SIK1在糖尿病肾病大鼠肾小球早期病变中的作用及机制研究
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摘要
[目的]
糖尿病肾病(DN)是糖尿病慢性并发症之一,也是导致终末期肾病的一个主要原因,在肾小球发生的细胞外基质(extracellular matrix, ECM)积聚等的纤维样病变提示了糖尿病肾病的发生和发展。在这一过程中,转化生长因子β1(Transforming growth factors-β1, TGF-β1)通过TGF-β1型受体即活化素受体样激酶5(the type Ⅰ activin receptor-like kinase (ALK)5, ALK5)进行信号转导,进而进一步激活ECM的主要成分纤连蛋白(fibronectin, FN)和可以诱导ECM降解的纤溶酶原激活物抑制剂1(plasminogen activator inhibitor type1, PAI-1)的表达,导致ECM的积聚和降解的减少。研究表明选择性抑制ALK5对与肾小球纤维化的这种慢性肾脏疾病是一种潜在治疗方法。盐诱导激酶1(Salt Inducible Kinase1, SIK1)是一种丝氨酸/苏氨酸激酶,SIK1可以与抑制型Smad7形成复合体并下调ALK5的表达而被认为是TGF-β的信号通路中的一个负向调控因子。虽然动物实验发现SIK1在大鼠肾脏高表达,但SIK1在糖尿病肾脏中表达和肾小球纤维化病变之间的关系还未完全阐明,而高糖是否影响SIK1表达也鲜有报道。
因此,本研究应用大鼠糖尿病模型和体外培养的大鼠肾系膜细胞,构建鼠SIK1基因的真核表达载体,从整体、细胞和分子水平,系统研究了糖尿病肾脏及高糖环境中系膜细胞SIK1蛋白表达情况及对ALK5信号通路的调控。
[方法]
1.SIK1在糖尿病肾病大鼠肾脏的表达和意义
将SPF级8周龄wistar大鼠随机分为正常对照组(normal组,10只)和糖尿病组(diabetic组,20只)。糖尿病模型采用腹腔注射链脲佐菌素(streptozotocin, STZ,溶于pH值4.5的0.1mol/L枸橼酸盐缓冲液中,60mg/kg),对照组只注射相当体积的枸橼酸盐缓冲液,72h后尾尖取血,血糖≥16.7mmol/L者确定为1型糖尿病模型。于12和16周龄糖尿病模型组取5只大鼠,乙醚麻醉大鼠,眼球内眦取血,离心分离血清,用于测定血糖；称重后于腹腔注射65mg/kg戊巴比妥钠麻醉,分离肾脏。于20周龄,将各组大鼠于代谢笼中,监测24h尿量,尿白蛋白及尿肌酐(Ucr)水平,并计算内生肌酐清除率(Ccr)和尿白蛋白排泄率(UAER)。麻醉取血,分离血清测定血糖,血肌酐及尿素氮水平。摘取肾脏,称重,计算肾脏指数。取部分肾组织置于4%多聚甲醛固定,用于HE, PAS染色观察其病理变化情况。
取肾组织制备切片进行免疫组化。分别提取肾组织及肾小球组织蛋白,应用免疫沉淀及Western blot方法以及观察糖尿病大鼠肾脏SIK1蛋白表达及磷酸化水平的影响。并应用Western blot方法观察20周龄大鼠肾小球ALK、FN. PAI-1表达情况,Masson染色观察20周龄大鼠肾小球胶原纤维及ECM聚集情况。
2.高糖刺激对系膜细胞SIK1的表达、活性、细胞内分布及ALK5信号通路的影响,SIK1表达质粒对高糖刺激下的系膜细胞ALK5信号通路及系膜细胞增殖的影响
大鼠肾系膜细胞HBZY-1细胞以含有10%胎牛血清、100KU/L青霉素及100mg/L链霉素的DMEM培养基培养,用高糖(30mmol/L, High glucose, HG)制备模型,于刺激后0、12、24和48h收集细胞,免疫沉淀、Western blot及半定量RT-PCR分别检测SIK1表达及磷酸化水平。构建pEGFP-SIK1表达质粒,激光共聚焦观察高糖刺激下细胞内生性及转染pEGFP空质粒及pEGFP-SIK1表达质粒后SIKl细胞内分布及核转运情况。Western blot及免疫细胞化学观察高糖对HBZY-1细胞ALK5信号通路的影响。
设计和构建针对SIK1基因的表达质粒,将对数生长期细胞接种于6孔培养板中,同步化后将细胞随机分为3组,空白细胞对照组、pCDF1空质粒对照组和pCDF1-SIK1质粒转染组,24h后细胞长至70-80%时开始转染,转染采用阳离子脂质体法。48h后荧光显微镜观察转染效率,终止培养进行蛋白、RNA提取。Western blot及半定量RT-PCR检测细胞内SIK-1蛋白和mRNA的表达。对各组转染成功细胞以高糖继续培养48h,终止培养后以Western blot及细胞免疫组化检测SIK1、ALK5、FN及PAI-1表达,MTT法检测细胞增殖情况。
[结果]
1.SIK1在糖尿病肾病大鼠肾脏的表达和意义
(1) wistar大鼠腹腔注射STZ成功建立1型糖尿病模型。与正常对照组比较,模型组表现出多饮、多食及体重减轻的特点。20周龄时,模型组餐后血糖水平升高,但血甘油三酯及胆固醇水平正常。与正常对照组相比,模型组出现糖尿病肾病的明显特征,各肾功能指标均表现出显著性差异。糖尿病模型组24h尿量、肾体重比、尿白蛋白分泌率、血肌酐,肌酐清除率,血尿素氮指标水平均明显升高,并出现肾小球肥大、纤维化,基底膜增厚等病理改变,肾小球面积及系膜增殖指数明显增大。
(2)免疫组织化学检测发现,SIK1在正常大鼠肾脏的肾小球及远曲小管表达明显,在近曲小管表达较少,且在各细胞的胞浆无着色,胞核无着色。在糖尿病模型组大鼠中,SIKl在肾小管的表达与正常对照组一致。而在肾小球的随病程月数的增加,SIK1的表达依次减少,呈时间依赖性。Western blot检测发现,在糖尿病大鼠肾组织SIK1的表达在三个时间点无明显变化,与正常对照组大鼠20周龄一致。而肾组织SIK1磷酸化水平在三个时间点却依次减少,且均明显低于正常对照组大鼠20周龄肾组织SIK1磷酸化水平。分离肾小球提取蛋白发现,糖尿病大鼠肾小球SIK1的表达在三个时间点表达却依次减少,均明显低于正常对照组大鼠20周龄肾小球SIK1的表达。
(3)与正常对照组大鼠相比,Western blot检测发现20周龄糖尿病大鼠肾小球ALK5、FN、PAI-1表达明显增高,Masson染色观察发现糖尿病大鼠肾小球肾小球胶原纤维及ECM明显聚集。
2.高糖刺激对系膜细胞SIKl的表达、活性、细胞内分布及ALK5信号通路的影响,SIK1表达质粒对高糖刺激下的系膜细胞ALK5信号通路及系膜细胞增殖的影响
