage (AD); (4) Aβ42 oligomer (2,5 nmol/10 µl) i.c.v. injection plus Cyclo-Z gavage (10 mg Zn/kg and 0,2 mg SHP/kg) (ADC). Aβ1–42 oligomers have been prepared according to the literature [ 27 ]. Aβ1–42 (Sigma-Aldrich, USA, product no: A9810) peptides were dissolved in DMSO to a concentration of 2 mmol. It was then diluted 8 times in sterile PBS and vortexed for 30 minutes at room temperature. It is then centrifuged at 15000xg for 1 hour at 4°C. Supernatants (250 µl) were aliquoted (25 µl) and frozen at -20°C. Aβ1–42 oligomers were used within 24 hours after leaving them at 4°C. Sham groups were given i.c.v. 10 µl of solution prepared with 1.25 µl of DMSO and 8.75 µl of PBS. Cyclo was purchased from Santa Cruz with the molecular formula C11H14N4O2 and a molecular weight of 234.26 g/mol (Santa Cruz Biotechnology, Europe). Zinc chloride with purity > 98% was purchased from Molar Kimya (MZK.100310.1000). Previous publications are referenced for Cyclo-Z dosage [ 28 ]. To obtain this dose, 50 mg of Cyclo and 2.5 g of Zn were dissolved in 1 L of water. Rats were anesthetized with a combination of ketamine (80 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Then, a rat model of AD was created by placing it in a standard stereotaxic device [ 29 , 30 ]. A mid-sagittal incision was made in the scalp and the skull was drilled bilaterally using a dental drill on the lateral ventricles (AP: -0.8 mm, ML:±1.4 mm, DV: -4.0 mm). Aβ-42 oligomers were injected into rats in AD and ADC groups, and a solution was prepared with DMSO + PBS in SH and SHC groups at a rate of 0.5 µl/min. The Hamilton syringe was removed 5 minutes after the injection. After the operation, the scalp was sutured closed. In addition, sulfamethoxazole was sprinkled over the suture to prevent infection, and penicillin (40,000 U) was injected intramuscularly once a day for 3 days. Gavage was applied for 21 days after i.c.v. injections. 2.2. Biochemical İnvestigations Pancreatic tissues were obtained for sandwich ELISA (n = 6–8) per group analyses. For biochemical measurements, pancreatic tissues were taken under anesthesia and animals were sacrificed. The excised pancreatic tissues were frozen in liquid nitrogen and stored at -80°C. Tissue samples were weighed and then homogenized on ice in PBS based on homogeneous tissue weight (PRO 200 Homogenizer, PRO Scientific Inc., Connecticut, USA). Homogenized samples were centrifuged at 2000–3000 RPM at 4 o C for 20 minutes. The resulting supernatants were separated and stored at -80 o C. 2.2.1. Measurement of AIPP Levels in Pancreatic Tissue Homogenates According to the manufacturer's instructions, AIPP levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-SRB-T-87752). AIPP was added to wells pre-coated with AIPP monoclonal antibody and then incubated. Biotin-labeled anti-AIPPantibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing Amylin. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of Amylin. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of AIPP is calculated with a standard curve plot. 2.2.2. Measurement of CER Levels in Pancreatic Tissue Homogenates According to the manufacturer's instructions, CER levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-4776). CER was added to wells pre-coated with CER monoclonal antibody and then incubated. Biotin-labeled anti-CER antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing CER. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of CER. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of CER is calculated with a standard curve plot. 2.2.3. Measurement of CERK Levels in Pancreatic Tissue Homogenates CERK levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-2001) by the manufacturer’s instructions. CERK was added to wells pre-coated with CERK monoclonal antibody and then incubated. Biotin-labeled anti-CERK antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to the color formation in the wells containing CERK. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of CERK. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of CERK is calculated with a standard curve plot. 2.2.4. Measurement of S1P Levels in Pancreatic Tissue Homogenates According to the manufacturer's instructions, S1P levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-1805). S1P was added to wells pre-coated with S1P monoclonal antibody and then incubated. Biotin-labeled anti-S1P antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing S1P. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of S1P. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of S1P is calculated with a standard curve plot. 2.2.5. Measurement of Total Oxidant Status (TOS) Levels in Pancreatic Tissue Homogenates The manufacturer's instructions measured TOS levels in the supernatants of pancreas homogenates using a commercially available sandwich ELISA kit (SunRed-201-11-1669). TOS was added to wells pre-coated with TOS monoclonal antibody and then incubated. Biotin-labeled anti-TOS antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing TOS. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of TOS. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of TOS is calculated with a standard curve plot. 2.2.6. Measurement of 4-Hydroxynonenal (4HNE) Levels in Pancreatic Tissue Homogenates According to the manufacturer's instructions, 4HNE levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-0813). 4HNE was added to wells pre-coated with 4HNE monoclonal antibody and then incubated. Biotin-labeled anti-4HNE antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing 4HNE. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of 4HNE. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of 4HNE is calculated with a standard curve plot. 2.2.7. Measurement of Caspase-3 Levels in Pancreatic Tissue Homogenates Caspase-3 levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-0281) according to the manufacturer’s instructions. Caspase-3 was added to wells pre-coated with Caspase-3 monoclonal antibody and then incubated. Biotin-labeled anti-Caspase-3 antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing Caspase-3. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of Caspase-3. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of Caspase-3 is calculated with a standard curve plot. 2.2.8. Measurement of Glutathione (GSH) Levels in Pancreatic Tissue Homogenates The manufacturer's instructions measured GSH levels in the supernatants of pancreas homogenates using a commercially available sandwich ELISA kit (SunRed-201-11-7122). GSH was added to wells pre-coated with GSH monoclonal antibody and then incubated. Biotin-labeled anti-GSH antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing GSH. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of GSH. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of GSH is calculated with a standard curve plot. 2.3. Statistical analysis Statistical ananalyses of the data obtained at the end of the study were performed with the SPSS 23.0 for Windows (SPSS, Chicago, IL, USA) program. Statistical comparisons of data obtained from biochemical analyses between groups were made using one-way ANOVA and post hoc Tukey test. Results are expressed as mean ± standard error. Significance levels were set at P < 0.05. All experimenters were blinded to animal experimental group membership during data collection and analysis. 3. Results 3.1. Pancreas AIPP Levels The mean values of pancreatic AIPP levels are given in Fig. 1 A. There was a statistically significant difference in pancreatic AIPP levels between groups [F(3,20) = 5.798, p < 0.01]. The pancreatic levels were significantly increased in the AD (47.35 ± 5.66 ng/g protein) versus the SH (26.44 ± 2.21 ng/g protein)(p < 0.01) and SHZ (31.94 ± 2.77 ng/g protein)(p < 0.05). However, a slight decrease was seen in the pancreatic levels of the ADC (36.32 ± 3,09 ng/g protein) group compared to the AD group. This decrement did not reach a significant level. However, the pancreatic level of the ADC group was not significantly different from the SH group. 3.2. Pancreas CER Levels The mean values of pancreatic CER levels are given in Fig. 1 B. There was a statistically significant difference in pancreatic CER levels between groups [F(3,20) = 17.704, p < 0.001]. The pancreatic CER levels were significantly increased in the AD (6242.78 ± 413,85 ng/g protein) versus the SH (2548,9 ± 237,69 ng/g protein)(p < 0.001). The pancreatic CER levels were significantly increased in the ADC (4412,8 ± 382,29 ng/g protein) versus the SH (p < 0.05). Although the CER level of the ADC group was significantly higher than that of the SH (p < 0.05) and SHC (3018,5±,497,93 ng/g protein) (p < 0.01) groups, it was significantly lower compared to the AD group (p < 0.05). 3.3. Pancreas CERK Levels The mean values of pancreatic CERK levels are given in Fig. 1 C. There was a statistically significant difference in pancreatic CERK levels between groups [F(3,20) = 14.157, p < 0.001]. The pancreatic CERK levels were significantly increased in the AD (10.90 ± 1.14 ng/mg protein) versus the SH (4.46 ± 0.38 ng/mg protein)(p < 0.001). No significant difference was found between the ADC (6.69 ± 0.69 ng/mg protein) group and the SH group. The pancreatic CERK levels were significantly decreased in the ADC versus the AD (p < 0.01). However, a slight increase was seen in the pancreatic CERK levels of the SHC (5.89 ± 0.46 ng/mg protein) group compared to the SH group. This increment did not reach a significant level. 3.4. Pancreas S1P Levels The mean values of pancreatic S1P levels are given in Fig. 1 D. There was a statistically significant difference in pancreatic S1P levels between groups [F(3,20) = 4.429, p < 0.05]. The pancreatic S1P levels were significantly increased in the AD (45.82 ± 4.82 ng/g protein) versus the SH (28.79 ± 2.52 ng/g protein) (p < 0.05). The pancreatic S1P levels were significantly increased in the ADC (47.03 ± 0.38 ng/g protein) versus the SH (p < 0.05). However, a slight increase was seen in the pancreatic S1P levels of the SHC (38.67 ± 3.25 ng/g protein) group compared to the SH group. This increment did not reach a significant level. No significant difference was found between the ADC group and the AD group. 3.5. Pancreas CER/S1P Ratio The mean values of pancreatic CER/S1P ratio are given in Fig. 1 E. There was a statistically significant difference in pancreatic CER/S1P ratio between groups [F(3,20) = 3.271, p < 0.05]. The CER/S1P ratio of the AD group (146.99 ± 20.70) rats tends to increase, although it is not significant, compared to the SH group (92.61 ± 13.03); but it is significantly higher than that of the SHP group (82.17 ± 16.01, p < 0.05). At the same time, it was found that the CER/S1P ratio of the ADC group(98.03 ± 12.58) tended to decrease compared to the AD group, although it was not significant. Additionally, the CER/S1P ratio of the ADC group was not found to be different from the SH group. 