糖尿病肾病大鼠肾小球叉头状转录因子O1的表达和作用

目的观察糖尿病肾病大鼠肾小球叉头状转录因子O1（FoxO1）的表达,探讨其与糖尿病肾病发生、发展的关系。方法8周龄健康雄性sD大鼠30只,链脲佐菌素（STZ）诱导建立糖尿病大鼠模型,采用随机数字表法分为糖尿病组（13只,普通饲料连续喂养4周）和糖尿病肾病组（13只,普通饲料连续喂养12周）。选取健康同龄大鼠18只作为正常对照组（NC组）。4、12周末检测体质量（BW）、血糖（BG）、血肌酐（Scr）、尿素氮（BUN）、24h尿蛋白（UPro／24h）及尿白蛋白定量（UAIb）。处死动物后计算肾重指数（KI）;分光光度计测定大鼠肾皮质丙二醛（MDA）含量和总超氧化物歧化酶（SOD）活性;免疫组化法检测肾小球FoxO1蛋白、胶原Ⅳ及纤连蛋白水平;RT—PCR检测肾皮质Fox01mRNA水平;光镜及电镜下观察肾脏组织形态学变化。组间比较采用独立样本t检验。结果糖尿病肾病组肾皮质MDA蛋白、肾小球胶原Ⅳ和纤连蛋白水平显著高于糖尿病组[分别为（3．49±0．31）VS（2．34±0．28）nmol／mgProt,20．1±1．3VS10．1±1．0,10．6±1．3VS6．3±1．0,t值分别为9．290、20．967、9．119,均P〈0．05];糖尿病肾病组肾皮质SOD活性蛋白、Fox01mRNA表达水平明显低于糖尿病组[分别为（23±8）VS（43±6）U／mgProt,0．20±0．06VS0．35±0．05,t值分别为7．069、6．717,均P〈0．05]。FoxO1蛋白表达水平各组问比较无显著差异（均P〉0．05）。结论糖尿病肾病大鼠肾皮质FoxO1 mRNA表达水平降低,其机制可能通过下调其抗氧化靶基因使。肾脏氧化应激反应增强,从而参与糖尿病肾病发生发展的过程。     

Sources of endogenous glucose production in the Goto-Kakizaki diabetic rat

Plasma glucose, insulin and glucose tolerance were quantified in diabetic Goto-Kakizaki (GK) rats (342+/-45 g, n = 5) and compared with weight-matched non-diabetic Wistars (307+/-30 g, n = 8). Compared to Wistars, GK rats had higher fasting plasma insulin (219+/-50 versus 44+/-14 pmol/l, P<0.002) and glucose (9.2+/-2.3 versus 5.5+/-0.5 mmol/l, P<0.025). GK rats showed impaired glucose tolerance (IPGTT 2 h plasma glucose=14+/-1.5 versus 6.4+/-0.1 mmol/l, P<0.001). Endogenous glucose production (EGP) from glycogenolysis, phosphoenolpyruvate (PEP) and glycerol after 6 hours of fasting was quantified by a primed infusion of [U-(13)C]glucose and (2)H(2)O tracers and (2)H/(13)C NMR analysis of plasma glucose. EGP was higher in GK compared to Wistar rats (191+/-16 versus 104+/-27 mumol/kg per min, P<0.005). This was sustained by increased gluconeogenesis from PEP (85+/-12 versus 35+/-4 mumol/kg per min, P<0.02). Gluconeogenesis from glycerol was not different (20+/-3 in Wistar versus 30+/-6 mumol/kg per min for GK), and glycogenolysis fluxes were also not significantly different (76+/-23 mumol/kg per min for GK versus 52+/-19 mumol/kg per min for Wistar). The Cori cycle accounted for most of PEP gluconeogenesis in both Wistar and GK rats (85+/-15% and 77+/-10%, respectively). Therefore, increased gluconeogenesis in GK rats is largely sustained by increased Cori cycling while the maintenance of glycogenolysis indicates a failure in hepatic autoregulation of EGP.     

