| Insulin resistance played an important role in type2diabetes (T2DM) and gestational diabetes mellitus (GDM). The explicit mechanism of insulin resistance is still not clear. Insulin resistance is currently regarded as the outcome of effects of inflammatory and hormonal factors, endoplasmic reticulum stress and excessive nutritional by-products accumulating in insulin-sensing tissues. Albumin, the main plasma protein, is responsible for binding and transporting a great variety of endogenous and exogenous compounds. It can present either insulin-like or insulin-inhibitory activity. Most serum insulin associates with serum proteins. Our previous studies proved that:1) first, there are two forms of insulin in the serum-the unassociated insulin as an active form and the associated insulin as a less active form;2) serum albumin serves as an insulin transporter and reservoir;3) albumin-insulin association and disociation regulates blood glucose and cell function. Based on these results, we propose a novel mechanism of insulin resistance. We hypothesize that unassociated insulin is immediately available for utilization while associated insulin is less active but can dissociate from protein to keep unassociated insulin level steady. Pathological conditions, such as hyperlipidemia and obesity, will change the balance. If serum albumin-insulin affinity increases, immediately available insulin decreases. In contrast, if the affinity decreases, immediately available insulin increases in postprandial state but decreases in fasting condition because of the short half-life of unassociated insulin. In either condition, the pancreas has to secrete more insulin to maintain normal levels of serum glucose in both pre-and postprandial states. If the compensation fails, a normal fasting glucose can not be achieved. The present study aimed to explore the unknown pathological factor that regulates albumin-insulin affinity, the mechanisms and the pathophysiological meanings.In first part of the study, we explored potential albumin-insulin association regulator using proteomics differential techniques. Sera from twelve patients with T2DM and ten matched controls as well as nine patients with GDM and eight matched controls were collected for two dimensional gel electrophoresis. The2D electrophoresis profiles were analyzed and matched. Four proteins changed similarly in the two profiles as identified by MALDI-TOF/TOF MS. The results showed465±11spots and423±15spots were detected in the profiles of T2DM and control groups, respectively.269protein spots were matched and the average matching rate is61%.19differentially changed proteins were detected among these matched spots,8up-regulated and11down-regulated in T2DM.325±18spots and342±10spots were detected in the profiles of GDM and control groups, respectively.197protein spots were matched and the average matching rate is59%.30differentially changed proteins were detected among these matched spots,19up-regulated and11down-regulated in GDM. In summary, we found two protein spots up-regulated and two down-regulated both in the sera of patients with T2DM and GDM. According to MALDI-TOF/TOF protein identification of these4spots, al-antitrypsin and angiotensinogen (serpin peptidase inhibitor, clade A, member8) were down-regulated (-3.82±0.73and-4.76±0.33vs control, respectively) while clusterin and human serum albumin in a complex with myristic acid and tri-iodobenzoic acid up-regulated (15.6±2.49and3.13±0.23vs control, respectively) in both diabetic sera.In second part of the study, the effect of al-antitrypsin on albumin-insulin association, blood glucose and their mechanism were observed. In part I, we found spots in both diabetic sera. Based on the molecular weights and serum concentrations of the four differentially expressed proteins found in part I, al-antitrypsin is the only protein with a sufficient serum molecule number to interact with albumin and insulin. We the performed co-immunoprecipitation analysis (co-IP) to determine if α1-antitrypsin binds to albumin and insulin in the serum. Anti-α1-antitrypsin antibody was used to precipitate proteins that bound to al-antitrypsin. Normal IgG was used as control. Our results showed both al-antitrypsin and albumin were detected when normal serum samples were immunoprecipitated by anti-al-antitrypsin antibody. al-antitrypsin was not detected by normal IgG. Insulin is not precipitated by anti-al-antitrypsin antibody.We then determined the impact of different concentrations of al-antitrypsin on albumin-insulin association in vitro. Compared to normal ratio of al-antitrypsin and albumin (2.58mg/ml vs3%), low ratio of the proteins increased albumin-associated/unassociated insulin ratio significantly (2.41±0.17vs1.76±0.20,.P=0.0024). The difference between low and high AAT/albumin ratio was more significant (2.41±0.17vs1.53±0.18, P=0.0004). The difference of insulin ratio between normal and high AAT/albumin ratio was not statistically significant (P=0.1406).The impact of introperitoneally injected