Analysis Of Copper,Zinc,Magnesium And Calcium In Type 1 Diabetic Mice Model And Patients With Type 1 Diabetes

BackgroundDiabetes has become one of the most serious endocrine and metabolic diseases in the world.Diabetes damages multiple organs to induce serious complications such as diabetic retinopathy,neuropathy,and nephropathy.Excessive calorie intake and improper consumption of nutrients are major driving forces behind escalating diabetes and appearance of epidemics worldwide.As essential components of daily diet intake,trace elements are essential for the occurrence and progression of diabetes.Alternations in trace elements status and increased oxidative stress in diabetes may lead to insulin resistance,development of diabetes and diabetic complications.On the other hand,development of diabetes can also lead to perturbation in metabolism and homeostasis of trace elements.Chemical or drug chaperones,such as 4-phenylbutyric acid(PBA)and tauroursodeoxycholic acid(TUDCA),can improve proteins folding and alleviate endoplasmic reticulum stress(ERS).Recently,the relationship between these molecular chaperones and trace elements has attracted great attention.This study aimed to investigate the metabolism and associations of trace elements such as copper,zinc,magnesium,and calcium in type 1 diabetic mice model and patients with type 1 diabetes,and the effect of TUDCA and PBA on metabolism of trace elements in type 1 diabetic mice model.Methods1.Preparation of type 1 diabetic mice modelMultiple low-dose streptozocin(STZ,40 mg/kg/d)was intraperitoneally injected to 8-10-week-old male FVB mice to induce diabetic mice model.STZ-treated mice with fasting blood glucose higher than 12 mmol/L were regarded as diabetic.After modeling,PBA treated diabetic mice were injected intraperitoneally with 500 mg/kg PBA twice-a-day;TUDCA treated diabetic mice were fed with 500 mg/kg TUDCA in drinking water daily.Blood glucose and body weight were measured at 2 weeks and 2 months after the onset of diabetes,respectively.The mice were sacrificed and heart,liver,kidney,muscle,spleen and whole blood were collected.2.Detection of biochemical indicatorsThe whole blood of mice was separated at 4?,12000r/min,centrifuged for 5 minutes.Glycosylated serum protein(GSP),blood urea nitrogen(BUN),creatinine(CRE),uric acid(UA),alkaline phosphatase(ALP),total cholesterol(CHO),triglyceride(TG),high density lipoprotein cholesterol(HDL)and low density lipoprotein cholesterol(LDL)were measured on automatic biochemical analyzer.3.Detection of trace elementsPreweighed pieces of heart,liver,kidney,muscle and spleen(approximately 0.5–1 g of tissue)from mice were digested separately in 1 ml 10% nitric acid in a 10-ml glass vial at room temperature and then heated to 110 ?for 8 h to facilitate digestion.A volume of 4 ml deionized water was added to each vial to increase the volume to 5 ml.The resulting clear liquid was used for analysis.A volume of 0.5 ml serum was digested separately in 2 ml 10% nitric acid,then dipped for 30 minutes,and subsequently heated to 100? for 2 h to facilitate digestion,heated to 150? for 10 h to facilitate digestion again.Deionized water was added to each vial to increase the volume to 15 ml.The resulting clear liquid was used for analysis.Copper,zinc,magnesium,and calcium concentrations were determined by inductively coupled plasma mass spectrometry(ICP-MS).4.Preparation,sectioning and staining of liver tissueLiver was fixed in 10% formalin,then immersed in 75%,85%,95%,100% ethanol in a proper order for dehydration,then immersed in xylene I,II,III for transparency,subsequently embedded in paraffin I,II and III at 60~63?,and routinely sectioned.The slides were stained for HE staining,Pexiodic Acid Schiff(PAS)staining,Sirius Red staining and TUNEL staining.5.Determination of biochemical markers and trace elements in patients with type 1 diabetesFrom January 2013 to December 2014,50 patients with type 1 diabetes diagnosed at the First Hospital of Jilin University were selected,and 50 healthy controls were included in the same period.Red blood cells and hemoglobin were measured by hematology analyzer and glycosylated hemoglobin A1c(HbA1c)was measured by automatic glycohemoglobin analyzer.Biochemical indicators such as blood glucose(GLU),BUN,CRE,CHO,TG,HDL,and LDL were measured by automatic biochemical analyzer,and copper,zinc,magnesium,and calcium in serum and urine were measured by ICP-MS.Result1.In the diabetic mice at 2 weeks and 2 months after the onset of diabetes,body weight was significantly lower,and blood glucose and GSP were significantly higher than control.Those items in PBA and TUDCA treated diabetic mice were improved,but still abnormal.2.Except UA and HDL,biochemical indicators such as BUN,CRE,ALP,CHO,TG,and LDL were significantly higher in diabetic mice than those of control.ALP and LDL in PBA treated diabetic mice were significantly lower,but were still higher than normal levels;PBA could