|Year : 2017 | Volume
| Issue : 3 | Page : 122-128
Role of autophagy and oxidative stress in experimental diabetes in rats
Asmaa H Okasha, Hanaa H Gaballah, Soha S Zakaria, Salwa M Elmeligy
Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||09-Jan-2017|
|Date of Acceptance||11-Apr-2017|
|Date of Web Publication||29-Nov-2017|
Asmaa H Okasha
Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Al-Geish Street, Tanta, 31527
Background Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia owing to defects in insulin secretion, insulin action, or both. The exact pathogenesis of β-cell failure in type 2 DM is still unclear. However, autophagy and oxidative stress have emerged presently as major contributing factors in the development of type 2 diabetes and β-cell failure.
Aim The aim is to assess the levels of beclin-1 (autophagy regulator) and some oxidative stress markers in animal models of high-fat diet-streptozotocin-induced diabetes and to study the effect of 4-phenylbutyrate (4-PBA) on them.
Materials and methods The study was conducted on 60 male albino rats that were divided randomly into three equal groups: group I (control group), group II (diabetic group), and group III (diabetic rats treated with 4-PBA). Pancreatic tissue levels of beclin-1 were measured by enzyme-linked immuosorbent assay, whereas pancreatic tissue levels of reduced glutathione (GSH) and plasma levels of advanced oxidation protein products (AOPPs) and glucose were measured spectrophotometrically.
Results The levels of beclin-1, AOPPs, and glucose were significantly increased in diabetic group compared with control group, whereas the levels of GSH were significantly decreased. Moreover, 4-PBA significantly decreased the levels of beclin-1, AOPPs, and glucose, whereas it increased GSH levels.
Conclusion Autophagy and oxidative stress are involved in the pathogenesis of type 2 DM. Moreover, 4-PBA (a chemical chaperone) attenuated autophagy and exhibited antioxidative stress effects in rats with high-fat diet-streptozotocin-induced diabetes.
Keywords: advanced oxidation protein products, autophagy, beclin-1, diabetes mellitus, reduced glutathione, oxidative stress
|How to cite this article:|
Okasha AH, Gaballah HH, Zakaria SS, Elmeligy SM. Role of autophagy and oxidative stress in experimental diabetes in rats. Tanta Med J 2017;45:122-8
|How to cite this URL:|
Okasha AH, Gaballah HH, Zakaria SS, Elmeligy SM. Role of autophagy and oxidative stress in experimental diabetes in rats. Tanta Med J [serial online] 2017 [cited 2018 Nov 15];45:122-8. Available from: http://www.tdj.eg.net/text.asp?2017/45/3/122/219438
| Introduction|| |
Type 2 diabetes mellitus (DM) has emerged as a global health problem as it affects the life quality and life expectancy of millions of affected individuals. In the past century, the incidence of obesity and type 2 DM has increased dramatically in developed as well as developing countries . By 2025, Egypt is expected to be among the top ten countries that would have the highest prevalence rates of diabetes in the world, notably type 2 DM . Diabetes is characterized by peripheral insulin resistance and reduced insulin secretion. The onset of type 2 DM is marked by failure of insulin target tissues such as muscle and adipose tissue to respond to insulin that eventually leads to pancreatic β-cell dysfunction and hyperglycemia . Autophagy and oxidative stress are profoundly implicated in β-cells dysfunction and in the pathophysiology of type 2 DM . Autophagy encompasses several intracellular lysosomal degradation pathways where its primary function is to allow cells to survive under stressful conditions . Autophagy is a double-edge sword that can either promote cell survival, suppressing apoptosis, or cell death, either in collaboration with apoptosis or as a back-up mechanism . The conditions under which autophagy provides protection or induces cell death depend on the intensity of the insult, the cell status, and the duration of autophagy . Defective β-cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy; however, whether this dysregulation represents autophagy augmentation or inhibition is unclear . An attempt was therefore made to determine the role of autophagy in type 2 diabetes through estimation of beclin-1 (an autophagy regulator) levels in pancreatic tissues of type 2 diabetic rats and to establish the relationship between autophagy and oxidative stress in this model. Beclin-1, a mammalian ortholog of yeast Atg6/Vps30, is the first identified mammalian autophagy effector. It