albumin and insulin on serum al-antitrypsin concentration was observed in mice. When initial albumin/α1-antitrypsin ratio in the blood is low, injected albumin increased serum α1-antitrypsin concentration. When the ratio is normal and higher, the injected albumin had no effect. In contrast, insulin injection decreased serum al-antitrypsin concentration when the ratio is low. Along with the results of in vitro studies, this result indicated that albumin decreased al-antitrypsin clearance and insulin increased al-antitrypsin clearance by competing with α1-antitrypsin in binding albumin.The impact of different concentrations of al-antitrypsin on albumin-insulin association was observed in mice. Injection of PBS solution containing0.64mg/ml al-antitrypsin and3%albumin significantly increased mouse blood glucose and prevented insulin-induced hypoglycemia. Increasing AAT concentration in the solution to2.58or10.30mg/ml exaggerated and prolonged insulin-induced hypoglycemia in a reverse dose-dependent manner. The effect of injecting albumin-free al-antitrypsin solution (0.64,2.58and10.30mg/ml, respectively) is similar to injecting the solutions with higher AAT/albumin ratio as expected. The results indicated that al-antitrypsin decreased blood glucose by competing with insulin in albumin association.In summary, we found, for the first time, that al-antitrypsin decreased insulin-albumin association and therefore increased immediately functionally available unassociated insulin. Low al-antitrypsin contributes to insulin resistance and diabetes. The circadian clock serves to organize behavior and many physiological functions. Circadian rhythms are generated by the transcription-translation feedback loops of the core circadian genes. Clock genes regulate many physical functions by regulating the clock controlled genes (CCGs). Many researches show that the circadian clock plays important roles in several pathological processes such as tumor and cardiovascular diseases besides physiological functions.Acute Myocardial Infarction (AMI) is the last stage of atherosclerosis. The occurrence of AMI shows a circadian rhythm with the peak in the morning hours. Although the precise mechanism underlying the phenomenon is still not clear, circadian clock may involve in the process. In this study, apoE knock-out (apoE-/-) mice were used to establish the atherosclerotic animal model. The expression changes of atherosclerosis-related genes:plasminogen activator inhibitor-1(Pai-1), tissue plasminogen activator (t-PA), tissue factor (TF), endothelin-1(ET-1), matrix metallo-proteinase (MMP-1,MMP-2,MMP-9) were analyzed to provide new insights for further study of atherosclerosis.Rhythm changes of atherosclerosis-related genes in the hearts of C57BL/6J mice and apoE-/-mice. apoE-/-mice were used to establish the atherosclerosis model of early stage and advanced stage.48male apoE-/-mice and24male C57BL/6J control mice were maintained on a light-dark(LD) cycle(12h light,12h dark) for4weeks, and apoE-/-mice were divided into two groups:mice of one group were fed normal chow,mice of the other group were fed with a western type diet (containing0.15%cholesterol and21%fat). According to Zeitgeber time(ZT; ZTO is defined as lights-on time and ZT12as lights-off time), mice were sacrificed at different time points including ZTO, ZT4, ZT8, ZT10, ZT12, ZT14, ZT16and ZT20. The serum was prepared for total cholesterol, LDL-CHO and HDL-CHO detection. The aorta roots were prepared for frozen sections. Lillie-Ashburnes oil red O staining method was used to detect atherosclerotic plagues. The hearts were removed, frozen in liquid nitrogen and stored at-70℃.The expression of atherosclerosis-related genes Pai-1, t-PA, TF, ET-1and MMP-1, MMP-2, MMP-9in the hearts was detected by Real-time PCR. The results suggested that no obvious plagues were found in apoE-/-mice fed with normal chow, but foam cells were observed under the endothelium. After feeding a western type diet for4weeks, atherosclerotic lesions formed in apoE-/-mouse aortas, oil red O staining showed obvious lipid deposition in the plaques. Thus, apoE-/-mice fed normal chow and western type diet were at the early stage and advanced stage of atherosclerosis respectively. Real-time PCR showed that the expression of Pai-1, t-PA, TF and ET-1peaked between ZT14and ZT16and bottomed at ZT10in C57BL/6J mice. Their expression in apoE-/-mice fed with normal diet lost circadian rhythm. Their expression in apoE-/-mice fed with high cholesterol diet peaked at ZT4, indicating a reverse circadian rhythm. Meanwhile, the expression of MMP-1、MMP-2and MMP-9showed no obvious circadian rhythm in the hearts of C57BL/6J mice and apoE-/-mice.In summary, we are the first to report that the expression rhythms and levels of atherosclerosis-related genes changed in apoE-/-mouse hearts. These changes may play significant roles in the pathological process of atherosclerosis, and the precise role and the mechanisms are to be elucidated. |
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