3.6. Pancreas Total Oxidant Status (TOS) Levels The mean values of pancreatic TOS levels are given in Fig. 2 A. There was a statistically significant difference in pancreatic TOS levels between groups [F(3,20) = 3.939, p < 0.05]. The pancreatic TOS levels were slightly increased in the AD (0.53 ± 0.07 nmol/mg protein) versus the SH (0.38 ± 0.03 nmol/mg protein). This increment did not reach a significant level. The pancreatic TOS levels were significantly decreased in the ADC (0.30 ± 0.03 nmol/mg protein) versus the AD (p < 0.05). No significant difference was found between the SHC (0.34 ± 0.04 nmol/mg protein) group and the SH group. 3.7. Pancreas 4-Hydroxynonenal (4HNE) Levels The mean values of pancreatic 4HNE levels are given in Fig. 2 B. There was a statistically significant difference in pancreatic 4HNE levels between groups [F(3,20) = 7.195, p < 0.01]. The pancreatic 4HNE levels were significantly increased in the AD (2.54 ± 0.33 pg/mg protein) versus the SH (1.15 ± 0.14 pg/mg protein) (p < 0.01) and SHC (1.57 ± 0.13 pg/mg protein) (p < 0.05). The pancreatic 4HNE levels were significantly increased in the ADC (2.09 ± 0.21 pg/mg protein) versus the SH (p < 0.05). The pancreatic 4HNE levels were slightly decreased in the ADC versus the AD. This decrement did not reach a significant level. No significant difference was found between the SHC group and the SH group. 3.8. Pancreas Caspase-3 Levels The mean values of pancreatic Caspase-3 levels are given in Fig. 2 C. There was a statistically significant difference in pancreatic Caspase-3 levels between groups [F(3,20) = 14.661, p < 0.001]. The pancreatic Caspase-3 levels were significantly increased in the AD (0.35 ± 0.04 ng/mg protein) versus the SH (0.16 ± 0.02 ng/mg protein) and SHC (0.11 ± 0.02 ng/mg protein). The pancreatic Caspase-3 levels were significantly decreased in the ADC (0.14 ± 0.008 ng/mg protein) versus the AD (p < 0.001 for all comparisons). No significant difference was found between the SHC group and the SH group. No significant difference was found between the ADC group and the SH group. 3.9. Pancreas Glutathione (GSH) Levels The mean values of pancreatic GSH levels are given in Fig. 2 D. There was a statistically significant difference in pancreatic GSH levels between groups [F(3,20) = 6.449, p < 0.01]. The pancreatic GSH levels were significantly increased in the AD (0.22 ± 0.01 mg/g protein) versus the SH (0.16 ± 0.01 mg/g protein). The pancreatic GSH levels were significantly increased in the SHC (0.21 ± 0.008 mg/g protein) versus the SH (p < 0.05 for all comparisons). The pancreatic GSH levels were significantly increased in the AHC (0.23 ± 0.01 mg/g protein) versus the SH (p < 0.01). No significant difference was found between the AHC group and the AH group. 3.10. Correlations between pancreas CER levels and pancreas AIPP levels Significant correlations were found between changes in the pancreas AIPP levels and pancreas CER levels (Fig. 3 A). There was a positive correlation between the pancreas AIPP and CER levels (Pearson r = 0.663, p < 0.001, N = 24). 3.11. Correlations between pancreas CER levels and pancreas CERK levels Significant correlations were found between changes in the pancreas CER levels and pancreas CERK levels (Fig. 3 B). There was a positive correlation between the pancreas CER and CERK levels (Pearson r = 0.754, p < 0.001, N = 24). 3.12. Correlations between pancreas S1P levels and pancreas GSH levels Significant correlations were found between changes in the pancreas S1P levels and pancreas GSH levels (Fig. 3 C). There was a positive correlation between the pancreas S1P and GSH levels (Pearson r = 0.624, p < 0.001, N = 24). 4. Discussion It is important to investigate the pathological changes seen in the early stages of AD disease [ 31 ]. Aβ can bind to the insulin receptor competitively and induce insulin resistance [ 32 ]. Studies have also found that hyperglycemia and hyperinsulinemia, which develop in the initial stages of AD, maybe early biomarkers of AD [ 33 , 34 ]. In patients with AD, insulin resistance in the periphery is positively associated with brain Aβ accumulation in the frontal and temporal regions [ 35 ]. Therefore, it is vital to learn the mechanisms that trigger peripheral insulin resistance in AD. The central infusion of AβOs leads to peripheral insulin resistance [ 36 ]. At the same time, it was found that there was a hyperinsulinemia state against hyperglycemia and peripheral insulin resistance that occurred in the AβO-induced early AD rat model [ 34 ]. Considering that chronic peripheral hyperinsulinemia reduces the amount of insulin transported to the brain by suppressing BBB insulin receptors, it is an expected result that patients with AD will reduce brain insulin concentration [ 37 , 34 ]. Many of the well-documented mechanisms that induce neuronal and synaptic degeneration in the AD brain are triggered and propagated due to the effects of soluble oligomers of the Aβ peptide on neurons and glia [ 38 ]. Therefore, our study aimed to examine the changes in the pancreas of the AβO-induced AD rat model. It has also been reported that 20-week-old mice have increased plasma Aβ levels, accompanied by systemic glucose intolerance, insulin resistance, and hyperinsulinemia, before the appearance of amyloid deposits in the brain [ 39 ]. It is known that Aβ levels also increase in the pancreas of AD patients [ 40 , 41 ]. Studies have shown that Aβ is deposited together with IAPP in the pancreas [ 42 , 40 , 43 ]. Pancreatic islet accumulation of Aβ is observed only in the presence of hIAPP [ 44 ]. Furthermore, it has been shown that Aβ deposited in the pancreas is not produced from pancreatic APP and probably originates from circulating amyloid peptides [ 31 ]. Therefore, increased AIPP in the pancreas of AD group rats may form a fibrillar structure in the pancreas together with plasma Aβ, and high circulating AIPP levels may increase the risk of AD [ 45 ]. As a matter of fact, in our study, it was determined that the amount of AIPP in the pancreas of AD group rats increased significantly compared to the SH group. In the progression of AD, accumulation of AIPP has been shown to induce hyperinsulinemia and insulin resistance in AD mice [ 46 , 47 ]. Therefore, it was concluded that restoring AIPP homeostasis in early AD could reduce AD pathology [ 48 ]. At the same time, AD-induced insulin resistance alters lipid homeostasis. Moreover, the role of lipid metabolism in AD progression is well-known [ 6 ]. Although the pathogenesis of AD has not yet been fully elucidated, the results of studies show that the bioactive SLs CER and S1P play a role in AD, starting from the earliest prodromal stages. It is well known that CER, the central metabolite of SL metabolism, is a pro-apoptotic molecule. It stimulates inflammation, autophagy, and oxidative stress with mitochondrial dysfunction. Its phosphorylated analog S1P stimulates cell survival, proliferation, and migration. S1P also plays a role in neurodevelopment, synaptic transmission, neuroinflammation, and autophagy [ 49 ]. Therefore, even minor changes in CER metabolism in response to various stimuli are decisive for cellular fate [ 50 ]. The imbalance of SL species due to dysregulation of SL metabolism is a common event in AD [ 49 ]. A study showed that brain CER levels of Alzheimer's patients increased compared to neurologically normal controls of the same age. Increased serum CER levels have also been reported in Alzheimer's patients with mild to moderate symptoms [ 50 ]. There is an increase in the serum CER level of AD patients, and in our study, it was observed that the CER level in the pancreatic tissue of AD rats increased significantly compared to the SH group. It also showed that the S1P levels of AD rats increased compared to the SH group. Our findings show that the level of bioactive metabolite S1P increased in the AD group due to increased CER production. However, the increase in the S1P level is very minor compared to the increase in the CER level. For this, the CER/S1P ratio was examined. It was found that the CER/S1P ratio of the AH group was significantly higher than that of the SH group. To evaluate this situation, the CER/S1P ratio was examined. It was found that the CER/S1P ratio of the AH group was significantly higher than that of the SH group. Since CER is the precursor molecule of S1P synthesis, a change in CER level is expected to affect the level of CER-derived metabolites [ 51 ]. It shows that SL metabolism in pancreatic tissue in the early stage of AD is regulated by the combined activation of pro-apoptotic and anti-apoptotic pathways. CER and C1P are antagonistic signals, and C1P can mimic many of the effects of S1P. The only enzyme known to induce the biosynthesis of C1P is CERK. Our study found that the CERK level of AD group rats was significantly increased compared to the SH group. It has been observed that, unlike relatively low concentrations of C1P that stimulate cell growth and inhibit apoptosis, relatively higher concentrations of C1P are toxic and can kill cells. This contradictory observation is explained by the fact that overexpression of CERK in the presence of abnormally high concentrations of CER will cause an excessive increase in intracellular C1P levels, thereby reaching C1P concentrations that are toxic to cells [ 7 ]. As a matter of fact, in our study, it was found that the increase in pancreatic CER levels had a significant correlation with the increase in CERK levels. Therefore, the overexpression of CERK at extremely high CER concentrations may explain the situation we encountered in the S1P results. It is well-established that apoptosis, or programmed cell death, occurs as a result of the accumulation of CER [ 52 ]. CER can initiate a series of deleterious changes that lead to apoptotic cell death by various mechanisms, such as increased mitochondria depolarization and permeability, increased ROS production, cytochrome-c release, Bcl-2 depletion, and caspase-3 activation [ 50 ]. Studies have shown that neuronal death due to CERs is typically linked to the mitochondrial pathway, which is regulated by caspase-9/caspase-3 [ 53 ]. In our study, we found that the level of caspase-3 in the pancreas of rats in the AD group was significantly higher than that in the SH group, indicating a higher incidence of apoptotic cell death in the pancreas of the AD group. The increase in CER levels also stimulates the generation of ROS in a concentration-dependent manner, which can cause oxidative stress and have detrimental consequences for neuronal survival. Oxidative stress occurs when the cellular antioxidant defenses are unable to keep ROS levels below the toxic threshold. In our study, we examined the effect of the significant increase in CER and CER/S1P levels of the AD group compared to the SH group on TOS levels. We observed that TOS levels in the pancreatic tissue of the AD group tended to increase compared to the SH group. Consistent with these findings, we observed a significant increase in the level of 4HNE in the pancreas of the AD group compared to the SH group. The typically toxic end product of ROS-induced lipid peroxidation is 4HNE [ 50 ]. Our study observed that the pancreatic 4HNE level of the AD group increased significantly compared to the SH group. Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and is the most important antioxidant within cells, providing tight control of the cellular redox state. Intracellular GSH depletion is an early sign in the progression of apoptosis. It has been previously reported that depletion of GSH leads to CER production and apoptosis [ 54 ]. GSH levels of the AD group were found to be significantly increased compared to the SH group. This increase in GSH levels indicated a compensatory mechanism, as we observed in the increases in S1P and C1P levels in the early stages of AD disease. It was observed that there was a significant correlation between pancreatic S1P and GSH levels of the AD group. Previous studies have suggested that relatively high concentrations of Zn + 2 are required for the normal function of pancreatic β cells. It also showed that Zn + 2 reduction in pancreatic islets of hIAPP-Tg mice was associated with hyperglycemia [ 19 ]. However, it has been shown that Zn + 2 chelation can reverse the expression level of Zn + 2 -induced IAPP, and also significantly reduces cell death and caspase-3 activity. Therefore, chelating Zn + 2 therapy is effective in reducing Zn + 2 -induced IAPP accumulation and beta-cell apoptosis [ 16 ]. Our study determined that the AIPP level of the ADC group tended to decrease compared to the AD group and was not significantly different from the SH group. Additionally, it was shown that there was a significant decrease in the caspase-3 level of the ADC group compared to the AD group and that Cyclo-Z treatment was effective in reducing pancreatic β-cell apoptosis. However, no difference was found in the AIPP and caspase-3 levels of the SHC group compared to the SH group. The CHP therapeutic agent has also been shown to reduce oxidative stress in the pancreas in an STZ-induced rat model [ 55 ]. In our study, pancreatic TOS levels of the ADC group decreased significantly compared to the AD group, and 4HNE levels tended to decrease. On the other hand, the increase in GSH levels shown by the AHC group compared to the AH group is compatible with the decrease in oxidative stress. Similarly, Cyclo-Z therapeutic agents administered to healthy rats also tended to reduce pancreatic caspase-3 and TOS levels of the SHP group compared to the SH group. At the same time, the GSH level of the SHP group increased significantly compared to the SH group. In a similar study, Zn + 2 supplementation was shown to increase GSH levels and reduce lipid peroxidation due to ischemia-reperfusion injury [ 56 ]. Based on these results, it was concluded that the Cyclo-Z agent reduces oxidative stress in the pancreas due to GSH, TOS, and 4HNE changes, and may also prevent pancreatic cell apoptosis. ROS modulates SL metabolism, including enzymes that generate CER and S1P. ROS activates CER-producing enzymes, inducing CER production, leading to apoptosis, and inhibiting S1P production, which promotes survival [ 57 ]. Therefore, it seems likely that there will be a change in the levels of CER, S1P, and C1P in pancreatic cells due to the reduction of oxidative stress due to Cyclo-Z treatment in the pancreas. Supporting this, in our study, it was found that the level of CER in the pancreas of the ADC group decreased significantly. At the same time, the pancreatic CERK level was significantly decreased in the ADC group compared to the AD group. Low expression of CERK in the presence of low levels of CER results in relatively low levels of C1P, which stimulates its intracellular growth and is known to inhibit apoptosis [ 7 ]. Additionally, cellular production of S1P depends on the activity of the ceramidase enzyme, and there is a Zn + 2 binding site in the active site of this enzyme. Zn + 2 is a necessary element for the full function of this enzyme [ 51 ]. A study showed that the mitogenic effects of S1P could be increased by a Zn + 2 -dependent mechanism [ 17 ]. In our study, it was observed that the Zn-chelating Cyclo-Z therapeutic agent tended to increase the S1P level of the ADC group in the AD group. At the same time, the ADC group had significantly higher S1P levels than the SH group. Also, Cyclo-Z tended to reduce the CER/S1P ratio of the ADC group compared to the AD group. However, the therapeutic effect of the ADC group of Cyclo-Z on SL biometabolites in SL metabolism was not observed in the SHC group. Therefore, it has been demonstrated that the therapeutic effect of Cyclo-Z agent is not only on the brain of AD rats but also can protect pancreatic cells from beta-cell apoptosis by regulating SL bioactive metabolites. The emergence of peripheral insulin resistance almost 15 years before Aβ accumulation in AD disease supports the contribution of AD biomarkers to the pathogenesis and reveals insights into the pathogenesis of AD. It is also known that chronic peripheral hyperinsulinemia causes the downregulation of insulin receptors in the BBB and reduces the amount of insulin transported to the brain [ 58 ]. Therefore, the therapeutic effect of Cyclo-Z on AD pancreatic β-cells is considered important for the early treatment of the disease. 5. Conclusions When pathological similarities and common pathogenic mechanisms between T2D and AD are investigated, the connection between both diseases becomes increasingly evident. For this reason, AD is also called “brain diabetes” or “T3D”. This condition has been described as a metabolic syndrome that can lead to progressive brain insulin resistance-related abnormalities resulting in disruption of central insulin signaling processes, neurotoxin accumulation, neuronal stress, and culminating in the process of neurodegeneration. Peripheral insulin resistance seen in the early stages of AD leads to decreased insulin signaling in the CNS and subsequent changes in brain metabolism. Therefore, treatment methods aimed at improving insulin sensitivity may also benefit patients at risk of AD in the early stages. IAPP forms amyloid plaques that play a role in impairing pancreatic islet function and mediating β-cell apoptosis. AIPP was found to accumulate in the pancreas of AD mice, causing hyperinsulinemia and insulin resistance. One of the most important reasons for trying to correct insulin resistance is that it can change lipid homeostasis in the pancreas. Accordingly, it determines the cellular fate by increasing the apoptosis or programmed cell death and oxidative stress of the pancreatic tissue. Therefore, how chronic peripheral hyperinsulinemia seen in the early stages of AD affects the pancreatic tissue and deriving effective therapeutic approaches to prevent it are considered very important for AD disease. Indeed, there are still many questions to be answered about therapeutic strategies derived from this topic. Declarations Funding This study was not supported by any funding. Conflict of Interest The authors report no conflicts of interest in this work. Ethics Approval Ethical approval for this work was obtained from Akdeniz University Local Committee on Animal Research Ethics (ethics approval date and number: 06.11.2023/2023.11.006). Availability of Data and Materials The data supporting this study's findings are available from the corresponding author, upon reasonable request. 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Cell Metab 5 (4):293–303. doi: 10.1016/j.cmet.2007.03.001 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Jun, 2024 Read the published version in Journal of Trace Elements in Medicine and Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3993800","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275354394,"identity":"8db4959b-a06a-4354-a732-9327f09197e5","order_by":0,"name":"Alev Duygu Acun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDCCAzAGewMzAw8DCDEwSBCnhecAihYDIrRIJDBD1BPSwncjx/g1T41d4nbJx48N3lTckZFvYD54m4fhTz4uLZI3csyseY4lJ+6cnWacOOfMMx7GBrZkax4GA8sGHFoMgFqMedgOJG64nWB8mLftMA/QeWbSQC04XQbR8g+o5ebxz4d5/x3mYWPg/0ZIi/Fj3jaglhs8xsm8DYd5eBh42PBqkTzzrIxxbl+y8YYzOcWGc44d5pFgZjO2nGNgjFML3/HkzR/efLOT3XD8+GaJNzWH7eXbmx/eeFMhhydiOMzQ4poZ7GDcGoAJ5fEHfNKjYBSMglEwChgAtmxTjS0ZUSQAAAAASUVORK5CYII=","orcid":"","institution":"Akdeniz University","correspondingAuthor":true,"prefix":"","firstName":"Alev","middleName":"Duygu","lastName":"Acun","suffix":""},{"id":275354395,"identity":"e1d4abe0-4c88-4b0d-81b5-2a583049587e","order_by":1,"name":"Deniz Kantar","email":"","orcid":"","institution":"Akdeniz University","correspondingAuthor":false,"prefix":"","firstName":"Deniz","middleName":"","lastName":"Kantar","suffix":""}],"badges":[],"createdAt":"2024-02-27 12:02:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3993800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3993800/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.jtemb.2024.127480","type":"published","date":"2024-06-01T11:56:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51970329,"identity":"13b013ee-a613-44e1-aa22-bf9bbac23340","added_by":"auto","created_at":"2024-03-04 18:47:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98916,"visible":true,"origin":"","legend":"\u003cp\u003eA: Pancreas AIPP levels in SH and experimental groups, n=6; B: Pancreas CER levels in SH and experimental groups, n=6; C: Pancreas S1P levels in SH and experimental groups, n=6; D: Pancreas CERK levels in SH and experimental groups, n=6; E: Pancreas CER/S1P ratio in SH and experimental groups, n=6. Results are presented as mean ±SEM. (*: Significant vs SH group, #: Significant vs SHZ group, δ: Significant vs AD group. *: p\u0026lt;0.05; **: p\u0026lt;0.01; ***: p\u0026lt;0.001, #: p\u0026lt;0.05; ##: p\u0026lt;0.01; ###: p\u0026lt;0.001, δ: p\u0026lt;0.05, δδ: p\u0026lt;0.01, δδδ: p\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-3993800/v1/4c248c1f9946effa0cbbb064.png"},{"id":51970327,"identity":"8ce26be1-ac25-4015-8cdb-6bc3bef9bb18","added_by":"auto","created_at":"2024-03-04 18:47:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78911,"visible":true,"origin":"","legend":"\u003cp\u003eA: Pancreas TOS levels in SH and experimental groups, n=6; B: Pancreas 4HNE levels in SH and experimental groups, n=6; C: Pancreas caspase-3 levels in SH and experimental groups, n=6; D: Pancreas GSH levels in SH and experimental groups, n=6. Results are presented as mean ±SEM. (*: Significant vs SH group, #: Significant vs SHZ group, δ: Significant vs AD group. *: p\u0026lt;0.05; **: p\u0026lt;0.01; ***: p\u0026lt;0.001, #: p\u0026lt;0.05; ##: p\u0026lt;0.01; ###: p\u0026lt;0.001, δ: p\u0026lt;0.05, δδ: p\u0026lt;0.01, δδδ: p\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-3993800/v1/4d6699c232645fadfe6047e6.png"},{"id":51970328,"identity":"2133888c-5053-40ec-81b8-44cbfaab8e5c","added_by":"auto","created_at":"2024-03-04 18:47:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54810,"visible":true,"origin":"","legend":"\u003cp\u003eA: The correlations between pancreas CER levels and pancreas AIPP levels (Pearson r= 0.663, p\u0026lt;0.001, N=24); B: The correlations between pancreas CER levels and pancreas CERK levels (Pearson r= 0.754, p\u0026lt;0.001, N=24); C: The correlations between pancreas S1P levels and pancreas GSH levels(Pearson r= 0.624, p\u0026lt;0.001, N=24).