Regulation of gluconeogenesis in liver, kidney and intestine by glucagon / 中国药理学通报

Aim To investigate the regulatory effect of glucagon on gluconeogenesis in liver, kidney and intes¬tine during different fasting periods and the underlying mechanism. Methods The 8-week-old male C57BIV 6J mice were randomly divided into six groups ( n = 6) :control group, control + glucagon group, fasting 18 h group, fasting 18 h + glucagon group, fasting 36 h group, and fasting 36 h + glucagon group. Glucose, triglyceride ( TG) and free fatty acids ( FFAs ) kits were used to detect their serum contents in mouse in-traperitoneal injection of glucagon at different fasting time points. Besides, liver/muscle glycogen assay kit and PAS staining were used to detect the glycogen con¬tents in liver tissue. RT-PCR method was used to observe the effects of glucagon on the gene expressions of peroxisome proliferators-activated receptor y coactivator la (PGC-1α), glucose-6-phosphatase (G6Pase) and phosphoenol pyruvate carboxykinase 1 (PEPCK) in liver, kidney and intestine of mice at different fasting time. Western blot was employed to detect the protein expressions of PGC-1α, G6Pase, PEPCK, phosphoryl-ase protein kinase A ( p-PKA) , protlein kinase A (PKA) , phosphorylase cAMp-response element binding protein (p-CREB) and cAMp-response element binding protein (CREB) in liver, kidney and intestine of mice were. Results (1) Glucagon increased the serum glucose level, reduced serum TG and FFAs levels, and reduced the hepatic glycogen content. (2) Glucagon promoted gluconeogenesis via upregulation of PGC-1α. On the stimulation of glucagon, PGC-1α gene and protein expressions in liver were significantly raised by glucagon when the mice were fasted 18 h and 36 h, while the gene and protein expressions of PGC-1α in kidney were obviously up-regulated by glucagon after fasting 18 h. However, PGC-1α gene and protein expressions in intestine were significantly elevated by glucagon at 36 h after fasting. (3 ) Glucagon induced gene and protein expressions of gluconeogenesis-related enzymes G6Pase and PEPCK in liver, kidney and intestine after fasting. (4 ) Glucagon upregulated p-PKA/PKA and p-CREB/CREB in liver. Conclusions Glucagon shows temporal difference in the gluconeo-genic response of liver, kidney and intestine in mice. Glucagon promotes the gene and protein expressions of key gluconeogenic enzymes G6Pase and PEPCK by increasing PGC-1α gene and protein expression, and thus increasing fasting blood glucose. Besides, glucagon promotes hepatic gluconeogenesis via PKA/CREB signaling pathway.

From the Origins of SARS-CoV-2 to the Correct Application of BLAST / 中国生物化学与分子生物学报

Basic local alignment search tool (BLAST) is one of the popular sequence similarity analysis tools. However, some students and researchers just blindly use the default parameters. Moreover, some students are confused about how to choose the right program. In a word, it is prone to be misused and researchers often draw conclusions incorrectly. In view of this, we traced back the internet hot topic in early 2020 - "MORDERATELY STRONG CONFIRMATION OF A LABORATORY ORIGIN OF COVID-19", and took it as teaching materials to guide the student to use BLAST currently through reanalyzing and reproducing the source of errors. Then we arranged an interesting experiment about fabricating dinosaur genes through modifying a chicken gene. In the experimental design to make the students grasp the BLAST tools better, one group fabricated the dinosaur gene and the other group decrypted the added bases. This instructional design could be conducive to cultivate students ' ability about distinguishing different viewpoints correctly, and we hope it can be enlightening and helpful to the teaching of BLAST tools.

The gene knockout of ATlaR improves insulin resistance induced by high-fat diet in rats / 中国药理学通报

Aim To explore the role of angiotensin U type 1 a reeeptor ( AT 1 aR ) , an important component of HAS, in obesity-induced insulin resistance.Methods Wild type ( WT) and ATlaR gene knockout (ATlaR ) SD rats were fed with normal diet and 60% high-fat diet for 12 weeks, respectively.After 12 weeks, blood was collected from the abdominal aorta of rats to obtain serum, and the serum insulin level was measured by ELISA.The epididvmal adipose tissue was obtained, and gene expressions of peroxisome pro- liferator-activated receptor -y ( PPAR7) and sterol reg¬ulator}' element binding protein lc (SREBP-lc) in ad¬ipose tissue were detected by RT-PCR method.The protein expressions of insulin signaling pathway and protein kinase C (PKC) in adipose tissue were detec¬ted by Western blot.Results ATI aR knockout signif¬icantly reduced HOMA-IR and improved insulin resist¬ance induced by high-fat diet.In ATlaR rats fed with high-fat, the protein expressions of insulin signa¬ling pathway were much higher than those of WT rats, indicating that ATlaR gene knockout improved the in¬sulin signaling pathway in high-fat diet.In addition, the PKCa, PKCe and PKCr| expressions of ATlaR rats were significantly lower than those of WT rats.And the gene expressions of PPAR-y and SREBP-lc, which promoted adipogenic differentiation, significantly increased in ATlaR rats fed with a high-fat diet, demonstrating that ATlaR knockout promoted adipo¬genic differentiation.Conclusions ATlaR knockout significantly improves high-fat diet induced 1R by en¬hancing protein expressions of insulin signaling path¬way, inhibiting PKC expression and promoting adipo¬genic differentiation.

