Effect And Mechanisms Of Zinc On Rats Of Experimental Diabetes

Objective:Having the diabetic rat with hyperlipemia as the subjects of the experiment, the study was to observe the effect of zinc on the serological index, anti-oxidation competence, trace elements and the physiopathology of the main organs, to explore the protective effect of zinc on the organ injury caused by diabetes and to study the underlying mechanisms how zinc induce metallothionein (MT) and phosphorylated protein kinase B (pAkt) work in diabetes mellitus and organ injury.Methods:Seventy five male Wistar rats with a body weight of 180~220 g were randomly divided into six groups: normal diet control (NC) group, high fat diet control (FC) group, high fat plus Zn (FZ) group, diabetes (DM) group, Zn intervention before STZ injection (ZnDM) group, Zn intervention after STZ injection (DMZn) group. NC group were always fed with normal forage and other groups with high fat forage. Thereafter, the rats were treated according to the time sequence. The rats in FZ and ZnDM groups were fed with Zn ions (Zn2+) 15mg?Kg-1?d-1 by giving gastric ZnSO4 solution, and the rats in other groups with equivalent volumes of physiological saline. Thirty-five days later, the high-fat animal model was identified by the serum detection.The rats fast for 12 hours, which were in DM, ZnDM and DMZn groups underwent a single intraperitoneal injection of streptozocin (STZ) 40mg·kg-1, the rats in other groups were given citrate buffer saline. Three days later, the fasting blood-glucose (FPG) was detected and the criterion for diagnosis of DM is FPG≥16.7 mM. The selected rats in FZ, ZnDM and DMZn groups were fed with Zn ions (Zn2+) 15mg?Kg-1?d-1 by giving gastric ZnSO4 solution daily and other groups with equivalent volumes of physiological saline.56 days after STZ injection, the rats were necrotized by intraperitoneal injection of 3% pentobarbital sodium 30 mg/kg, carotid artery was exposed and cannulated for the monitoring on the heart rate, systolic pressure, diastolic pressure, left ventricular pressure (LVP), left ventricular end-diastolic pressure (LVEDP) and±dp/dt. The serum was collected by the abdominal venous for detecting the function of liver and kidney, blood glucose, blood lipid (T-CHOL, TG and LDL-C), insulin, SOD, MDA, and GSH-Px, T-AOC, trace elements (Ca,Fe,Zn,Cu). The heart, liver and kidney were harvested and weighted, then they were fixed with 10% formaldehyde solution for use in the histology and immunohistochemistry, and then a portion of the heart were stored in -80°C for use in Western blotting and trace elemental analysis. Left myocardium with a volume of 1 mm3 was fixed with 2.5% glutaraldehyde and sent for electronic scanning microscopy. Bilateral myocardium of 0.6 g was baked dry and the trace elements were detected. Organs of 0.5 g was homogenized and precipitated by centrifugation, and then the supernatant was harvested for detection.The methods for the detection were as following. The daily food taking and water drinking was recorded for each cage. The body weight and the organ weight were measured by an electronic balance, calculating the organ index. The FPG was detected by a glucometer, the blood lipid, the BUN and CRE (renal function markers), the AST/ALT (hepatic function markers) and the trace elements in serum were detected by an autobiochemical analyzer. The blood insulin was detected by a radioimmunoassay. The anti-oxidation competence (serum SOD, GSH-Px, MDA and T-AOC) was detected by kits; the myocardial zinc content and other metal element contents were detected by an atomic absorption method. The MT level was determined by 109Cd hemoglobin saturation method in serum and tissue. The pathohistological change of liver, heart, and kidney were observed in light microscope; the pAkt and LC3-II levels in organs were investigated by immunohistochemical methods. The levels of LC3-Ⅱand GRP78 in the heart, liver and kidney were detected by Western blotting. In the statistical processing, SPSS12.0 software was used , and the data were expressed with x±S. The multiple groups comparison made use of ANOVA and Wilcoxon Rank sum test and the relation between a portion of factors was determined by Pearson correlation.Results:1. The rats fed with high fat forage induced the high lipid state (the LDL-C levels increased significantly P< 0.05) in 35 days to construct the animal model with hyperlipemia. The high-fat model rats underwent a single intraperitoneal injection of 40mg·kg-1 STZ and then presented the symptoms of diabetes mellitus, hyperglycemia (FPG≥16.7 mM; P<0.01 versus the controls), polydipsia (P< 0.05), polyphagia (P< 0.05) and weight loss (P < 0.05). Gastric zinc supplementation alleviated the diabetic symptoms (ZnDM and DMZn versus DM), the daily food-intaking, water-drinking and weight were improved significantly (P<0.05, P>0.05, P<0.05 respectively).2. Fifty six days after the STZ injection, the FPG in DM, ZnDM and DMZn groups was increased significantly (P< 0.01); the FPG level of DMZn group was significantly decreased (P< 0.05 versus DM), and the FPG of ZnDM group also trend to decrease (P> 0.05). For the groups fed with high fat forage, the serum T-CHOL, TG and LDL-C were significantly increased (P<0.01 versus NC). For the STZ groups (DM, ZnDM and DMZn), the serum T-CHOL, TG and LDL-C levels were significantly higher than NC, FC and FZ groups (P< 0.05); the serum T-CHOL and LDL-C levels of DMZn group were significantly decreased than DM (P<0.05). There was no significant difference in the serum insulin (ISI) level between STZ groups (P> 0.05), but the ISI of STZ groups was significantly lower than NC, FC and FZ (P<0.01). The results demonstrated that the diabetic rats with hyperlipemia presented sugar and lipid metabolic disorder 56 days after injection of STZ, and gastric zinc obviously improved the serum FPG and lipid levels although it could not cease completely. Zinc has the insulinomimetic activity.3. The MDA level was significantly increased and the serum T-AOC level was significantly decreased (P< 0.05, FC versus NC). The serum SOD, GSH-Px and T-AOC levels reduced significantly and the MDA level trend to increase (DM versus FC, P< 0.01 and P> 0.05, respectively). The serum SOD, GSH-Px and T-AOC levels was significantly increased (P< 0.05 ZnDM versus DM) and the serum GSH-Px and T-AOC levels was significantly increased (P< 0.05 DMZn versus DM). The MDA level of ZnDM and DMZn groups trend to decrease (P> 0.05 versus DM). The results demonstrated the rats with hyperlipemia and the diabetic rats with hyperlipemia presented the lipid peroxidative injury and a decrease in the anti-oxidation competence; gastric zinc could improve the anti-oxidation competence obviously.4. The organ index of heart, liver and kidney rose significantly (P< 0.05) in the diabetic rats, and gastric zinc improved the organ index significantly (P< 0.05).5. The LVP and±dp/dt max were significantly decreased (P< 0.01 versus control), the LVEDP was significantly increased (P< 0.05) in the diabetic rats. It indicated that the high fat diabetes injured myocarudium causing the hypofunction of heart contraction and diastole. Gastric zinc improved the contract and diastolic function of the heart (P< 0.05 and P< 0.01 versus DM).6. The serum ALT/AST and BUN/CRE levels were significantly increased (P<0.01) in DM rats. It indicated the high fat diabetes injured the liver and the kidney. Gastric zinc improved the renal function significantly (P< 0.05), the hepatic function trend to improve (P> 0.05).7. Histopathology showed that for diabetic hyperlipidemia model, myocardial cells showed vacuolar degeneration, cardiac hypertrophy, distortion, disorganized, large hyperchromatic nuclei and irregular nuclear type, it presented focal myocardial necrosis and fibrosis, pink collagen fibers, increased cell gap, inflammatory cell infiltration and fibroblast invasion. The rats suffered severe liver cell injury and had severe water-like degeneration and fatty degeneration and point or focal necrosis, irregular liver cell arrange. Liver cells were irregular in shape, a portion of the nucleus was extruded deviation from the center. Lobule were irregular. Liver cells around the central lobule shrink and disappeared, and lymphocytic infiltration occurred in some parts. Glomerular volume decreased, part of the glomeruli was lobulated, mesangial area was widened, mesangial cell proliferation was more obviously, mesangial matrix increased, a large number of renal tubular epithelial cells were swelling, vacuolar degeneration and luminal narrowing, hyaline cast existed. Some tubular epithelial cell was necrosis and shedding, it presented interstitial fibrosis. In the diabetic group of zinc intervention, the heart, liver and kidney histopathology was improved obviously. Zinc intervention prevented organ damage in diabetic rats.8. The serum Zn and Cu and Zn/Fe levels were significantly decreased (P< 0.05 or P<0.01 FC versus NC). The serum Zn and myocardial Zn levels was significantly decreased (P< 0.05 DM versus NC), the serum Cu and Fe levels was significantly increased (P<0.01), the Zn/Cu and Zn/Fe levels was significantly decreased (P<0.01 versus NC); the myocardial Cu and Fe levels was also significantly decreased (P<0.05). Gastric zinc improved the serum and myocardial Zn level (P< 0.05 versus DM). It suggested that the high fat rats and the diabetic high fat rats presented the trace elemental disorder, gastric zinc could improve the serum and myocardial Zn level obviously.9. The MT content was determined by 109Cd hemoglobin saturation method. The MT content in the serum and liver were significantly increased (P< 0.05 FC versus NC). The MT content in the liver was