improve CRE level to normal.ALP and TG in TUDCA treated diabetic mice were significantly lower,but were still higher than normal levels;TUDCA could make CHO and LDL normal.3.HE staining showed that PBA and TUDCA can ameliorate infiltration of inflammatory cell and fibrosis in the mice.4.PAS staining showed that PBA and TUDCA can improve accumulation of glycogen in hepatocytes of mice.5.Sirius Red staining was used to detect the collagen content in mice liver.The results showed PBA and TUDCA can significantly reduce fibrosis of liver tissue in diabetic mice.6.TUNEL staining was used to detect the number of apoptotic cells in mice liver.The results demonstrated that PBA and TUDCA can decrease apoptosis of hepatocytes in diabetic mice.7.The levels of magnesium in heart of diabetic mice at 2 weeks and 2 months after the onset of diabetes were both significantly decreased.However,the level of cardiac copper was only declined in diabetic mice at 2 months after the onset of diabetes.8.The levels of copper,zinc and magnesium in liver of diabetic mice at 2 weeks and 2 months after the onset of diabetes were significantly decreased.PBA can restore hepatic copper levels to normal in diabetic mice at 2 weeks and 2 months after the onset of diabetes and it can increase hepatic zinc levels to normal in diabetic mice at 2 weeks after the onset of diabetes.However,PBA can enhance hepatic zinc levels at 2 months after the onset of diabetes,but still abnormal.TUDCA could restore hepatic magnesium levels in diabetic mice.9.In the diabetic mice at 2 weeks and 2 months after the onset of diabetes,the levels of renal calcium and copper in diabetic mice were significantly elevated,while magnesium levels were significantly decreased.Both PBA and TUDCA could restore magnesium to normal in diabetic mice,and TUDCA made calcium levels return in diabetic mice.10.Muscular calcium levels were significantly elevated in diabetic mice at 2 weeks and 2 months after the onset of diabetes.The level of muscular zinc significantly elevated in diabetic mice at 2 months after the onset of diabetes.PBA reduced it in diabetic mice at 2 weeks after the onset of diabetes,but it was still higher than control.PBA can restore muscular calcium levels to normal in diabetic mice at 2 months after the onset of diabetes.TUDCA reduced muscular calcium in diabetic mice,but still abnormal.11.In the spleens of diabetic mice at 2 weeks and 2 months after the onset of diabetes,only the calcium levels decreased when the levels of copper,magnesium,and zinc did not change.12.Serum copper levels in diabetic mice at 2 weeks and 2 months after the onset of diabetes increased significantly,while magnesium and calcium levels decreased significantly.TUDCA restored serum magnesium and calcium to normal levels.In diabetic mice at 2months after the onset of diabetes,both PBA and TUDCA can restore serum zinc levels,whereas PBA can return serum copper levels to normal.13.There was a positive correlation between copper levels and zinc concentrations,magnesium levels and calcium levels in heart,liver,kidney,muscle,spleen and serum.14.The serum copper,urinary zinc,urinary magnesium,and urinary calcium concentrations in patients with type 1 diabetes were significantly elevated,and serum zinc,serum calcium,serum magnesium,urinary copper,and Zn/Cu ratios were significantly decreased.15.In patients with type 1 diabetes,serum copper was positively correlated with serum zinc and Hb,and negatively correlated with blood glucose;Urinary copper was positively correlated with urinary zinc and negatively correlated with cholesterol;Serum zinc was positively correlated with serum copper,serum calcium,and CRE,and negatively correlated with blood glucose;Urinary zinc was positively correlated with urinary copper and negatively correlated with eGFR;Serum magnesium was positively correlated with serum zinc,serum calcium,TG,and BUN;Serum calcium was positively correlated with serum zinc,serum magnesium,BUN and TG,and negatively correlated with HDL;Urinary calcium was positively correlated with urinary magnesium and LDL.Conclusions1.The abnormal metabolism of copper,zinc,calcium,and magnesium in heart,liver,kidney,muscle,spleen,and serum exist in type 1 diabetic mice.2.The levels of copper in heart,liver,kidney,muscle,spleen and serum of type 1 diabetic mice are positively correlated with zinc levels,and magnesium levels are positively correlated with calcium levels.3.In type 1 diabetic mice,PBA could restore hepatic copper and renal magnesium levels,whereas TUDCA could return hepatic magnesium,renal magnesium and calcium,serum magnesium and calcium levels to normal.4.The metabolism of serum copper,zinc,calcium and magnesium in patients with type 1 diabetes is abnormal.5.There is a positive correlation between serum copper levels and zinc levels,magnesium and calcium levels,urinary copper and zinc levels,urinary calcium and magnesium levels in patients with type 1 diabetes.