Effect Of Iron Regulatory Proteins 2 Deletion On Glucose Homeostasis And Its Mechanism

Objectives: The prevalence of diabetes which arises from the interactions of multiple genes and lifestyle factors,continues to rise worldwide causing serious health problems and imposing a substantial economic burden on societies.According to the epidemiological study of2010 China Noncommunicable Disease Surveillance Group,the estimated prevalence of diabetes among a representative sample of Chinese adults was11.6% and the prevalence of prediabetes was 50.1%.Projections based on sample weighting suggest this may represent up to 113.9 million Chinese adults with diabetes and 493.4 million with prediabetes.These findings indicate the importance of diabetes as a public health problem in China.Researchers believe that etiology of diabetes is multifactorial,with genetic,environmental,socioeconomic,and behavioral or psychological influences.Recently,many evidences indicated that excess iron is likely to be an important risk factor for diabetes.Iron is one of the essential trace elements,the strict iron metabolism regulation can balance the iron in human body.Iron overload may cause many diseases,including diabetes.Iron-regulatory proteins 2（IRP2）,a kind of polypeptide in cells,is one of the iron sulfur clusters isomerases,expressed in a wide variety of tissues/cells.IRP2 plays an important role in regulating iron metabolism,therefore,knocking out IRP2 gene can led to iron deposit.It has been reported that iron verload can affect the preverance of diabetes,but its mechanism is not yet clarified.To better understand the relationship between iron overload and abnormal glucose metabolism,we utilized a IRP2 knockout(IRP2^-/-)mouse model（endogenous iron overload model）as a powerful tool to examine the functional roles of iron overload on glucose homeostasis and its underlying molecular mechanism.Methods: Twenty-four healthy male IRP2^-/-mice（15-month-old,C57BL/6,weight about 30g）were selected randomly and housed under specific pathogen-free conditions in the Animal Facility of Hebei Normal University.15-month-old wild-type（WT）healthy male mice（IRP2+/+,C57BL/6,n=24）as control.They were maintained with freely provided standard rodent chow and tap water.After 3 days adaptive feed,we performed a series of studies.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 deposition was measured by atomic absorption,synchrotron radiation and immunohistochemistry.The levels of SOD and MDA in liver and skeletal muscle were detected.Apoptotic cells in liver and skeletal muscle were identified by terminal dUTP nick-end labeling（TUNEL）staining.The expression of glucose transporter 4（Glut4）in skeletal muscle and insulin receptor substrate 2（IRS2）in liver was investigated by Real-time PCR and Western-blot.Data were analyzed by SPSS 20.0 software.Results:1 Compared with wild-type control,abnormal glucose metabolism(Glucose values at the indicated time points following IPGTT:0min 6.62±0.79 mmol/L vs 5.08±0.27 mmol/L,p^0min=0.022;15min16.32±3.33 mmol/L vs 13.06±0.81 mmol/L,p^15min=0.093;30min 17.64±2.71mmol/L vs 13.78±0.85 mmol/L,p^30min=0.07;60min 13.96±1.61 mmol/L vs9.86±0.89 mmol/L,p^60min=0.001;120min 10.90±1.67 mmol/L vs 6.26±0.36mmol/L,p^120min=0.036)and decreased insulin sensitivity(Glucose values at the indicated time points following ITT: 0min 6.56±0.59 mmol/L vs5.10±0.33 mmol/L,p^0min=0.001;15min 4.82±0.19 mmol/L vs 4.28±0.38mmol/L,p^15min=0.021;30min 4.58±0.13 mmol/L vs 2.52±0.15 mmol/L,p^30min=0.001;60min 3.40±0.23 mmol/L vs 2.74±0.15 mmol/L,p^60min=0.001;120min 5.82±0.47 mmol/L vs 4.34±0.48 mmol/L,p^120min=0.001)was observed in IRP2^-/-mice.The fasting and first-phase insulin secretion had no significant differences between IRP2^-/-mice and their control littermates(0min33.15±1.41 uIU/ml vs 31.45±1.41uIU/ml,p^0min=0.308;30min 70.99±1.22uIU/ml vs 77.70±6.56 uIU/ml,p^30min=0.156);2 Atomic absorption andsynchrotron radiation showed that iron was mainly deposited in liver（540.91±131.41 μg/g vs 270.99±64.85 μg/g,p < 0.001）,skeletal muscle（（43.28,73.47）vs（28.49,57.80）μg/g,p < 0.05）and pancreas（172.69±7.94 μg/g vs 119.03±11.50 μg/g,p<0.001）;3Immunohistochemistry and prussian blue reaction conformed that iron was mainly deposited in pancreatic acinar cells;4 Compared with WT mice,the levels of SOD were significantly decreased(（0.47,0.52）vs（0.49,0.63）U/mgprot,p^liver<0.001;0.81±0.11 U/mg prot vs 1.00±0.13 U/mg prot,p^muscle<0.05),whereas MDA was significantly increased in IRP2^-/-mice(6.03±1.25 nmol/mg prot vs 4.13±0.14 nmol/mg prot,p^liver < 0.05;1.38±0.20 nmol/mg prot vs 1.00±0.10 nmol/mg prot,p^muscle<0.01);5 The number of TUNEL-positive cells in liver（≈ 10 times）,skeletal muscle（≈ 12times）and pancreatic acinar cells（≈ 3 times）was significantly increased in the IRP2^-/-mice compared with that of WT mice;6 The mRNA and protein expression of IRS2 in liver(（0.40,0.84）vs（0.54,2.05）,p^mRNA <0.001;0.98±0.24 vs 2.48±0.19,p^protein<0.001)and Glut4 in skeletal muscle(（0.01,0.82）vs（0.79,1.51）,p^mRNA<0.001;1.04±0.27 vs 1.80±0.15,pprotein<0.001)was significantly decreased in IRP2^-/-mice compared with their control littermates.Conclusions:1 Iron regulatory protein 2 gene knockout can lead to abnormal glucose metabolism,which may not due to impaired insulin secretion,but insulin resistance.2 Iron regulatory protein 2 gene knockout can lead to iron deposition in liver and skeletal muscle which were the target orgens of insulin,causing oxidative stress disorder,apoptosis,and decline expression of IRS2 and Glut4,which resulted in insulin resistance and ultimately abnormal glucose metabolism.