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.     

Antihyperglycemic and blood pressure-reducing effects of stevioside in the diabetic Goto-Kakizaki rat

Stevioside, a glycoside present in the leaves of the plant, Stevia rebaudiana Bertoni (SrB), has acute insulinotropic effects in vitro. Its potential antihyperglycemic and blood pressure-lowering effects were examined in a long-term study in the type 2 diabetic Goto-Kakizaki (GK) rat. Rats were fed 0.025 g x kg(-1) x d(-1) of stevioside (purity > 99.6%) for 6 weeks. An intra-arterial catheter was inserted into the rats after 5 weeks, and conscious rats were subjected to arterial glucose tolerance test (2.0 g x kg(-1)) during week 6. Stevioside had an antihyperglycemic effect (incremental area under the glucose response curve [IAUC]): 985 +/- 20 (stevioside) versus 1,575 +/- 21 (control) mmol/L x 180 minutes, (P <.05), it enhanced the first-phase insulin response (IAUC: 343 +/- 33 [stevioside] v 136 +/- 24 [control] microU/mL insulin x 30 minutes, P <.05) and concomitantly suppressed the glucagon levels (total AUC: 2,026 +/- 234 [stevioside] v 3,535 +/- 282 [control] pg/mL x 180 minutes, P <.05). In addition, stevioside caused a pronounced suppression of both the systolic (135 +/- 2 v 153 +/- 5 mm Hg; P <.001) and the diastolic blood pressure (74 +/- 1 v 83 +/- 1 mm Hg; P <.001). Bolus injections of stevioside (0.025 g x kg(-1)) did not induce hypoglycemia. Stevioside augmented the insulin content in the beta-cell line, INS-1. Stevioside may increase the insulin secretion, in part, by induction of genes involved in glycolysis. It may also improve the nutrient-sensing mechanisms, increase cytosolic long-chain fatty acyl-coenzyme A (CoA), and downregulate phosphodiesterase 1 (PDE1) estimated by the microarray gene chip technology. In conclusion, stevioside enjoys a dual positive effect by acting as an antihyperglycemic and a blood pressure-lowering substance; effects that may have therapeutic potential in the treatment of type 2 diabetes and the metabolic syndrome.     

Effect of phospholipid methylation on beta-adrenergic receptors in the normal and hypertrophied rat myocardium

Abdominal aortic constriction in rats results in mild cardiac hypertrophy (20% increase in left ventricular weight compared to sham-operated controls) which is associated with increased numbers of beta-adrenergic receptors (123 +/- 7.3 fmol/mg protein (mean +/- SE) vs. 87 +/- 4.3 fmol/mg in controls, P < 0.01) without changes in their affinities for dihydroalprenolol. In vitro synthesis of phosphatidylcholine through successive methylation of phosphatidylethanolamine by S-adenosyl-L-methionine is enhanced in the hypertrophied myocardium) 0.38 +/- 0.03 nmol/mg per 30 minutes vs. 0.23 +/- 0.03 nmol/mg per 30 minutes in controls, P < 0.01). In both experimental groups, methyltransferase activity has a high affinity for S-adenosyl-L-methionine (Km = 6.8 microM), depends on Mg2+, is optimal around pH 9.0, and is inhibited by S-adenosyl-L-homocysteine (ki = 8.3 microM). The possible relationship between phospholipid methylation and changes in myocardial beta-adrenergic receptors was studied in both normal and hypertrophied hearts. Preincubation of cardiac membranes with S-adenosyl-L-methionine increased the numbers of b eta-adrenergic receptors in proportion to the duration of incubation and the concentration of S-adenosyl-L-methionine. In both groups, S-adenosyl-homocysteine, but not 5'-AMP or L-methionine, attenuated the increase in beta-adrenoreceptors. These results indicate that phospholipid methylation may be an important mechanism for regulation of beta-adrenergic mechanisms in both normal and hypertrophied myocardium.     