\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-3993800/v1/d582ba4d12923e810980b116.png"},{"id":58285024,"identity":"51567d28-115a-4808-86e1-129148fd5614","added_by":"auto","created_at":"2024-06-13 11:56:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":931111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3993800/v1/3a0b5b47-b4ee-4554-8638-4c8ac9d57608.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulation of Oxidative Stress and Apoptosis by Alteration of Bioactive Lipids in The Pancreas, and Effect of Zinc Chelation in a Rat Model of Alzheimer's Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eType-2 diabetes (T2D) increases the risk of Alzheimer's disease (AD) type dementia and the conversion from mild cognitive impairment to dementia. However, the mechanisms linking T2D to AD are largely unknown. It is well established that impaired glucose metabolism, insulin resistance, inflammation, oxidative stress; amyloid, amylin (IAPP) and advanced glycosylation end-product accumulation, and mitochondrial dysfunction are biological events that occur in both T2D and AD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This is why AD has recently been called \"type-3 diabetes\" [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] or \"diabetes of the brain\" [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the existence of common molecular mechanisms underlying these events and the involvement of possible common triggers remain largely unknown. It has recently been thought that lipotoxicity may contribute to neurological dysfunction and neurodegeneration, possibly representing the missing link in the pathogenetic mechanisms linking T2D to AD. Lipotoxicity can lead to insulin resistance, oxidative stress, ceramide (CER), and amyloid accumulation, which are common biological events in the pathogenesis of T2D and AD. Interestingly, the pathways of lipotoxicity-induced damage are very similar in all tissues involved in the pathogenesis of both T2D and AD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, elucidating the common molecular pathways activated by lipotoxicity in T2D and AD may lead to new therapeutic perspectives.\u003c/p\u003e \u003cp\u003eCERs are considered potential lipotoxic molecules with the ability to modulate cellular metabolism [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pancreatic β-cell lipotoxicity caused by CER accumulation was first demonstrated in 1998 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. CER catabolism is controlled by ceramidases, which reduce CER to free fatty acids and sphingosine that can be further phosphorylated by sphingosine kinase and produce sphingosine-1 phosphate (S1P). CER and S1P are bioactive lipids downstream of the sphingolipid pathway and play important roles in cell signaling. In physical conditions, they play opposite roles in cellular metabolism. Although CER is known to be involved in stress-related cellular responses and apoptosis; S1P stimulates cell survival, proliferation, and tissue regeneration. Therefore, cells need to maintain the balance between CER and S1P [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At the same time; phosphorylation of CER by CER kinase (CERK) produces CER 1-phosphate (C1P), which has the opposite effects of CER and is a key regulator of cell homeostasis. To date, the only enzyme identified to induce C1P biosynthesis is CERK [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, it has been noticed that there are changes in CER levels in pathological conditions such as AD and T2D [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Changes in SL metabolism enzymes observed at different stages of AD lead to an increase in CER levels (pro-apoptotic) and a decrease in S1P and C1P (anti-apoptotic). In this case, it may determine or promote cell fate in the pathophysiology of AD [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CER deposition-induced lipotoxicity is a mechanism also involved in the pathogenesis of AD. In the early stages of Alzheimer's disease, increased CER levels have been observed in the brains of AD patients [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and high serum CER levels may increase the risk of developing AD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, insulin resistance, mainly caused by lipotoxicity, is known to be actively involved in the pathophysiology of AD. Peripherally synthesized CERs can cross the blood-brain barrier (BBB), alter insulin signaling, and induce insulin resistance in the brain. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It has been shown that the increase in CERs in rat pancreatic islets leads to impaired insulin secretion as well as a decrease in the proliferative capacity of β-cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Pancreatic beta cells are characterized by low expression of antioxidant enzymes, so they are more vulnerable to oxidative stress. At the same time, many studies show that lipotoxicity induces β-cell oxidative stress, thereby impairing β-cell function and survival [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, reducing CER levels in the pancreas can be considered an important therapeutic target in the pathogenesis of AD disease.\u003c/p\u003e \u003cp\u003eAt the same time, the increase in insulin secretion demand due to insulin resistance leads to the overproduction of pancreatic islet amyloid polypeptide (IAPP), also known as amylin, which accumulates and tends to form aggregates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. IAPP, a pancreatic β-cell hormone co-secreted with insulin, is expressed mainly in the pancreas. Although not expressed in the brain, people with AD have increased concentrations of IAPP in the blood, cerebrospinal fluid (CSF), and brain parenchyma. It has been found that intravenously injected IAPP enters the mouse brain and is taken up by neurons and microglia, contributing to AD pathology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. İnsulin resistance and the resulting increase in demand for insulin secretion leads to overproduction of IAPP and tends to accumulate and form aggregates [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Remarkably, IAPP aggregates can induce oxidative stress and apoptosis, thus being cytotoxic to beta-cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Increased apoptotic rate of β-cells is also associated with increased CER levels in pancreatic islets [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, we can conclude that AIPP accumulation, which develops due to CER accumulation in the pancreas, causes pancreatic β-cells and further exacerbates insulin resistance.\u003c/p\u003e \u003cp\u003eIt has been shown that Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e chelation can reverse Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-induced expression level and accumulation and is also effective in reducing cell apoptosis cell death and oxidative stress [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, it has been shown that the mitogenic effects of S1P can be enhanced by a Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-dependent mechanism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Cyclo (His-Pro) (CHP) has a chelating effect on Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e and assists Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e transport via an intestinal CHP transport mechanism independent of the normal elemental Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e transport system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CHP is a metabolite of thyrotropin-releasing hormone (TSH) containing the amide chemical formula PGlu-His-Pro [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CHP is ubiquitous in the CNS, blood, and gastrointestinal tract as well as several body fluids [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and is a cyclic form of two amino acids, L-histidine and proline [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This cyclic form is also required for active transport through the gut [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and the BBB [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While the therapeutic application of Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e or CHP alone in the control of blood glucose levels is minimally effective; Cyclo-Z, the form in which CHP and Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e are applied together, has been very effective [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Cyclo-Z treatment given to amyloid-beta (Aβ) oligomer (AβO)-induced Alzheimer rats has been shown to have a positive effect on peripheral insulin resistance and brain insulin pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The effect of Cyclo-Z treatment on pancreatic lipotoxicity, defined as the common molecular mechanism of T2D and AD disease, is unknown. Therefore, it is crucial to elucidate the effect of Cyclo-Z treatment on bioactive lipids in the pancreas and the resulting insulin resistance and oxidative stress mechanisms.