is essential for the initiation of autophagy by forming the beclin-1-interacting complex . It is a 60 kDa coiled-coil protein that was discovered as a direct interactor of the anti-apoptotic B-cell lymphoma-2 protein and was therefore given the name B-cell lymphoma-2-interacting myosin-like coiled-coil protein (beclin-1) . Beclin-1 is involved in many biological processes, including development, differentiation, stress adaptation, inflammation, tumorigenesis, aging, and cell death . Oxidative stress occurs when the production of oxidants surpasses the antioxidant capacity in living cells . Oxidative damage of proteins leads to loss of their specific function and is considered to be among the molecular mechanisms leading to the progression and development of diabetes and its complications . Advanced oxidation protein products (AOPPs) are proteins damaged by oxidative stress, mostly albumin and its aggregates. AOPPs are primarily generated by chlorinated oxidants, including hypochloric acid and chloramines, which result from myeloperoxidase activity . Reduced glutathione (GSH) is known to protect the cellular system against the toxic effects of lipid peroxidation . It also plays a core role in maintaining endoplasmic reticulum (ER) oxidoreductases in a reduced state to catalyze reduction or isomerization reactions . It has been widely reported that feeding animals with high-fat diet (HFD) renders them insulin resistant, and the slight insult by low dose of STZ (streptozotocin) compromise the β-cell function . Therefore, in the present study, HFD along with low-dose STZ treatment was used to induce type 2 diabetes in male albino rats. Using this model, this study aimed to investigate the potential ameliorative effect of 4-phenylbutyrate (4-PBA) on the levels of beclin-1 as well as some oxidative stress markers in pancreatic tissues or plasma of type 2 diabetic rats. The chemical chaperone 4-PBA is a low molecular weight fatty acid that has been used for the treatment of urea cycle disorders, sickle cell disease, and Mediterranean anemia for many years . Recent studies have shown that 4-PBA regulates ER stress, attenuates cell damage, and mediates cytoprotection . Moreover, it prevents the occurrence of cerebral and hepatic ischemia by inhibiting ER stress-mediated apoptosis. It also affects apoptosis in certain malignant cells .
| Materials and methods|| |
Most chemicals including STZ and 4-PBA were purchased from Sigma–Aldrich Chemical Co., St. Louis, Missouri, USA.
This study was carried out on 60 male albino rats weighing 100–150 g. During the whole period of the study (6 weeks), animals were housed in wire mesh cages and were allowed free access to water and to either a standard rat chew or high-caloric diet rich in fat. They were kept under constant environmental conditions (temperature 23±2°C and 12 h dark-light cycle).
After being acclimatized for 1 week, the studied animals were divided randomly into three groups equally as follows:
- Group I (control group): this group included 20 rats given intraperitoneal injection of physiological saline in a dose of 1 ml/kg body weight.
- Group II (diabetic group): this group included 20 diabetic rats. Diabetes was experimentally induced by a combination of feeding HFD for 2 weeks followed by intraperitoneal injection of STZ in a single dose of 35 mg/kg body weight . Induction of diabetes was assessed by measuring of fasting plasma glucose level.
- Group III (diabetic rats treated with 4-PBA): this group included 20 diabetic rats treated with 4-PBA by gavage in a dose of 500 mg/kg body weight daily for 20 days .
Induction of experimental diabetes
Experimental diabetes was induced in both second and third groups by a combination of feeding house-prepared HFD (58% of total kcal as fat, 25% of total kcal as carbohydrate, and 17% of total kcal as protein ad libitum) for 2 weeks followed by administration of STZ intraperitoneally in a single dose of 35 mg/kg body weight . Every 5 days, ingredients for diets were mixed, formed into dough with water, rolled into pellets, wrapped with plastic wrap, and stored at −20°C until use to minimize oxidation. A single dose of STZ (35 mg/kg body weight) is dissolved in cold citrate buffer 0.1 M (pH=4.5) and injected intraperitoneally. STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. Within 1 week, rats with fasting blood glucose levels of 250 mg/dl or above were considered diabetic rats . The rats were allowed to continue to feed on their respective diets until the end of the study (6 weeks).