(1)高糖刺激后0、12、24、48h,经免疫沉淀及Western blot检测发现,SIK1的表达及磷酸化水平依次递减,呈时间依赖性,有统计学意义。细胞免疫荧光后激光共聚焦观察发现SIK1在HBZY-1细胞的核内外均匀表达,高糖刺激下表达减少并向细胞内转运。转染pEGFP-SIKl表达质粒后,激光共聚焦观察发现GFP荧光信号在高糖刺激下由核外向核内转移。
(2)高糖刺激后0、12、24、48h,经Western blot检测发现,ALK5、FN、PAI-1表达水平依次递增,呈时间依赖性,有统计学意义。相对于高糖刺激后0h,细胞免疫组化发现在高糖刺激48h后FN、PAI-1表达水平明显增高。
(3)经酶切鉴定和测序分析,质粒符合设计要求,命名为pCDF1-SIK1。转染HBZY-1细胞24h后荧光倒置显微镜可见绿色荧光蛋白表达,48h表达量达60%以上。相对于正常对照组及pCDFl空质粒对照组,半定量RT-PCR及Western blot检测提示pCDFl-SIK组mRNA水平分别增加1.81和1.86倍,蛋白水平分别增加1.81和2.06,继续高糖刺激48h后,Western blot检测提示pCDF1-SIK组ALK5蛋白水平降低37.5%和39%,FN、PAI-1表达水平亦明显下降,并且MTT检测提示pCDFl-SIK可明显抑制高糖对HBZY-1细胞的增殖作用。
[结论]
(1) Wistar鼠腹腔注射STZ建立的1型糖尿病模型,在20周龄时出现糖尿病肾病的明显特征有,明显的ECM聚集；(2)SIK1主要在正常大鼠肾脏的远曲小管和肾小球表达明显,在近曲小管表达较少。随着糖尿病病程进展,肾脏SIK1表达无明显变化,但肾脏SIK1磷酸化水平及肾小球SIK1的表达逐渐减少。而在20周龄时肾小球ALK5,FN及PAI-1表达明显,提示SIK1在肾小球表达减少可能参与肾小球纤维化早期病变的发生和发展；(3)在细胞模型上,高糖可抑制SIK1的182位点磷酸化水平及表达水平,导致SIK1向细胞核内转运。同时高糖激活ALK5信号通路,提示SIK1的182位点磷酸化的抑制是高糖下调SIK1的主要原因,受抑制的SIK1可能导致ALK5信号通路的激活,进而导致细胞内的ECM聚集和细胞增殖。(4)重组质粒pCDF1-SIK能够激活体外培养的大鼠系膜细胞SIK1蛋白表达,下调ALK5信号通路,减少细胞内ECM聚集,抑制高糖诱导的细胞增殖。
(Objective]
Diabetic nephropathy is one of the most important causes of end-stage renal disease. Increasing evidence has shown that diabetic nephropathy in experimental animals, as well as in diabetic patients, develop fibrotic changes such as extracellular matrix (ECM) accumulation in glomeruli, which manifest as overt nephropathy. Transforming growth factors-β (TGF-β) plays a central role in the progression of glomerular fibrosis and transduces its signal through the type I activin receptor-like kinase (ALK)5, and then specifically induces expression of fibronectin (FN), the plasminogen activator inhibitor type1(PAI-1), and ECM accumulations. Several studies have shown that selective inhibit ALK5could be a potential therapy for chronic renal disease. Salt-inducible kinase1(SIKl), a serine/threonine protein kinase, is originally found in the adrenal glands of rats fed a high salt diet. SIK1belongs to a family of AMP-activated protein kinase (AMPK) due to the acid sequence closely related to catalytic a subunit of AMPK. Topical study also has shown that SIKl is identified as an inducible gene target of TGF-β/Smad signaling, and that SIK1forms complexes with the ALK5, the inhibitory Smad7and Smurf2to down-regulate ALK5as well as negative regulation of TGF-β signaling pathway. Although animal experiment reported that SIK mRNA is found in the highest levels in rat kidney, the specific role of SIK1in the kidney, especially in the glomeruli, of diabetic rats have not been evaluated yet, and there was limited knowledge concerning the specific rule of SIK1which involved in glucose metabolism at cellular level.