Angiotensin II promotes the expression of TXNIP through angiotensin II type 1 receptor in islet β cells / 生理学报

This study investigated the effect of angiotensin II (Ang II) on apoptosis and thioredoxin-interacting protein (TXNIP) expression in INS-1 islet cells and the underlying mechanism. INS-1 cells cultured in vitro were treated with different concentration of Ang II for different time, and the viability was measured using cell counting kit-8 (CCK-8). After treatment with 1 × 10 mol/L Ang II for 24 h, flow cytometry and Western blot were used to measure the cell apoptosis, and Western blot was used to analyze the protein expression of TXNIP, carbohydrate response element-binding protein (ChREBP) and angiotensin II type 1 receptor (AT1R). Real-time PCR was used to detect TXNIP and ChREBP mRNA expression. IF/ICC was used to observe the TXNIP, ChREBP and AT1R expression. The results showed that Ang II reduced cell viability and induced the expression of TXNIP in a dose- and time-dependent manner (P < 0.05, n = 6) compared with the control group. Ang II induced apoptosis and up-regulated the expression of ChREBP and AT1R (P < 0.05, n = 6). AT1R inhibitor, telmisartan (TM), blocked Ang II-induced TXNIP and ChREBP overexpression (P < 0.05, n = 6) and inhibited Ang II-induced apoptosis. Taken together, Ang II increased ChREBP activation through AT1R, which subsequently increased TXNIP expression and promoted cell apoptosis. These findings suggest a therapeutic potential of targeting TXNIP in preventing Ang II-induced INS-1 cell apoptosis in diabetes.

Adenovirus-mediated overexpression of thioredoxin interaction protein inhibits INS-1 islet β cell proliferation / 生理学报

Diabetes can cause a significant increase in the expression of thioredoxin (Trx)-interacting protein (TXNIP), which binds to Trx and inhibits its activity. The present study was aimed to investigate the effect of TXNIP on proliferation of rat INS-1 islet β cells and the underlying mechanism. TXNIP overexpressing adenovirus vectors (Ad-TXNIP-GFP and Ad-TXNIPc247s-GFP) were constructed and used to infect INS-1 cells. Ad-TXNIPc247s-GFP vector carries a mutant C247S TXNIP gene, and its expression product (TXNIPc247s) cannot attach and inhibit Trx activity. The expression of TXNIP was detected by real-time PCR and Western blot. EdU and Ki67 methods were used to detect cell proliferation. Protein phosphorylation levels of ERK and AKT were detected by Western blot. The results showed that both TXNIP and TXNIPc247s protein overexpressions inhibited the proliferation of INS-1 cells, and the former's inhibitory effect was greater. Moreover, both of the two kinds of overexpressions inhibited the phosphorylation of ERK and AKT. These results suggest that TXNIP overexpression may inhibit the proliferation of INS-1 cells through Trx-dependent and non-Trx-dependent pathways, and the mechanism involves the inhibition of ERK and AKT phosphorylation.

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy / 中国病理生理杂志

AIM:To explore whether the angiotensin II type 1 receptor autoantibodies(AT1-AA)induces islet β-cell apoptosis and whether autophagy is involved in the process.METHODS:The INS-1 cells treated with AT1-AA at 10-6mol/L for 24 h,and then the apoptosis was analyzed by flow cytometry,Western blot and Hoechst 33258 staining.In addition,the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot.The effects of AT1-AA on the apoptosis,autophagy and viability of INS-1 cells with or without 3-methyladenine(3-MA;a com-mon autophagy inhibitor)or telmisartan(an angiotensin Ⅱ type 1 receptor blocker)pretreatment, were detected by flow cytometry,Western blot and CCK-8 assay.RESULTS: Treatment with AT1-AA at 10 -6mol/L for 24 h significantly re-duced the cell viability(P<0.05).Compared with the negative IgG control group,the apoptotic cells increased after incu-bation with AT1-AA for 12 h,24 h and 36 h,respectively(P<0.05).Moreover,the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time,and the elevation of apoptosis and autophagy were blocked by telmisartan.After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone.CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet βcells by upregulating autophagy via the angiotensin Ⅱtype 1 receptor pathway.

Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells / 生理学报

Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

Effects of Indomethacin on Proliferation and Senescence of Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of indomethacin on proliferation and senescence of leukemia cells and to analyze whether non-steroidal anti-inflammatory drugs have any adjunctively therapeutic effects on leukemia.</p><p><b>METHODS</b>Three kinds of leukemia cell lines (U266, K562 and U937) were treated with either indomethacin (final concentration 30 µmol/L) or solvent DMSO (control) for 7 days. Cell viability was determined by trypan blue, cell apoptosis and cell cycle were determined by flow cytometry, mRNA expression of hyperplastic suppression gene P21 and P27 was determined by RT-PCR, and cell senescence rate was assayed at day 0, 4, 7 after treatment.</p><p><b>RESULTS</b>After treatment with indomethacin, the cell viability in U266 and U937 groups decreased significantly, while it was not changed in K562 group. Cell cycle in U266 and U937 groups was blocked at G(2)/M phase, but the blooking effect was not found in K562 cells. The cell apoptotic rate was enhanced in 3 treated groups (P < 0.01). The mRNA expression of P21 and P27 was significantly increased after indomethacin treatment in U266 and K562 cell lines except for the P27 mRNA expression of U937. The expression levels of P21 and P27 mRNA in U266 and K562 increased obviously. The cell senescence rate in K562 and U937 group also increased.</p><p><b>CONCLUSION</b>The indomethacin possesses inhibitory effects on leukemia cells. However, its mechanisms are varied from different types of leukemia cells. It suggests that non-steroidal anti-inflammatory drugs can play a role in adjuvant therapy for leukemia, while its application would be adjusted according to different types of leukemia.</p>

Effect of adenovirus-mediated TXNIP overexpression on apoptosis and injury of H9C2 cardiomyocytes / 生理学报

Adenovirus transfection technique was used in the current study to show if thioredoxin-interacting protein (TXNIP) overexpression can induce cell apoptosis and injury in H9C2 cardiomyocytes cultured in normal glucose condition. And the mechanisms were then investigated. Briefly, H9C2 cardiomyocytes in logarithmic growth phase were randomly divided into three groups: normal cultured group, empty adenovirus vector group (Ad-eGFP) and TXNIP overexpression group (Ad-TXNIP-eGFP). All cells were cultured in DMEM containing normal concentration of glucose (5 mmol/L) and lipid. 72 h after adenovirus transfection, cells and culture mediums were collected for further assay. The results showed that Ad-eGFP and Ad-TXNIP-eGFP adenovirus transfected H9C2 cells successfully, and the transfection efficiency reached the peak at 72 h. Compared with Ad-eGFP group, Ad-TXNIP-eGFP transfection significantly increased TXNIP mRNA (P < 0.05) and protein expression level (P < 0.01). TXNIP overexpression induced remarkable cell apoptosis and injury as evidenced by increased caspase-3 activity (P < 0.05), apoptotic rate (P < 0.01) and LDH activity (P < 0.01). To further analysis the mechanisms of TXNIP-induced cell apoptosis, we also determined Trx activity, Trx related free radical injury and p38 kinase activation, which are involved in free radical induced apoptosis. The results showed that, compared with those in Ad-eGFP group, Trx activity was significantly decreased (P < 0.01), while malondialdehyde (MDA), 3-nitrotyrosine contents and p38 kinase activity were significantly increased (P < 0.01) in TXNIP overexpression group. These results suggest that TXNIP overexpression alone can induce severe apoptosis and injury in H9C2 cardiomyocytes even they are cultured in normal glucose and lipid concentration conditions. The mechanism involved is that overexpressed TXNIP can bind and inhibit Trx, impairs its antioxidative and antiapoptotic function, and then increases free radical induced injury and p38 kinase dependent apoptosis.

The change of thioredoxin system in myocardial tissue of type 2 diabetic rats undergoing myocardial injury / 生理学报