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal preparation\u003c/h2\u003e \u003cp\u003e 3 months male albino Wistar rats weighing 250\u0026ndash;300 g were obtained from Akdeniz University Animal Care Unit. Groups of 4 rats per cage were housed in stainless steel cages and given food and water ad libitum. Animals were kept at a constant temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and subjected to a 12-hour light-dark cycle. The animals were divided into four groups with 8 animals in each group. 1) sham surgery (1.25 \u0026micro;l DMSO\u0026thinsp;+\u0026thinsp;8.75 \u0026micro;l PBS/10\u0026micro;l) intracerebroventricular (i.c.v) injection plus water gavage (SH); (2) sham surgery (1,25 \u0026micro;l DMSO\u0026thinsp;+\u0026thinsp;8,75 \u0026micro;l PBS/10\u0026micro;l) i.c.v. injection plus Cyclo-Z gavage (10 mg Zn/kg and 0,2 mg SHP/kg) (SHC); (3) Aβ42 oligomer (2,5 nmol/10 \u0026micro;l) i.c.v. injection plus water gavage (AD); (4) Aβ42 oligomer (2,5 nmol/10 \u0026micro;l) i.c.v. injection plus Cyclo-Z gavage (10 mg Zn/kg and 0,2 mg SHP/kg) (ADC).\u003c/p\u003e \u003cp\u003eAβ1\u0026ndash;42 oligomers have been prepared according to the literature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Aβ1\u0026ndash;42 (Sigma-Aldrich, USA, product no: A9810) peptides were dissolved in DMSO to a concentration of 2 mmol. It was then diluted 8 times in sterile PBS and vortexed for 30 minutes at room temperature. It is then centrifuged at 15000xg for 1 hour at 4\u0026deg;C. Supernatants (250 \u0026micro;l) were aliquoted (25 \u0026micro;l) and frozen at -20\u0026deg;C. Aβ1\u0026ndash;42 oligomers were used within 24 hours after leaving them at 4\u0026deg;C. Sham groups were given i.c.v. 10 \u0026micro;l of solution prepared with 1.25 \u0026micro;l of DMSO and 8.75 \u0026micro;l of PBS. Cyclo was purchased from Santa Cruz with the molecular formula C11H14N4O2 and a molecular weight of 234.26 g/mol (Santa Cruz Biotechnology, Europe). Zinc chloride with purity\u0026thinsp;\u0026gt;\u0026thinsp;98% was purchased from Molar Kimya (MZK.100310.1000). Previous publications are referenced for Cyclo-Z dosage [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To obtain this dose, 50 mg of Cyclo and 2.5 g of Zn were dissolved in 1 L of water.\u003c/p\u003e \u003cp\u003eRats were anesthetized with a combination of ketamine (80 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Then, a rat model of AD was created by placing it in a standard stereotaxic device [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A mid-sagittal incision was made in the scalp and the skull was drilled bilaterally using a dental drill on the lateral ventricles (AP: -0.8 mm, ML:\u0026plusmn;1.4 mm, DV: -4.0 mm). Aβ-42 oligomers were injected into rats in AD and ADC groups, and a solution was prepared with DMSO\u0026thinsp;+\u0026thinsp;PBS in SH and SHC groups at a rate of 0.5 \u0026micro;l/min. The Hamilton syringe was removed 5 minutes after the injection. After the operation, the scalp was sutured closed. In addition, sulfamethoxazole was sprinkled over the suture to prevent infection, and penicillin (40,000 U) was injected intramuscularly once a day for 3 days. Gavage was applied for 21 days after i.c.v. injections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Biochemical İnvestigations\u003c/h2\u003e \u003cp\u003ePancreatic tissues were obtained for sandwich ELISA (n\u0026thinsp;=\u0026thinsp;6\u0026ndash;8) per group analyses. For biochemical measurements, pancreatic tissues were taken under anesthesia and animals were sacrificed. The excised pancreatic tissues were frozen in liquid nitrogen and stored at -80\u0026deg;C. Tissue samples were weighed and then homogenized on ice in PBS based on homogeneous tissue weight (PRO 200 Homogenizer, PRO Scientific Inc., Connecticut, USA). Homogenized samples were centrifuged at 2000\u0026ndash;3000 RPM at 4\u003csup\u003eo\u003c/sup\u003eC for 20 minutes. The resulting supernatants were separated and stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Measurement of AIPP Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, AIPP levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-SRB-T-87752). AIPP was added to wells pre-coated with AIPP monoclonal antibody and then incubated. Biotin-labeled anti-AIPPantibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing Amylin. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of Amylin. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of AIPP is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Measurement of CER Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, CER levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-4776). CER was added to wells pre-coated with CER monoclonal antibody and then incubated. Biotin-labeled anti-CER antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing CER. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of CER. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of CER is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Measurement of CERK Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eCERK levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-2001) by the manufacturer\u0026rsquo;s instructions. CERK was added to wells pre-coated with CERK monoclonal antibody and then incubated. Biotin-labeled anti-CERK antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to the color formation in the wells containing CERK. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional\u003c/p\u003e \u003cp\u003eto the amount of CERK. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of CERK is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Measurement of S1P Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, S1P levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-1805). S1P was added to wells pre-coated with S1P monoclonal antibody and then incubated. Biotin-labeled anti-S1P antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing S1P. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of S1P. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of S1P is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Measurement of Total Oxidant Status (TOS) Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eThe manufacturer's instructions measured TOS levels in the supernatants of pancreas homogenates using a commercially available sandwich ELISA kit (SunRed-201-11-1669). TOS was added to wells pre-coated with TOS monoclonal antibody and then incubated. Biotin-labeled anti-TOS antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing TOS. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of TOS. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of TOS is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6. Measurement of 4-Hydroxynonenal (4HNE) Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, 4HNE levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-0813). 4HNE was added to wells pre-coated with 4HNE monoclonal antibody and then incubated. Biotin-labeled anti-4HNE antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing 4HNE. The substrate solution reacts with the HRP enzyme, resulting in color formation in the wells proportional to the amount of 4HNE. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of 4HNE is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7. Measurement of Caspase-3 Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eCaspase-3 levels in the supernatants of pancreas homogenates were measured using a commercially available sandwich ELISA kit (SunRed-201-11-0281) according to the manufacturer\u0026rsquo;s instructions. Caspase-3 was added to wells pre-coated with Caspase-3 monoclonal antibody and then incubated. Biotin-labeled anti-Caspase-3 antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing Caspase-3. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of Caspase-3. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of Caspase-3 is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.8. Measurement of Glutathione (GSH) Levels in Pancreatic Tissue Homogenates\u003c/h2\u003e \u003cp\u003eThe manufacturer's instructions measured GSH levels in the supernatants of pancreas homogenates using a commercially available sandwich ELISA kit (SunRed-201-11-7122). GSH was added to wells pre-coated with GSH monoclonal antibody and then incubated. Biotin-labeled anti-GSH antibodies were added to combine with streptavidin-HRP. After incubation and washing, unbound components are removed and substrate solutions A and B are added. The substrate solution reacts with the HRP enzyme, leading to color formation in the wells containing GSH. The substrate solution reacts with the HRP enzyme resulting in color formation in the wells proportional to the amount of GSH. The reaction is terminated by adding an acidic stop solution. OD is measured spectrophotometrically at 450 nm. The sample amount of GSH is calculated with a standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical ananalyses of the data obtained at the end of the study were performed with the SPSS 23.0 for Windows (SPSS, Chicago, IL, USA) program. Statistical comparisons of data obtained from biochemical analyses between groups were made using one-way ANOVA and post hoc Tukey test. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Significance levels were set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All experimenters were blinded to animal experimental group membership during data collection and analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Pancreas AIPP Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic AIPP levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. There was a statistically significant difference in pancreatic AIPP levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;5.798, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01]. The pancreatic levels were significantly increased in the AD (47.35\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66 ng/g protein) versus the SH (26.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 ng/g protein)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and SHZ (31.