| Objectives: Diabetes mellitus is characterized by chronic hyperglycemia resulting from deficiency of insulin secretion and/or insulin resistance.It showed that iron overload might be an independent risk factor for diabetes.Iron is one of the most abundant essential trace metals in the organism.Iron balance is important for cell signalling and homeostasis.Ceruloplasmin(Cp)is synthesized mostly in liver.As a ferroxidase,Cp can catalyze iron from ferrous to the ferric form,and promote iron binding to transferrin.Cp gene(Cp-/-)knock out can led to iron deposit and impaired glucose tolerance,but its mechanism is not yet clarified.Tea polyphenols(TP),as a natural antioxidant,have iron chelating properties,researches have shown that TP could reduce blood glucose level,but the mechanism of TP on glycometabolism is not yet clarified.We utilized Cp-/-mice as iron overload model,and treated with TP through intragastric administration,to detect the underlying molecular mechanism of iron overload on glucose homeostasis and the effect of TP on pancreatic ? cell secretory function and insulin action,in order to provide new treatment strategy for patients with diabetes.Methods: 12 healthy female Cp-/-BALB/c JŚ129SvJ mice(10-month-old)were selected as experiment group,10-month-old healthy female Cp+/+ mice(n=12)as control.Then experiment and control group were divided into TP sub-group(TPG,n=6,Gavage Tea Polyphenols)and NS sub-group(NSG,n=6,Gavage Normal saline),respectively.After four weeks of TP intervention,glucose tolerance and insulin sensitivity were performed by Intraperitoneal Glucose Tolerance Test(IPGTT)and Insulin Tolerance Test(IPITT).Insulin levels were detected by radioimmunoassay.Tissue iron content was measured by colorimetry.Insulin immunohistochemistry and Prussian blue staining were performed to detect iron deposition in the pancreas.The levels of SOD and MDA in liver were detected.Apoptotic cells in liver were ident ified by terminal d UTP nick-end labeling(TUNEL)staining.The expression of glucose transporter 2(GLUT2)and insulin receptor substrate 2(IRS2)in liver was investigated by Real-time PCR and Western-blot,respectively.Results:1 Compared with Cp+/+ NSG,abnormal glucose metabolism(Glucose values at the indicated time points following IPGTT: 0 min 6.80±0.91 mmol/L vs 4.98±0.71 mmol/L,P0min=0.004;15 min 14.88±0.69 mmol/L vs 12.70±0.67 mmol/L,P15min=0.011;30 min 15.45±0.97 mmol/L vs 8.85±0.79 mmol/L,P30min<0.001;60 min 10.30±1.61 mmol/L vs 7.75±0.37 mmol/L,P60min=0.004;120 min 8.00±0.70 mmol/L vs 5.68±0.34 mmol/L,P120min<0.001)and decreased insulin sensitivity(Glucose values at the indicated time points following ITT: 0 min 7.15±1.20 mmol/L vs 5.13±0.54 mmol/L,P0min=0.001;15 min 5.58±0.87 mmol/L vs 4.18±0.49 mmol/L,P15min=0.014;30 min 3.55±0.60 mmol/L vs 2.80±0.28 mmol/L,P30min=0.013;60 min 2.30±0.22 mmol/L vs 1.63±0.25 mmol/L,P60min<0.001)were observed in Cp-/-NSG mice.There were no significant differences between Cp+/+ TPG and Cp+/+ NSG in glucose tolerance(Glucose values at the indicated time points following IPGTT: 0min 4.35±0.68 mmol/L vs 4.98±0.71 mmol/L,P0min=0.245;15min 12.70±1.44 mmol/L vs 12.70±0.67 mmol/L,P15min=1.000;30min 8.23±0.75 mmol/L vs 8.85±0.79 mmol/L,P30min=0.450;60min 7.68±0.54 mmol/L vs 7.75±0.37 mmol/L,P60min=0.919;120min 4.90±0.78 mmol/L vs 5.68±0.34 mmol/L,P120min=0.091)and insulin sensitivity(Glucose values at the indicated time points following ITT: 0min 4.95±0.24 mmol/L vs 5.13±0.54 mmol/L,P0min=0.726;15min 4.03±0.73 mmol/L vs 4.18±0.49 mmol/L,P15min=0.763;30min 2.33±0.26 mmol/L vs 2.80±0.28 mmol/L,P30min=0.089;60min 1.43±0.10 mmol/L vs 1.63±0.25 mmol/L,P60min=0.167).Compared with Cp-/-NSG,improved glucose metabolism(Glucose values at the indicated time points following IPGTT: 0min 5.65±0.53 mmol/L vs 6.80±0.91 mmol/L,P0min=0.044;15min 13.23±1.08 mmol/L vs 14.88±0.69 mmol/L,P15min=0.042;30min 12.70±1.74 mmol/L vs 15.45±0.97 mmol/L,P30min=0.005;60min 7.23±1.08 mmol/L vs 10.30±1.61 mmol/L,P60min=0.001;120min 5.45±0.47mmol/L vs 8.00±0.70 mmol/L,P120min<0.001)and insulin sensitivity(Glucose values at the indicated time points following ITT: 0min 5.10±0.35 mmol/L vs 7.15±1.20 mmol/L,P0min=0.001;15min 4.13±0.61 mmol/L vs 5.58±0.87 