Therefore, in the current study, we assessed the possible role of SIK1in glomeruli of Streptozotocin (STZ)-induced type1diabetic rat. We were also using rat glomerular mesangial cells line (HBZY-1cells) to examine the modulation of SIK1protein under the condition of high glucose. Furthermore, we have constructed two specific SIK1expression plasmid and transfected them into HBZY-1cells respectively to explore whether SIK1play a beneficial role during mesangial cell fibrogenesis by regulating ALK5signaling pathway.
[Methods]
1.The expression of SIK1in the kidney of STZ-induced diabetic rats.
Thirty male Specific-pathogen-free (SPF) Wistar rats were housed in temperature (23±3℃)and humidity-controlled (60±5%) room with a12:12-hour light-dark cycle and fed with the standard diet. After1week adaptation, rats aged8weeks were randomized into2groups, ten rats were chosen randomly as normal group and the rest twenty rats as a model of diabetic mellitus. The STZ-induced rats were injected intraperitoneally with60mg/kg body weight STZ in50mM sodium citrate solution (pH4.5) to make type1diabetic model, meanwhile, rats in the normal group were injected with50mM sodium citrate solution. Type1diabetic were defined by fasting serum glucose level>16.7Mm after72h of STZ injection. All animals were given with equal volume distilled water every day. At the age of12,16weeks, diabetic rats were fasted for12h and anesthetized with intraperitoneal sodium pentobarbital (65mg/kg), its blood was then collected and its kidneys excised. After12weeks of administration, each last rat aged20was placed in a metabolic cage for24-h urine collection, then rats were weighed and sacrificed, the left kidney was removed and weighed. Glomeruli were isolated from rat kidneys by a modified procedure. Serum glucose was measured by an automatic biochemical analyzer. Urea and creatinine (Cr) were analyzed using enzymatic kits. Urinary microalbumin was measured by immunoturbidimetric assay using mALB kit. Albumin excretion rate (AER) was assessed as an index for kidney injury.
Renal tissue samples for light microscopy were fixed in4%buffered paraformaldehyde,4-um-thick paraffin-embedded samples were used and Routine Hematoxylin-eosin (HE) staining and periodic acid Schiff (PAS) staining was performed, the glomerular tuft area and mesangial matrix index were measured. The lysates extracted from kidney and glomeruli tissues were used for Western blot and immunoprecipitation to examine the expression of SIK1and pT186-SIKl. The expression ALK5, FN and PAI-1expression were also examined of rats at the age of20weeks. Meanwhile, Kidney glomerular fibrosis is predominantly featured as the production of extracellular matrix (ECM) and Masson staining was used to observe the accumulation of ECM.