significantly increased (P< 0.05 DM versus NC). Gastric zinc increased significantly the MT content in serum, the heart, liver and kidney (P<0.01). The liver had the highest rise and the kidney changed the least.10. The immunohistochemical assay showed the pAkt-positive rate of the heart, liver and kidney cells were significantly increased (P< 0.01 DM versus NC). Gastric zinc improved the pAkt-positive rate of the heart, liver and kidney (P< 0.05).11. The immunohistochemical assay showed DM group had the highest percentage of LC3-II-positive cells, ZnDM and DMZn groups had less, the non-STZ group had the least. The Western blotting showed a similar result with the immunohistochemical assay, NC group had no expression of LC3-II, and the high fat groups and the STZ groups increased the LC3-II expression to some extent. It indicated that the high fat rats and the diabetic rats presented an increased autophagy level in organ cells and zinc supplementation inhibited or alleviated the genesis of autophagy.12. The Western blotting showed the expression of GRP78 was very low in the normal condition and the ERS was at a very low level. The high-fat forage and diabetes mellitus triggered the severe ERS, especially the latter (P< 0.05). The zinc supplementation inhibited or alleviated the genesis of autophagy.13. The correlation of trace elements was analyzed between myocardiun and serum. The serum zinc was positively correlated with myocardial Zn and the serum copper was negatively correlated with myocardial copper. The linear correlation analysis was performed between myocardial MT content and myocardial trace elements. The myocardial MT content was positively correlated with myocardial Zn, Zn/Cu and Zn/Fe. The relationship between the gray value of pAkt-positive cells of the heart, liver and kidney and the MT content of the corresponding tissues by the linear correlation analysis. The results showed that the myocardial and hepatic pAkt was positively correlated with MT.Conclusion:1. The hyperlipidemia models could be established by feeding male Wistar rats with high fat forage for 35 days. With a single intraperitoneal injection of STZ 40mg/kg to hyperlipidemia rats, diabetic hyperlipidemia model with polydipsia, polyphagia and weight loss could be induced.2. FC and DM animal models existed to varying degrees lipid disorders, trace element imbalance, lipid peroxidation and antioxidant capacity decrease. Gastric ZnSO4 (Zn2+ 15 mg·kg-1·d-1) improved the level of serum zinc, play an insulin-like role, and increased the sensitivity to insulin; in both FC and DM rat models, the lipid disorders had been significantly improved, and the anti-oxidant capacity and the abilities to resist lipid peroxidation damage was increased.3. DM rats at 56 d suffered from diabetic myocardial damage, presenting with increased heart index, systolic and diastolic cardiac dysfunction, myocardial histopathological change, myocardial trace element imbalance, and apparently increased cardiac autophagy and endoplasmic reticulum stress. Zinc intervention can improve myocardial zinc levels, improve cardiac systolic and diastolic function, and reduce the pathological changes of myocardial tissue, inhibiting the genesis of autophagy and endoplasmic reticulum stress.4. DM rats at 56 d suffered from liver damage, manifested as increased liver index, liver function abnormalities, liver histopathological change, and apparently increased liver autophagy and endoplasmic reticulum stress. Zinc intervention can slightly improve liver function, and reduce the pathological changes of liver tissue, inhibiting the genesis of autophagy and endoplasmic reticulum stress.5. DM rats at 56 d suffered from diabetic kidney damage, presenting with increased kidney index, renal dysfunction, renal histopathological change, and apparently increased renal cell autophagy and endoplasmic reticulum stress. Zinc intervention can obviously improve renal function and reduce the renal pathological changes, inhibiting the genesis of autophagy and endoplasmic reticulum stress.6. Zinc intervention with DM rats at 56 d obviously improved the MT contents in serum, heart, liver and kidney; there, MT contents increased the most in the liver, the least in the kidney; Zinc intervention with DM rats at 56 d obviously improved the pAkt levels in the heart, liver and kidney tissues; there, MT content was positively correlated with pAkt levels in the heart and liver.7. Gastric zinc improved the MT protein synthesis, the MT increase induced the phosphorylation (activation) of Akt, which relieved the excessive autophagy caused by the turbulence of lipid metabolism. At the same time, the MT increase and the resulting decrease of oxidative injury reduced the ERS-induced tissue injury and autophagy. Zinc interacting with MT improved the organ function of diabetic rat through multiple pathways.