Treatment with 4-phenylbutyric acid
After induction of diabetes in group III, 4-PBA was given in a dose of 500 mg/kg body weight daily by gavage for 20 days. The 4-PBA solution was freshly prepared by titrating equimolar amounts of 4-PBA (molecular weight=164.2) and sodium hydroxide (molecular weight=40) to pH 7.4 and dissolved in an appropriate amount of physiologic saline.
Blood and tissue sampling
At the end of the experimental period, all rats were fasted overnight, and then killed by cervical decapitation. Blood samples were collected in sterile EDTA-containing tubes. After immediate centrifugation at 5000 rpm for 20 min, plasma was separated. Plasma-containing tubes were used immediately for measurement of glucose levels and then were frozen immediately at −20°C until used. Then, the abdomen and thorax were opened; pancreatic tissues were dissected carefully, washed three times with ice cold saline to remove extraneous materials, weighed, and stored at −70°C till use. Care of the animals as well as the experimental procedures was performed in Medical Biochemistry Department, Faculty of Medicine, Tanta University, Egypt, in accordance with guidelines of the ethical committee of Faculty of Medicine, Tanta University, Egypt (approval code 2806/10/14).
- Measurement of pancreatic tissue levels of beclin-1 was done using a commercial enzyme-linked immuosorbent assay kit supplied by Glory Science Co. Ltd. (catalog no. 32392; Del Rio, TX, USA), according to the manufacturer’s instructions. The levels were expressed as pg/mg tissue protein .
- Measurement of pancreatic tissue levels of GSH spectrophotometrically was done using a commercially available kit obtained from Bio Diagnostic (catalog no. GR 2511; Cairo, Egypt). The method is based on the reduction of 5,5′dithiobis (2-nitrobenzoic acid) with GSH to produce a yellow compound, whose absorbance is measured at 405 nm. Results were expressed as mg/g pancreatic tissue .
- Measurement of plasma AOPPs levels spectrophotometrically was done using semiautomated method on a microplate reader, and the readings were calibrated with chloramines-T solutions (Sigma, St. Louis, Missouri, USA) in the presence of potassium iodide at 340 nm. The results were expressed as micromoles per liter of chloramine-T equivalents .
- Measurement of fasting plasma glucose levels by enzymatic colorimetric glucose oxidase method according to Trinder, 1969, was done using a commercial kit obtained from Elitech Diagnostics Company (Washington, USA). The results were expressed as mg/dl .
- Protein content was measured according to the method of Bradford. This method involves the binding of the dye Coomassie Brilliant Blue G-250 to proteins. A standard curve was plotted, and the protein content of the unknown samples was read from the constructed curve at wave length of 595 nm .
Statistical presentation and analysis of the results of the present study was conducted, and the data were presented as mean±SD using statistical package for the social sciences, version 20.0 for Windows (SPSS; SPSS Inc., Chicago, Illinois, USA). One-way analysis of variance and Tukey tests were calculated for multiple comparisons to evaluate the statistical significance between the experimental groups. The correlation study was calculated using Pearson’s correlation. P value less than 0.05 was considered significant.
| Results|| |
[Table 1] summarized the comparative statistics of studied parameters between all groups. Using the multiple comparison test (Tukey’s test), there were statistically significant increases in pancreatic tissue beclin-1 levels, plasma AOPPs levels as well as plasma glucose levels in diabetic group when compared with control group, with significant decreases in these levels in 4-PBA-treated group compared with diabetic group, reflecting its proactive role. However, there was a significant decrease in pancreatic tissue GSH level in diabetic group when compared with control group with significant increase in its level in 4-PBA-treated group compared with diabetic group. However, there was no significant difference of all parameters between control group compared with diabetic group.
|Table 1 Comparative statistics of the studied parameters between all groups|
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[Table 2] showed correlation between all studied parameters in all groups. The pancreatic tissue levels of beclin-1 showed a significant positive correlation with plasma levels of AOPPs and glucose (r=0.869, 0.830, respectively, P<0.001). Also, a significant positive correlation between plasma levels of AOPPs and glucose was found (r=0.622, P<0.001). However, the pancreatic tissue levels of GSH showed a significant negative correlation with pancreatic tissue levels of beclin-1 and plasma levels of AOPPs and glucose (r=−0.835, −0.682, −0.941, respectively, P<0.001).