2. High glucose significantly suppresses the expression and activity of SIK1, regulates the nuclear redistribution of SIK1and induces activation of the ALK5signaling pathway in HBZY-1cells, Overexpression of SIK1in HBZY-1cells resulted in downregulation of ALK5and its target gene FN and PAI-1, and reversed the high glucose-induced HBZY-1cellular overproliferation.
HBZY-1cells, a rat glomerular mesangial cells line, were maintained at37℃in a humidified atmosphere of5%CO2and cultured in Dulbecco's modified Eagle's medium supplemented with10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin. Cells were grown to-80%confluence and then incubated in serum-free medium for24h. To investigate the effects of glucose on protein expression of SIK1and pT186-SIK1, serum-deprived HBZY-1cells were cultured in DMEM containing30mM glucose (high glucose) for0,12,24and48h. At the end of each time, SIK1, pT186-SIK1, ALK5, FN and PAI-1expression were examined. In order to examine the direct effects of high glucose on the specific cellular localization of SIK1, HBZY-1cells were transfected with expression vectors for GFP-fused full-length SIK1(pEGFP-SIK1) and cultured with high glucose in glass-bottomed culture dishes for0and48h, and then observed and imaged by a Olympus confocal microscope. To examine SIK1negatively regulate ALK5signaling pathway, HBZY-1cells were randomly divided into three groups:untransfection normal group, empty vector control group and pCDFl-SIKl group. At48h after transfection, HBZY-1cells were collected and analyzed for SIK1expression, and were continuing cultured with high glucose for further48h to analyze SIK1, ALK5, FN and PAI-1expression by Western blot and immunocytochemistry analyses, MTT assays for HBZY-1cellular overproliferation analysis. Transient transfection of HBZY-1cells was carried out using Lipofectamine2000according to the manufacturer's instruction. At48h after transfection, fluorescent microscopy was used to examine GFP expression and then the cells were collected for the extraction of protein.
[Results]
Part1
(1). Twelve weeks after STZ injection, serum blood glucose level and systolic blood pressure were significantly higher in the type1diabetic group than in the normal group at the age of20weeks. The body weight of the rats in the diabetic groups was reduced in comparison with the normal group significantly. On the contrary, the ratio of kidney-to-body weight (KW/BW) was significantly higher in diabetic groups than in the normal group. The urine volume, AER, Cr, Ccr and Urea in the diabetic group were significantly increased compared with the normal group, indicating a damaged renal function in diabetic rats. Twelve wks after STZ injection, based on the results of HE and PAS staining, diabetic rats exhibited larger glomerular tuft area and an increased mesangial matrix index by two-fold compared with rats in normal group. Kidneys of the type1diabetic rats exhibited profound extracellular matrix deposition inside glomeruli. These indicating the pathological abnormalities were noteworthy in kidney of diabetic rats.
(2). The expression of SIK1protein was investigated by immunohistochemistry and result showed that expression of the endogenous SIK1was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. To our surprise, gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. To further prove this finding and to investigate the activity changes of SIK1during diabetic nephropathy, we explored kidney SIK1protein expression and SIK1activity (Thr-182phosphorylation, pT182) by immunoprecipitation and Western blot. We found that there was no significant difference of the kidney SIK1expression among diabetic rats at the age of12,16and20weeks and normal rats at the age of20weeks. Nevertheless, SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Activity of SIK1(pT182), with the diabetic state extending, was decrease rapidly in kidney from diabetic rats. Without any other process, Glomeruli-enriched tissues isolated from rat kidneys were lysed for Western blot analysis immediately. We found that the level of SIK1protein expression in the glomeruli isolated from kidneys of diabetic rat at the age of12,16and20weeks was significantly decreased than that of normal rats at the age of20weeks, glomerular SIK1expression of diabetic rat at the age of16and20weeks also markedly decreased than that of diabetic rat at the age of12weeks, but there was a negligible difference between diabetic rat at the age of16and20weeks.
(3). To clarify the possible relationship between SIK1and ALK5signaling pathway under the state of diabetic glomerulopathy, Western blot analysis of ALK5, FN and PAI-1in glomeruli was performed. Of diabetes rats at the age of20weeks, as expected, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks, in agreement with this, our further Masson staining showing the increased accumulation of glomerular ECM of diabetic rats compared with normal rats at the age of20weeks.