叉头状转录因子O1对糖尿病大鼠足细胞影响的实验研究

目的 探讨叉头状转录因子O1（Fox01）对糖尿病大鼠足细胞的影响.方法 链脲佐菌素（STZ）诱导构建糖尿病大鼠模型90只,并采用简单随机抽样法分为糖尿病（DM）＋空慢病毒载体（LV-pSC-GFP）感染组（LV-NC组,n=30）,DM＋大鼠结构性活性FoxO1慢病毒载体（LV-CA-FoxO1）感染组（LV-CA组,n=30）,DM组（n=30）,另设对照组（NG组,n=30）注射相应体积的柠檬酸钠-柠檬酸缓冲液.于慢病毒感染后的2、4、8周末,测大鼠尿白蛋白、体重、血糖、血肌酐、尿素氮,光镜和透射电镜观察肾小球及其足细胞结构变化,实时定量聚合酶链反应（RT-PCR）和Western blotting法检测大鼠肾皮质中FoxO1、足盂蛋白（PCX）、nephrin mRNA和蛋白的表达.多组间比较采用单因素方差分析.结果 与LV-NC、DM组相比,LV-CA组大鼠肾脏中FoxO1 mRNA、蛋白表达水平明显升高（F8W值分别为10 919.75、3 867.34,均P＜0.05）,尿白蛋白、血肌酐、尿素氮明显降低（2周除外）（F8W值分别为132.72、187.68、503.69,均P＜0.05）,肾脏中PCX、nephrin mRNA和蛋白水平明显升高（mRNA F8w值分别为778.94、478.10;蛋白F8W值分别为393.64、255.79,均P＜0.05）,肾脏病理学变化也明显改善,可见肾小球体积减小,系膜细胞及基底膜增生程度降低,足突融合也有一定程度改善.结论 通过靶向注射慢病毒载体来上调FoxO1的表达可改善DM大鼠足细胞的损伤.     

The behavior of some enzymes of the hypertrophied and postnatally developing myocardium of the rat

Summary The present study was undertaken in order to investigate the behavior of lactate dehydrogenase (LDH) and hexokinase (HK) in the mechanically overloaded and the postnatally developing left ventricle of the rat.In Goldblatt rats (GBR) 2 to 4 months after operation total LDH activity was decreased by 7%, accompanied by an isoenzyme shift towards a higher M proportion. Swimming rats (SR) showed an 11% increase in LDH activity. HK activity was increased by 14% in GBR and unaltered in SR.Histochemical investigations revealed no indication of heterogeneous alterations in various areas of the myocardium.During postnatal development, a gradual increase of activity and H subunit proportion of LDH were observed. HK activity was increased after one week, but underwent a slow decrease thereafter. These changes are interpreted as processes of adaptation to the conditions of extrauterine life.

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Das Verhalten einiger Enzyme im Rattenmyokard bei Hypertrophie und während der postnatalen Entwicklung

Zusammenfassung Aktivitätsveränderungen der Lactatdehydrogenase (LDH) und der Hexokinase (HK) im linken Ventrikel der Ratte bei chronischer mechanischer Mehrbelastung und während der postnatalen Entwicklung waren Gegenstand der vorliegenden Untersuchung. Goldblattratten (GBR) zeigten 2 bis 4 Monte nach der Operation eine Abnahme der LDH-Aktivität um 7%, eine Zunahme des Anteils der M-Untereinheiten an der Gesamtaktivität um 13% sowie eine um 14% erhöhte HK-Aktivität. Schwimmratten (SR) hingegen wiesen nach 2- bis 4monatigem Training eine um 11% gesteigerte LDH-Aktivität auf, während die Aktivität der HK unverändert blieb. Histochemische Untersuchungen ergaben keinerlei Hinweise auf uneinheitliche Veränderungen in einzelnen Myokardarealen.Im Verlaufe der postnatalen Entwicklung wurde ein Anstieg der LDH-Aktivität und des relativen Anteils der H-Untereinheiten an dieser festgestellt. Die HK-Aktivität stieg zunächst an, um anschließend wieder allmählich abzufallen. Diese Veränderungen werden als Ausdruck von Anpassungsvorgängen an die extrauterinen Lebensbedingungen interpretiert.