The aim of the present study is to investigate the change of thioredoxin (Trx) system in myocardial tissue of type 2 diabetic rats after myocardial injury and the underlying mechanism. Adult Sprague Dawley rats were randomly divided into two groups: normal control (NC) group and diabetes (DM) group. Rats in DM group were subjected to high-sugar, high-fat diet and streptozotocin (STZ) injection. Rats in NC group were only given normal diet and equal amount of citric acid buffer injection. At week 1, 2, 4, 12, 21 after STZ injection, plasma glucose concentration and the concentrations of insulin, creatine kinase MB (CK-MB), cardiac troponin I (cTnI) in serum were measured. Myocardial Trx and thioredoxin reductase (TR) activities, as well as caspase-3 activity, were determined by respective assay methods. Protein and mRNA levels of Trx, TR, Trx interacting protein (TXNIP) were determined by Western blot and real time PCR, respectively. The results showed that type 2 diabetic rat model was successfully established at week 1 after STZ injection, and myocardial injury was induced from week 2. Moreover, caspase-3 activity was significantly increased at week 4, 12 in diabetic rats. The activities of myocardial Trx and TR in diabetic rats was decreased from week 2, and continually aggravated as the disease developed. Compared with those in NC group, the mRNA levels of Trx1, Trx2, TR1, TR2 in DM group decreased at week 4, and then increased in week 12. In DM group, the protein levels of Trx1, Trx2, TR1 and TR2 increased significantly at week 12. The mRNA expressions of myocardial TXNIP in diabetic rats were significantly increased at week 4, 12, 24 and protein expression was increased at week 12. These results suggest diabetes can decrease myocardial Trx, TR activity, inducing myocardial cell apoptosis and heart injury. The inhibitory effect of diabetes is mainly associated with TXNIP up-regulation and Trx nitration.

Detection of serum autoantibodies against AT₁A-receptor during the development of the four types of hypertensive rat models / 生理学报

Using two-kidney one-clip renal hypertensive (2K1C group), stress-induced hypertensive (neural group), DOCA-salt treated hypertensive (DOCA group) and spontaneously hypertensive rats (SHR group), to investigate the change in AT(1A)-receptor autoantibodies (AT(1A)-AAs) during the development of the four types of hypertension. The biological activities of AT(1A)-AAs were examined. It was shown that the frequency of occurrence and titres of AT(1A)-AAs increased significantly during the development of hypertension. In the four hypertensive groups studied, the occurrence of AT(1A)-AAs was most prominent in SHR, 2K1C and neural groups. The biological effects of AT(1A)-AAs were shown to increase the beating frequency of cultured neonatal myocardial and vascular contractile tension. It is suggested that autoimmune mechanisms are involved the pathogenesis of different types of hypertension and the AT(1A)-AAs may be one of the mechanisms leading to cardiac hypertrophy.

Comparative studies of vasodilating effects of angiotensin-(1-7) on the different vessels / 生理学报

The purpose of this study was to compare the vasodilating effects of angiotensin-(1-7) [Ang-(1-7)] on the different vessels and to clarify its mechanisms by using relaxing responses of preconstricted vascular rings. The results showed: (1) Ang-(1-7) dose-dependently induced vasorelaxation in all the vessels studied. However, there is apparent heterogeneity in the responsiveness of vessels from different origin. (2) The Ang-(1-7)-induced vasorelaxation was endothelium dependent and largely mediated by NO system. (3) The vasodilator action of Ang-(1-7) was not mediated by AT1 or AT2 receptor subtypes. It is suggested that the Ang-(1-7)-induced vasorelaxation is endothelium dependent by some other unclarified angiotensin receptor subtypes and is largely mediated by NO system.

Effect of losartan on produce of sera autoantibodies to angiotensin II-1 receptor in renovascular hypertension rats / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>The effects of losartan (after operation 2 week to 10 week, 5 mg/kg d ig) on generation of AT1R-AA in sera were observed during development of hypertension in rats. The renovascular hypertension (RVH) model was established by two-kidney one-clip method, a synthetic peptide corresponding to amino acid sequence 165-191 of the second extracellular loop of the angiotensin II-1 receptor (AT1R) was used as antigen, SA-ELISA were used to examine sera AT1R autoantibody (AT1R-AA).</p><p><b>RESULTS</b>The frequencies and titres of AT1R-AA after operation one week rats were significantly increased (P < 0.05). The treatment with losartan not only inhibited structural and functional changes, but also the frequencies and titres of AT1R-AA was significantly lower (P < 0.05) than RVH group.</p><p><b>CONCLUSION</b>It is suggested that the losartan significantly inhibits generation of the AT1R-AA.</p>

Changes in autoantibody against cardiovascular AT1-receptor during development of renovascular hypertension in rats / 生理学报

The aim of this study was to observe the change in angiotensin II receptor subtype 1 (AT(1)) autoantibody during the development of renovascular hypertension (RVH). The Goldblatt renovascular hypertension model was established by the two-kidney one-clip method, and a synthetic peptide corresponding to amino acid sequence 165-191 of the second extracellular loop of the AT(1)-receptor was used as the antigen. Sera AT(1)-receptor autoantibody was detected by SA-ELISA. It was shown that two weeks after operation both the frequency of occurrence and the titre of autoantibodies to AT(1)-receptor were significantly increased as compared with the pre-treatment control. The increase in autoantibodies lasted several weeks and then decreased gradually to the pre-clipping level at 12 weeks. It is suggested that autoimmune mechanisms are involved in the pathogenesis of renovascular hypertension and the AT(1) autoantibodies may be one of the mechanisms leading to cardiac hypertrophy.