|Table 2 Pearson’s correlation between all studied parameters in all groups|
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| Discussion|| |
Type 2 DM is a worldwide health issue with potential for significant negative health outcomes . It remains a leading cause of cardiovascular disorders, blindness, end-stage renal failure, amputations, and hospitalizations. It is also associated with increased risk of cancer, serious psychiatric illness, cognitive decline, chronic liver disease, accelerated arthritis, and other disabling or deadly conditions . New insights about the role of and autophagy in the pathogenesis of type 2 diabetes and β-cell failure are evoked nowadays. This could result from the metabolic milieu of diabetes, i.e. elevated blood glucose and lipids and inflammation that might cause oxidative stress, thereby impairing protein folding. Proinsulin misfolding stimulates autophagy while the diabetic milieu may eventually impair rather than stimulate autophagy. Nevertheless, once stimulated, autophagy may alleviate stress, prevent β-cell apoptosis, and improve diabetes . The present study displayed that beclin-1 levels were significantly elevated in pancreatic tissues of the diabetic group compared with those of the control group, indicating initiation of autophagosomes formation and triggering the cellular autophagic process in HFD-STZ-induced type 2 diabetes. This finding concurs with the study carried out by Sun et al.  who demonstrated that beclin-1 gene expression levels were significantly upregulated in pancreatic islet cells of HFD-fed rats compared with the normal diet-fed control rats. This finding could be further bolstered by the study done by Chu et al.  who concluded that HFD increases autophagic flux in pancreatic beta cells in vivo and ex vivo in mice. There may be biological plausibility for the finding herein reported that autophagy is enhanced in type 2 diabetic rats, as its activation could be mediated by the mammalian target of rapamycin complex (mTOR) pathway. mTOR regulates cell function mainly through regulation of protein synthesis . Along this line, diabetic milieu contributes to production of excess ROS which are essential for eliciting autophagy. ROS induce the activation of poly (ADPribose) polymerase (PARP-1) which plays an important role in repairing damaged DNA through an energetically expensive process, causing rapid depletion of cellular NAD+ and failure in ATP production . Given that the major regulator of mTOR is AMPK, it is tempting to speculate that ROS-induced PARP-1 activation promotes autophagy through AMPK activation, probably by suppression of mTOR pathway . In addition, hyperglycemia associated with lack of insulin was found to inhibit mTOR pathway, triggering autophagy in STZ-administrated rats . Alternatively, the removal of damaged proteins through autophagy is particularly important following ER stress . Several lines of evidence support a role for ER stress pathway within the islet β cells in induction and activation of autophagy in diabetes. In mammalian cells, unfolded protein response regulator and ER chaperone glucose-regulated protein-78 knock down inhibited autophagosome formation induced by ER stress, indicating that glucose-regulated protein-78 is required for stress-induced autophagy . Protein kinase-like endoplasmic reticulum kinase–eukaryotic initiation factor-2α and inositol-requiring enzyme-1α-C-Jun N-terminal kinases signaling pathways are also required for autophagy activation after ER stress . Interestingly, other studies suggest precisely the opposite where Zhao et al.  showed that chronic high glucose induced β-cell injury through autophagy down-regulation in pancreatic β cells in type 2D rats. Meanwhile, infusion of bone marrow-derived mesenchymal stem cells enhanced formation of autophagosomes and autolysosomes, combined with reduced β-cell apoptosis and increased number of insulin granules, thus result in improvement of β-cell function and survival . Nevertheless, examination of human islets from organ donors demonstrated accumulation of autophagic vacuoles and autophagolysosomes in type 2 diabetic donors compared with control . This was associated with an increase in dead β cells with a massive vacuole overload, together suggesting altered autophagy. However, autophagic flux was not examined in this study, so it is not possible to conclude whether these changes are due to autophagic activation or impaired flux. These conflicting results could be viewed on the basis that the alterations in autophagy seen in diabetes are complex, vary in different tissues, and are not yet fully understood. Interpretation of animal studies in this area is more complex, as manipulation of autophagy in one target tissue can result in significant alterations in whole-body metabolism and compensatory mechanisms in other target tissues . Further study is clearly required in this area, particularly focusing on changes in autophagy in human tissues in type 2 diabetes. Furthermore, the current study showed that 4-PBA treatment caused significant decline in beclin-1 levels in pancreatic tissues of the treated diabetic group compared with the nontreated diabetic group. 