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Paper, presented at the Erwin Riesch Symposium, Tübingen, April 3–7, 1979

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With 3 figures

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A part of this paper is taken from the unpublished doctoral thesis ofU. Koehler.     

Genetic lesions in MYC and STAT3 drive oncogenic transcription factor overexpression in plasmablastic lymphoma

The mutational profile of plasmablastic lymphoma has not been described. We performed a targeted, exonic next-generation sequencing analysis of 30 plasmablastic lymphoma cases with a Bcell lymphoma-dedicated panel and fluorescence in situ hybridization for the detection of MYC rearrangements. Complete phenotyping of the neoplastic and microenvironmental cell populations was also performed. We identified an enrichment in recurrent genetic events in MYC (69% with MYC translocation or amplification and three cases with missense point mutations), PRDM1/Blimp1 and STAT3 mutations. These gene mutations were more frequent in Epstein-Barr virus (EBV)-positive disease. Other genetic events included mutations in BRAF, EP300, BCR (CD79A and CD79B), NOTCH pathway (NOTCH2, NOTCH1 and SGK1) and MYD88pL265P. Immunohistochemical analysis showed consistent MYC expression, which was higher in cases with MYC rearrangements, together with phospho-STAT3 (Tyr705) overexpression in cases with STAT3 SH2 domain mutations. Microenvironmental cell populations were heterogeneous and unrelated to EBV, with enrichment of tumor-associated macrophages (TAM) and PD1-positive T cells. PD-L1 was expressed in all cases in the TAM population but only in the neoplastic cells in five cases (4 of 14 EBV-positive cases). HLA expression was absent in the majority of cases of plasmablastic lymphoma. In summary, the mutational profile of plasmablastic lymphoma is heterogeneous and related to EBV infection. Genetic events in MYC, STAT3 and PRDM1/Blimp1 are more frequent in EBV-positive disease. An enrichment in TAM and PD1 reactive T lymphocytes is found in the microenvironment of plasmablastic lymphoma and a fraction of the neoplastic cells express PD-L1.     

Stage-specific differential activation of mitogen-activated protein kinases in hypertrophied and failing rat hearts

Mitogen-activated protein kinases (MAPKs) are involved in the early development of cardiac hypertrophy, but their roles in chronic left ventricular hypertrophy (LVH) are unclear. We studied the angiotensin (Ang) II-induced cardiac MAPK activation of the hypertensive Dahl salt-sensitive (DS) rats in the subacute developing LVH stage, the chronic compensated LVH stage, and the congestive heart failure (CHF) stage. In the isolated, coronary-perfused heart preparation, Ang II infusion (1x10(-6)mol/l) activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38-MAPK in the LV myocardium. No substantial differences were observed in the Ang II-induced ERK activation between the normotensive control DS rats and the hypertensive DS rats in either stage. In contrast, the Ang II-induced activation of JNK and p38-MAPK was augmented in the subacute LVH stage of the hypertensive DS rats, but then progressively attenuated in the chronic LVH and CHF stages. Chronic treatment with an angiotensin converting enzyme inhibitor, temocapril (20 mg/kg/day), ameliorated the responsiveness of the JNK/p38-MAPK activation, suggesting that the decreased JNK/p38-MAPK activation is a consequence of negative feedback regulation for the activated cardiac renin-angiotensin system in chronic LVH and CHF. Thus, the Ang II-induced activation of multiple cardiac MAPK pathways are differentially regulated, depending on the stages of chronic hypertrophic process. The JNK and p38-MAPK activation may be involved in the early development of adaptive LVH. However, the responsiveness of the cardiac JNK/p38-MAPK pathways progressively decreased in chronic LVH and CHF under the chronic activation of tissue renin-angiotensin system.