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77 ng/g protein)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, a slight decrease was seen in the pancreatic levels of the ADC (36.32\u0026thinsp;\u0026plusmn;\u0026thinsp;3,09 ng/g protein) group compared to the AD group. This decrement did not reach a significant level. However, the pancreatic level of the ADC group was not significantly different from the SH group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Pancreas CER Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic CER levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. There was a statistically significant difference in pancreatic CER levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;17.704, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. The pancreatic CER levels were significantly increased in the AD (6242.78\u0026thinsp;\u0026plusmn;\u0026thinsp;413,85 ng/g protein) versus the SH (2548,9\u0026thinsp;\u0026plusmn;\u0026thinsp;237,69 ng/g protein)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The pancreatic CER levels were significantly increased in the ADC (4412,8\u0026thinsp;\u0026plusmn;\u0026thinsp;382,29 ng/g protein) versus the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the CER level of the ADC group was significantly higher than that of the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and SHC (3018,5\u0026plusmn;,497,93 ng/g protein) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) groups, it was significantly lower compared to the AD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Pancreas CERK Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic CERK levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. There was a statistically significant difference in pancreatic CERK levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;14.157, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. The pancreatic CERK levels were significantly increased in the AD (10.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 ng/mg protein) versus the SH (4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 ng/mg protein)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant difference was found between the ADC (6.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 ng/mg protein) group and the SH group. The pancreatic CERK levels were significantly decreased in the ADC versus the AD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, a slight increase was seen in the pancreatic CERK levels of the SHC (5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 ng/mg protein) group compared to the SH group. This increment did not reach a significant level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Pancreas S1P Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic S1P levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD. There was a statistically significant difference in pancreatic S1P levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;4.429, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. The pancreatic S1P levels were significantly increased in the AD (45.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82 ng/g protein) versus the SH (28.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52 ng/g protein) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The pancreatic S1P levels were significantly increased in the ADC (47.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 ng/g protein) versus the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, a slight increase was seen in the pancreatic S1P levels of the SHC (38.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25 ng/g protein) group compared to the SH group. This increment did not reach a significant level. No significant difference was found between the ADC group and the AD group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Pancreas CER/S1P Ratio\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic CER/S1P ratio are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. There was a statistically significant difference in pancreatic CER/S1P ratio between groups [F(3,20)\u0026thinsp;=\u0026thinsp;3.271, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. The CER/S1P ratio of the AD group (146.99\u0026thinsp;\u0026plusmn;\u0026thinsp;20.70) rats tends to increase, although it is not significant, compared to the SH group (92.61\u0026thinsp;\u0026plusmn;\u0026thinsp;13.03); but it is significantly higher than that of the SHP group (82.17\u0026thinsp;\u0026plusmn;\u0026thinsp;16.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the same time, it was found that the CER/S1P ratio of the ADC group(98.03\u0026thinsp;\u0026plusmn;\u0026thinsp;12.58) tended to decrease compared to the AD group, although it was not significant. Additionally, the CER/S1P ratio of the ADC group was not found to be different from the SH group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Pancreas Total Oxidant Status (TOS) Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic TOS levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. There was a statistically significant difference in pancreatic TOS levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;3.939, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. The pancreatic TOS levels were slightly increased in the AD (0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 nmol/mg protein) versus the SH (0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nmol/mg protein). This increment did not reach a significant level. The pancreatic TOS levels were significantly decreased in the ADC (0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nmol/mg protein) versus the AD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant difference was found between the SHC (0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 nmol/mg protein) group and the SH group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Pancreas 4-Hydroxynonenal (4HNE) Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic 4HNE levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. There was a statistically significant difference in pancreatic 4HNE levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;7.195, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01]. The pancreatic 4HNE levels were significantly increased in the AD (2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 pg/mg protein) versus the SH (1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 pg/mg protein) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and SHC (1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 pg/mg protein) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The pancreatic 4HNE levels were significantly increased in the ADC (2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 pg/mg protein) versus the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The pancreatic 4HNE levels were slightly decreased in the ADC versus the AD. This decrement did not reach a significant level. No significant difference was found between the SHC group and the SH group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Pancreas Caspase-3 Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic Caspase-3 levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. There was a statistically significant difference in pancreatic Caspase-3 levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;14.661, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. The pancreatic Caspase-3 levels were significantly increased in the AD (0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ng/mg protein) versus the SH (0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ng/mg protein) and SHC (0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ng/mg protein). The pancreatic Caspase-3 levels were significantly decreased in the ADC (0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 ng/mg protein) versus the AD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all comparisons). No significant difference was found between the SHC group and the SH group. No significant difference was found between the ADC group and the SH group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Pancreas Glutathione (GSH) Levels\u003c/h2\u003e \u003cp\u003eThe mean values of pancreatic GSH levels are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. There was a statistically significant difference in pancreatic GSH levels between groups [F(3,20)\u0026thinsp;=\u0026thinsp;6.449, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01]. The pancreatic GSH levels were significantly increased in the AD (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g protein) versus the SH (0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g protein). The pancreatic GSH levels were significantly increased in the SHC (0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 mg/g protein) versus the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons). The pancreatic GSH levels were significantly increased in the AHC (0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g protein) versus the SH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No significant difference was found between the AHC group and the AH group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Correlations between pancreas CER levels and pancreas AIPP levels\u003c/h2\u003e \u003cp\u003eSignificant correlations were found between changes in the pancreas AIPP levels and pancreas CER levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was a positive correlation between the pancreas AIPP and CER levels (Pearson r\u0026thinsp;=\u0026thinsp;0.663, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, N\u0026thinsp;=\u0026thinsp;24).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.11. Correlations between pancreas CER levels and pancreas CERK levels\u003c/h2\u003e \u003cp\u003eSignificant correlations were found between changes in the pancreas CER levels and pancreas CERK levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). There was a positive correlation between the pancreas CER and CERK levels (Pearson r\u0026thinsp;=\u0026thinsp;0.754, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, N\u0026thinsp;=\u0026thinsp;24).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.12. Correlations between pancreas S1P levels and pancreas GSH levels\u003c/h2\u003e \u003cp\u003eSignificant correlations were found between changes in the pancreas S1P levels and pancreas GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). There was a positive correlation between the pancreas S1P and GSH levels (Pearson r\u0026thinsp;=\u0026thinsp;0.624, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, N\u0026thinsp;=\u0026thinsp;24).