4-PBA is a chemical chaperone reported to improve protein misfolding and ER stress in pancreatic β-cells . In harmony with this finding, Kim et al.  demonstrated that 4-PBA alleviated autophagy, as indicated by the lowered gene expression levels of beclin-1 and LC-3II (as two autophagy markers) and reduced autophagic vacuoles in human gingival cells, signifying inhibition of ER stress-induced cell death and autophagy. As ER stress is critical for autophagy, it seems plausible that 4-PBA alleviated autophagy possibly though inhibiting ER stress. Oxidative stress plays a major role in the pathogenesis of both types of DM . Data obtained in the present study provided unequivocal evidence for an altered redox status in type 2 diabetic rats, as represented by significant elevation of AOPPs plasma levels accompanied by significant reduction in GSH levels in pancreatic tissues of type 2 diabetic group as compared with the nondiabetic control group. These findings are not without precedence, having been previously noted by Adebiyi et al.  as well as Tiwari et al.  who revealed that plasma levels of AOPPs were significantly upregulated in diabetic rats compared with the nondiabetic control rats. Meanwhile, Gradinaru et al.  pointed out that AOPPs and oxidative stress markers increase in adults with type 2 diabetes with and without microvascular/macrovascular complications. Therefore, it can be concluded that AOPPs might serve as efficient markers to estimate the level of protein damage mediated by oxidants in diabetic patients. Concomitantly, the decline in the GSH concentrations in the pancreatic tissue of type 2 diabetic rats, observed in the present study, is consistent with numerous previous studies which portrayed the decreased levels of GSH as a sign for glucolipotoxicity-mediated oxidative stress and pancreatic β-cell dysfunction during HFD-STZ-induced type 2 diabetes in rats ,. Conceivably, unfolded protein response signaling in the ER can result in augmenting oxidative stress as ER provides an exclusive oxidizing environment to the proteins to facilitate disulfide bond formation . ROS may be generated when accumulation of unfolded proteins in the ER elicits Ca2+ leakage into the cytosol through inositol trisphosphate receptor. The perturbed cytoplasmic calcium levels evoke influx of Ca2+ in the nuclei and mitochondria resulting in generation of ROS . In this regard, it has been hypothesized that oxidative stress causes autophagy dysregulation. A major consequence of impaired autophagy is accumulation of damaged mitochondria whose turnover is mainly dependent on autophagy , resulting in mitochondrial membrane permeabilization, ATP decrease, and overproduction of reactive oxygen species . Noteworthy, the decreased GSH concentrations observed in pancreas of type 2 diabetic rats may be attributable to the utilization of nonprotein thiols by increased oxygen-free radicals generated in sustained hyperglycemic conditions and ER stress . Taken in conjunction, these findings might validate the theory that diabetic milieu leads to imbalances in the antioxidant capacity within the cell resulting in oxidative/nitrosative stress and suggest an intimate relationship between autophagy and ROS production as major pathogenic mechanisms of diabetes. Moreover, treatment with 4-PBA resulted in significant improvement in the antioxidant status of pancreas with elevated levels of GSH and an associated reduction in plasma levels of AOPPs in the treated diabetic group as compared with the nontreated diabetic group. In agreement to this finding, Luo et al.  demonstrated that treatment with 4-PBA significantly inhibits the process and development of diabetic nephropathy in rats through the regulation of ER stress-oxidative activation. This observed ameliorative effect of 4-PBA could be credited to its action as chemical molecular chaperone which can suppress oxidative stress by attenuating ER stress .
| Conclusion|| |
The present study signifies that uncontrolled autophagy as well as augmented oxidative stress in pancreas acted as major contributing factors for the pathogenesis of type 2 diabetes in experimental rats. It also shed some light on the potential therapeutic value of 4-PBA in type 2 diabetes, thus hinting new perspectives for the use of chemical chaperones in pancreatic diseases. 4-PBA was found to be effective in protecting the pancreas of diabetic rats by ameliorating autophagy and oxidative stress-mediated cell damage. However, further studies are warranted to justify its therapeutic efficacy before considering it as potential drug candidates for treating type 2 diabetes and related disorders in humans.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]