mmol/L,P15min=0.011;30min 2.53±0.15 mmol/L vs 3.55±0.60 mmol/L,P30min=0.002;60min 1.48±0.17 mmol/L vs 2.30±0.22 mmol/L,P60min<0.001)were observed in Cp-/-TPG mice;2 Compared with Cp+/+ NSG,fasting insulin secretion was significantly higher,whereas first-phase insulin secretion had no significant difference in Cp-/-NSG mice(37.93±3.36 uIU/ml vs 33.91±2.38 uIU/ml,P0min=0.036;79.31±7.84 uIU/ml vs 68.81±6.89 u IU/ml,P30min=0.068).There were no significant differences between Cp+/+ TPG and Cp+/+ NSG in fasting insulin and first-phase insulin level(31.58±2.09 u IU/ml vs 33.91±2.38 uIU/ml,P0 min =0.198;69.11±8.38 uIU/ml vs 68.81±6.89 uIU/ml,P30min=0.956).After 4 weeks of TP treatment,decreased fasting insulin secretion was observed in Cp-/-TPG mice when compared with Cp-/-NSG,whereas no significant difference of first-phase insulin secretion between Cp-/-TPG and Cp-/-NSG(33.88±1.44 uIU/ml vs 37.93±3.36 uIU/ml,P0 min =0.035;71.35±6.37 uIU/ml vs 79.31±7.84 uIU/ml,P30min=0.154);3 Liver non-haem iron determinations showed that severe iron deposition was observed in Cp-/-NSG mice as compared with Cp+/+ NSG(21.47 ±3.53 mg/gprot vs 3.04±0.61 mg/gprot,P<0.001).After TP treatment,iron deposition(10.77±1.13 mg/gprot vs 21.47±3.53 mg/gprot,P<0.001)was alleviated in Cp-/-TPG than Cp-/-NSG.There was no significant difference between Cp+/+ TPG and Cp+/+ NSG(2.68±0.55 mg/gprot vs 3.04±0.61 mg/gprot,P=0.827)in liver non-haem iron;4 Immunohistochemistry and prussian blue reaction conformed that iron was mainly deposited in pancreatic acinar cells;5 Compared with Cp+/+ NSG mice,the level of SOD was significantly decreased(1.70±0.27 U/mgprot vs 2.11±0.09 U/mgprot,P=0.005),whereas the level of MDA was significantly increased(5.46±0.51 nmol/mgprot vs 4.41±0.43 nmol/mgprot,P=0.001)in Cp-/-NSG mice.After 4 weeks of TP treatment,the level of SOD was significantly increased(2.46±0.21U/mgprot vs 1.70±0.27U/mgprot,P<0.001),whereas the level of MDA was significantly decreased(4.75±0.48 nmol/mgprot vs 5.46±0.51 nmol/mgprot,P=0.019)in Cp-/-TPG when compared with Cp-/-NSG.Moreover,compared with Cp+/+ NSG,the level of SOD was significantly increased(2.53±0.14 U/mgprot vs 2.11±0.09 U/mgprot,P=0.004),whereas the level of MDA was significantly decreased(3.76±0.25 nmol/mgprot vs 4.41±0.43 nmol/mgprot,P=0.031)in Cp+/+ TPG.6 The number of TUNEL-positive cells in liver was higher in Cp-/-NSG than that of Cp+/+ NSG(67.00±4.58 vs 13.67±2.08,P<0.001).After TP treatment,the number of TUNEL-positive cells was decreased in Cp-/-TPG than Cp-/-NSG(38.33±5.03 vs 67.00±4.58,P<0.001).There was no significant difference in TUNEL-positive cells between Cp+/+ TPG and Cp+/+ NSG(12.67±2.52 vs 13.67±2.08,P=0.754).7 The mRNA and protein expressions of IRS2 [(0.09,0.29)vs(0.42,0.78),PmRNA=0.011;(0.52±0.05 vs 0.70±0.07),Pp rotein=0.024] and GLUT2 [(0.03±0.02 vs 0.97±0.13),PmRNA<0.001;(0.45±0.05 vs 0.81±0.06),Pp rotein<0.001] in liver were significantly decreased in Cp-/-NSG mice when compared to Cp+/+ NSG mice.All these decreasing were partly reversed by TP treatment.Compared with Cp-/-NSG mice,the mRNA and protein expressions of IRS2 [(0.46,0.88)vs(0.09,0.29),PmRNA=0.006;(0.69±0.05 vs 0.52±0.05),Pp rotein=0.029] and GLUT2 [(0.96±0.23 vs 0.03±0.02),PmRNA<0.001;(0.75±0.06 vs 0.45±0.05),Pp rotein<0.001] in liver were significantly increased in Cp-/-TPG mice as compared with Cp-/-NSG mice.No significant differences were detected between Cp+/+ TPG and Cp+/+ NSG in the expressions of IRS2 [(0.74,1.48)vs(0.42,0.78),PmRNA=0.077;(0.72±0.12 vs 0.70±0.07),Pp rotein=0.759] and GLUT2 [(1.02±0.22 vs0.97±0.13),PmRNA=0.755;(0.84±0.04 vs 0.81±0.06),Pp rotein=0.478] in the liver.Conclusions:1 Ceruloplasmin gene knockout can lead to abnormal glucose metabolism,which may not due to impaired insulin secretion,but insulin resistance.2 Ceruloplasmin gene knockout can lead to iron deposition in liver,which is the target organ of insulin,causing oxidative stress disorder,apoptosis,and decline expression of IRS2 and GLUT2,resulting insulin resistance and ultimately abnormal glucose metabolism.3 Tea polyphenols alleviate iron deposition and oxidative stress disorder in liver,ultimately improving abnormal glucose metabolism caused by Cp knockout. |
PDF Full Text Request