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIt is important to investigate the pathological changes seen in the early stages of AD disease [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Aβ can bind to the insulin receptor competitively and induce insulin resistance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Studies have also found that hyperglycemia and hyperinsulinemia, which develop in the initial stages of AD, maybe early biomarkers of AD [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In patients with AD, insulin resistance in the periphery is positively associated with brain Aβ accumulation in the frontal and temporal regions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, it is vital to learn the mechanisms that trigger peripheral insulin resistance in AD. The central infusion of AβOs leads to peripheral insulin resistance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. At the same time, it was found that there was a hyperinsulinemia state against hyperglycemia and peripheral insulin resistance that occurred in the AβO-induced early AD rat model [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Considering that chronic peripheral hyperinsulinemia reduces the amount of insulin transported to the brain by suppressing BBB insulin receptors, it is an expected result that patients with AD will reduce brain insulin concentration [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Many of the well-documented mechanisms that induce neuronal and synaptic degeneration in the AD brain are triggered and propagated due to the effects of soluble oligomers of the Aβ peptide on neurons and glia [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, our study aimed to examine the changes in the pancreas of the AβO-induced AD rat model. It has also been reported that 20-week-old mice have increased plasma Aβ levels, accompanied by systemic glucose intolerance, insulin resistance, and hyperinsulinemia, before the appearance of amyloid deposits in the brain [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It is known that Aβ levels also increase in the pancreas of AD patients [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Studies have shown that Aβ is deposited together with IAPP in the pancreas [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Pancreatic islet accumulation of Aβ is observed only in the presence of hIAPP [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, it has been shown that Aβ deposited in the pancreas is not produced from pancreatic APP and probably originates from circulating amyloid peptides [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, increased AIPP in the pancreas of AD group rats may form a fibrillar structure in the pancreas together with plasma Aβ, and high circulating AIPP levels may increase the risk of AD [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. As a matter of fact, in our study, it was determined that the amount of AIPP in the pancreas of AD group rats increased significantly compared to the SH group. In the progression of AD, accumulation of AIPP has been shown to induce hyperinsulinemia and insulin resistance in AD mice [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, it was concluded that restoring AIPP homeostasis in early AD could reduce AD pathology [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the same time, AD-induced insulin resistance alters lipid homeostasis. Moreover, the role of lipid metabolism in AD progression is well-known [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although the pathogenesis of AD has not yet been fully elucidated, the results of studies show that the bioactive SLs CER and S1P play a role in AD, starting from the earliest prodromal stages. It is well known that CER, the central metabolite of SL metabolism, is a pro-apoptotic molecule. It stimulates inflammation, autophagy, and oxidative stress with mitochondrial dysfunction. Its phosphorylated analog S1P stimulates cell survival, proliferation, and migration. S1P also plays a role in neurodevelopment, synaptic transmission, neuroinflammation, and autophagy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Therefore, even minor changes in CER metabolism in response to various stimuli are decisive for cellular fate [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The imbalance of SL species due to dysregulation of SL metabolism is a common event in AD [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A study showed that brain CER levels of Alzheimer's patients increased compared to neurologically normal controls of the same age. Increased serum CER levels have also been reported in Alzheimer's patients with mild to moderate symptoms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. There is an increase in the serum CER level of AD patients, and in our study, it was observed that the CER level in the pancreatic tissue of AD rats increased significantly compared to the SH group. It also showed that the S1P levels of AD rats increased compared to the SH group. Our findings show that the level of bioactive metabolite S1P increased in the AD group due to increased CER production. However, the increase in the S1P level is very minor compared to the increase in the CER level. For this, the CER/S1P ratio was examined. It was found that the CER/S1P ratio of the AH group was significantly higher than that of the SH group. To evaluate this situation, the CER/S1P ratio was examined. It was found that the CER/S1P ratio of the AH group was significantly higher than that of the SH group. Since CER is the precursor molecule of S1P synthesis, a change in CER level is expected to affect the level of CER-derived metabolites [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. It shows that SL metabolism in pancreatic tissue in the early stage of AD is regulated by the combined activation of pro-apoptotic and anti-apoptotic pathways. CER and C1P are antagonistic signals, and C1P can mimic many of the effects of S1P. The only enzyme known to induce the biosynthesis of C1P is CERK. Our study found that the CERK level of AD group rats was significantly increased compared to the SH group. It has been observed that, unlike relatively low concentrations of C1P that stimulate cell growth and inhibit apoptosis, relatively higher concentrations of C1P are toxic and can kill cells. This contradictory observation is explained by the fact that overexpression of CERK in the presence of abnormally high concentrations of CER will cause an excessive increase in intracellular C1P levels, thereby reaching C1P concentrations that are toxic to cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As a matter of fact, in our study, it was found that the increase in pancreatic CER levels had a significant correlation with the increase in CERK levels. Therefore, the overexpression of CERK at extremely high CER concentrations may explain the situation we encountered in the S1P results.\u003c/p\u003e \u003cp\u003eIt is well-established that apoptosis, or programmed cell death, occurs as a result of the accumulation of CER [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. CER can initiate a series of deleterious changes that lead to apoptotic cell death by various mechanisms, such as increased mitochondria depolarization and permeability, increased ROS production, cytochrome-c release, Bcl-2 depletion, and caspase-3 activation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Studies have shown that neuronal death due to CERs is typically linked to the mitochondrial pathway, which is regulated by caspase-9/caspase-3 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In our study, we found that the level of caspase-3 in the pancreas of rats in the AD group was significantly higher than that in the SH group, indicating a higher incidence of apoptotic cell death in the pancreas of the AD group. The increase in CER levels also stimulates the generation of ROS in a concentration-dependent manner, which can cause oxidative stress and have detrimental consequences for neuronal survival. Oxidative stress occurs when the cellular antioxidant defenses are unable to keep ROS levels below the toxic threshold. In our study, we examined the effect of the significant increase in CER and CER/S1P levels of the AD group compared to the SH group on TOS levels. We observed that TOS levels in the pancreatic tissue of the AD group tended to increase compared to the SH group. Consistent with these findings, we observed a significant increase in the level of 4HNE in the pancreas of the AD group compared to the SH group. The typically toxic end product of ROS-induced lipid peroxidation is 4HNE [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our study observed that the pancreatic 4HNE level of the AD group increased significantly compared to the SH group. Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and is the most important antioxidant within cells, providing tight control of the cellular redox state. Intracellular GSH depletion is an early sign in the progression of apoptosis. It has been previously reported that depletion of GSH leads to CER production and apoptosis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. GSH levels of the AD group were found to be significantly increased compared to the SH group. This increase in GSH levels indicated a compensatory mechanism, as we observed in the increases in S1P and C1P levels in the early stages of AD disease. It was observed that there was a significant correlation between pancreatic S1P and GSH levels of the AD group.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that relatively high concentrations of Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e are required for the normal function of pancreatic β cells. It also showed that Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e reduction in pancreatic islets of hIAPP-Tg mice was associated with hyperglycemia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, it has been shown that Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e chelation can reverse the expression level of Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-induced IAPP, and also significantly reduces cell death and caspase-3 activity. Therefore, chelating Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e therapy is effective in reducing Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-induced IAPP accumulation and beta-cell apoptosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our study determined that the AIPP level of the ADC group tended to decrease compared to the AD group and was not significantly different from the SH group. Additionally, it was shown that there was a significant decrease in the caspase-3 level of the ADC group compared to the AD group and that Cyclo-Z treatment was effective in reducing pancreatic β-cell apoptosis. However, no difference was found in the AIPP and caspase-3 levels of the SHC group compared to the SH group. The CHP therapeutic agent has also been shown to reduce oxidative stress in the pancreas in an STZ-induced rat model [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In our study, pancreatic TOS levels of the ADC group decreased significantly compared to the AD group, and 4HNE levels tended to decrease. On the other hand, the increase in GSH levels shown by the AHC group compared to the AH group is compatible with the decrease in oxidative stress. Similarly, Cyclo-Z therapeutic agents administered to healthy rats also tended to reduce pancreatic caspase-3 and TOS levels of the SHP group compared to the SH group. At the same time, the GSH level of the SHP group increased significantly compared to the SH group. In a similar study, Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e supplementation was shown to increase GSH levels and reduce lipid peroxidation due to ischemia-reperfusion injury [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Based on these results, it was concluded that the Cyclo-Z agent reduces oxidative stress in the pancreas due to GSH, TOS, and 4HNE changes, and may also prevent pancreatic cell apoptosis.\u003c/p\u003e \u003cp\u003eROS modulates SL metabolism, including enzymes that generate CER and S1P. ROS activates CER-producing enzymes, inducing CER production, leading to apoptosis, and inhibiting S1P production, which promotes survival [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, it seems likely that there will be a change in the levels of CER, S1P, and C1P in pancreatic cells due to the reduction of oxidative stress due to Cyclo-Z treatment in the pancreas. Supporting this, in our study, it was found that the level of CER in the pancreas of the ADC group decreased significantly. At the same time, the pancreatic CERK level was significantly decreased in the ADC group compared to the AD group. Low expression of CERK in the presence of low levels of CER results in relatively low levels of C1P, which stimulates its intracellular growth and is known to inhibit apoptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, cellular production of S1P depends on the activity of the ceramidase enzyme, and there is a Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e binding site in the active site of this enzyme. Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e is a necessary element for the full function of this enzyme [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. A study showed that the mitogenic effects of S1P could be increased by a Zn\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-dependent mechanism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our study, it was observed that the Zn-chelating Cyclo-Z therapeutic agent tended to increase the S1P level of the ADC group in the AD group. At the same time, the ADC group had significantly higher S1P levels than the SH group. Also, Cyclo-Z tended to reduce the CER/S1P ratio of the ADC group compared to the AD group. However, the therapeutic effect of the ADC group of Cyclo-Z on SL biometabolites in SL metabolism was not observed in the SHC group. Therefore, it has been demonstrated that the therapeutic effect of Cyclo-Z agent is not only on the brain of AD rats but also can protect pancreatic cells from beta-cell apoptosis by regulating SL bioactive metabolites. The emergence of peripheral insulin resistance almost 15 years before Aβ accumulation in AD disease supports the contribution of AD biomarkers to the pathogenesis and reveals insights into the pathogenesis of AD. It is also known that chronic peripheral hyperinsulinemia causes the downregulation of insulin receptors in the BBB and reduces the amount of insulin transported to the brain [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, the therapeutic effect of Cyclo-Z on AD pancreatic β-cells is considered important for the early treatment of the disease.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWhen pathological similarities and common pathogenic mechanisms between T2D and AD are investigated, the connection between both diseases becomes increasingly evident. For this reason, AD is also called \u0026ldquo;brain diabetes\u0026rdquo; or \u0026ldquo;T3D\u0026rdquo;. This condition has been described as a metabolic syndrome that can lead to progressive brain insulin resistance-related abnormalities resulting in disruption of central insulin signaling processes, neurotoxin accumulation, neuronal stress, and culminating in the process of neurodegeneration. Peripheral insulin resistance seen in the early stages of AD leads to decreased insulin signaling in the CNS and subsequent changes in brain metabolism. Therefore, treatment methods aimed at improving insulin sensitivity may also benefit patients at risk of AD in the early stages. IAPP forms amyloid plaques that play a role in impairing pancreatic islet function and mediating β-cell apoptosis. AIPP was found to accumulate in the pancreas of AD mice, causing hyperinsulinemia and insulin resistance. One of the most important reasons for trying to correct insulin resistance is that it can change lipid homeostasis in the pancreas. Accordingly, it determines the cellular fate by increasing the apoptosis or programmed cell death and oxidative stress of the pancreatic tissue. Therefore, how chronic peripheral hyperinsulinemia seen in the early stages of AD affects the pancreatic tissue and deriving effective therapeutic approaches to prevent it are considered very important for AD disease. Indeed, there are still many questions to be answered about therapeutic strategies derived from this topic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was not supported by any funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this work was obtained from Akdeniz University Local Committee on Animal Research Ethics (ethics approval date and number: 06.11.2023/2023.11.006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.D.A. and D.K. generated the rat models. A.D.A. analyzed ELISA\u0026apos;s data. A.D. and D.K. wrote the main manuscript text and A.D.A.prepared figures 1-3. \u0026nbsp; A.D.A. and D.K. were involved in data interpretation, critically reviewed and provided their final approval of the manuscript, and agreed to be accountable for the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMarrano N, Biondi G, Borrelli A, Rella M, Zambetta T, Di Gioia L, Caporusso M, Logroscino G, Perrini S, Giorgino F, Natalicchio A (2023) Type 2 Diabetes and Alzheimer's Disease: The Emerging Role of Cellular Lipotoxicity. 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Int J Mol Sci 23 (7). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms23074010\u003c/span\u003e\u003cspan address=\"10.3390/ijms23074010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSano H, Eguez L, Teruel MN, Fukuda M, Chuang TD, Chavez JA, Lienhard GE, McGraw TE (2007) Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane. Cell Metab 5 (4):293\u0026ndash;303. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2007.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2007.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's Disease, Cyclo-Z, Ceramide, Amylin, Apoptosis, Oxidative Stress","lastPublishedDoi":"10.21203/rs.3.rs-3993800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3993800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). It is known that peripheral insulin resistance in the early stages of AD precedes and is a precursor to amyloid-β (Aβ) deposition. Although it is known that improving the CNS insulin sensitivity of AD patients is an important therapeutic goal and that the majority of insulin in the brain comes from the periphery, there has been little attention to the changes that occur in the pancreatic tissue of AD patients. Therefore, it is crucial to elucidate the mechanisms affecting insulin resistance in pancreatic tissue in AD. It is known that zinc (Zn+2) chelation is effective in reducing peripheral insulin resistance, cell apoptosis, cell death, and oxidative stress. Aims: This study aimed to determine the bioactive lipids in the pancreas in the Aβ oligomer-induced rat model to determine the changes in amylin (AIPP), oxidative stress, and apoptosis in pancreatic cells and to reveal the therapeutic effect of the Cyclo-Z agent on them. AD and ADC rats were intracerebroventricular (i.c.v.) Aβ1-42 oligomers. Cyclo-Z gavage was applied to ADC and SHC rats for 21 days. First of all, the effects of AIPP, bioactive ceramides, apoptosis and oxidative stress on the pancreatic tissue of AD group rats were evaluated. Then, the effect of Cyclo-Z treatment on these was examined. ELISA kit was used in biochemical analyses. AIPP and ceramide (CER) levels and CER/ sphingosine-1 phosphate (S1P) ratio were increased in the pancreatic tissue of AD rats. It also increased the level of CER kinase (CERK), which is known to increase the concentration of CER 1-phosphate (C1P), which is known to be toxic to cells in the presence of excessive CER concentration. Due to the increase in CER level, it was observed that apoptosis and oxidative stress increased in the pancreatic cells of AD group rats. Cyclo-Z, which has Zn+2 chelating properties, reduced AD model rats' AIPP level and oxidative stress and could prevent pancreatic apoptosis. Similar therapeutic effects were not observed in the pancreatic tissue of Cyclo-Z administered to the SH group. For this reason, it is thought that Cyclo-Z agent may have a therapeutic effect on the peripheral hyperinsulinemia observed in the early stages of AD disease and the resulting low amount of insulin transported to the brain, by protecting pancreatic cells from apoptosis and oxidative stress by regulating their bioactive metabolites.\u003c/p\u003e","manuscriptTitle":"Modulation of Oxidative Stress and Apoptosis by Alteration of Bioactive Lipids in The Pancreas, and Effect of Zinc Chelation in a Rat Model of Alzheimer's Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-04 18:47:20","doi":"10.21203/rs.3.rs-3993800/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e228cf43-6690-4e8e-89f7-a4dc6d760672","owner":[],"postedDate":"March 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T11:56:53+00:00","versionOfRecord":{"articleIdentity":"rs-3993800","link":"https://doi.org/10.1016/j.jtemb.2024.127480","journal":{"identity":"journal-of-trace-elements-in-medicine-and-biology","isVorOnly":true,"title":"Journal of Trace Elements in Medicine and Biology"},"publishedOn":"2024-06-01 11:56:53","publishedOnDateReadable":"June 1st, 2024"},"versionCreatedAt":"2024-03-04 18:47:20","video":"","vorDoi":"10.1016/j.jtemb.2024.127480","vorDoiUrl":"https://doi.org/10.1016/j.jtemb.2024.127480","workflowStages":[]},"version":"v1","identity":"rs-3993800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3993800","identity":"rs-3993800","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}