Part2
(1). HBZY-1cells were stimulated with high glucose, and the activity resulting from Thr-182phosphorylation of the SIK1gene was monitored. The HBZY-1cells were cultured in media containing30mM glucose for0,12,24, or48h. Immunoprecipitation followed by Western blot analysis showed that SIK1activity decreased after glucose treatment in a time-dependent manner. The levels of Thr-182phosphorylation at12,24, and48h were24.8%,58%, and58.4%, respectively, compared to0h. The level of phospho-Thr-182is important to maintain the SIK1protein level, which was also true in the HBZY-1cells treated with high glucose. The density ratios of SIKl/β-actin at12,24, and48h were18.2%,61.2%, and72%, respectively, of that at0h. Further semi-quantitative RT-PCR analysis also demonstrated equivalent results at the transcriptional level under high glucose conditions at each time point. These results showed that high glucose concentrations induced the decreased activity and expression of SIK1. Because the intracellular distribution of SIK1reflects its functional activity we decided to examine the localization of SIK1protein in HBZY-1cells, immuno-positive signals for SIK1were detected in the cytoplasm and nucleus of unstimulated HBZY-1cells. After stimulation with high glucose, the SIK1signals gradually redistributed into the nucleus with decreased intensity.
To confirm our results from immunocytochemical analysis, green fluorescence protein (GFP)-tagged SIK1protein was expressed in HBZY-1cells and the subcellular translocation of GFP signals was monitored before and after the addition of high glucose. In unstimulated cells, the GFP-SIK1signal was observed in both the nuclear and cytoplasmic compartments. When the cells were treated with high glucose, the green fluorescence signal was subsequently translocated into the nuclei from the cytoplasm. These results indicated that high glucose induced the nuclear import of SIK1, which might be a result from the reduction in SIK1kinase activity.
(2). Western blot analysis using HBZY-1cells also showed that the level of ALK5was upregulated by high glucose with1.82-and2.92-fold inductions at24and48h, respectively (normalized with β-actin), whereas no significant difference in the ALK5level was observed between0and12h. In contrast to ALK5, protein levels for FN and PAI-1, downstream targets of ALK5, were also increased by high glucose in a time-dependent manner. Immunocytochemical analysis of FN and PAI-1protein in HBZY-1cells also provided consistent results at48h, suggesting that high glucose induces the activation of the ALK5signaling pathway, which may be attributed to the downregulation of SIK1.
(3). To examine the involvement of the reduced levels of SIK1in the upregulation of ALK5signaling in glomerular mesangial cells, we decided to perform paradoxical assays through forced expression of SIK1in HBZY-1cells. The transformation efficiency, monitored by the control copGFP-expression, was approximately60%. When the SIK1expression plasmid was transformed into HBZY-1cells, the levels of SIK1protein and mRNA increased approximately1.8-and1.9-fold, respectively, compared to non-or mock-transfected cells. After stimulation of the cells with high glucose for48h, a negative correlation between the levels of SIK1protein and ALK5, FN, and PAI-1proteins was observed. When the SIK1expression plasmid was transformed into HBZY-1cells, the ALK5protein expression decreased to approximately38%in pCDFl-SIK1transfection cells compared to control cells. Immunocytochemical analysis of FN and PAI-I also supported the negative correlation between SIK1and ALK5signaling. A significant reduction of proliferation of HBZY-1cells from overexpression of SIK1was also observed.
[Conclusions]
(1).STZ-induced type1diabetes rat model to demonstrate the possible role of SIK1in early diabetic nephropathy. Twelve weeks later, diabetic rats developed diabetic nephropathy showing typical disorder features which developed severe hyperglycemia, albuminuria and renal pathological changes such as the expansion of mesangial matrix and the accumulation of ECM, all of those are characteristic of early diabetic nephropathy, and demonstrated that an animal model of diabetic nephropathy was successfully established。
(2). Endogenous SIKl was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. Gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Of diabetes rats at the age of20weeks, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks. Our results indicated that decreasing expression and activity of SIK1in glomerular cells of diabetic rats may play an important role associated with abnormal fibrosis processing.
(3). High glucose induces the depression of the activity and expression of SIK, the activation of the ALK5signaling pathway in HBZY-1cells and regulates the specific localization and distribution of SIKl in HBZY-1cells. But, forced expression of SIK1induces the degradation of ALK5and reduces the ECM production and reversed the high glucose-induced overproliferation in HBZY-1cells.
糖尿病肾病(DN)是糖尿病慢性并发症之一,也是导致终末期肾病的一个主要原因,在肾小球发生的细胞外基质(extracellular matrix, ECM)积聚等的纤维样病变提示了糖尿病肾病的发生和发展。在这一过程中,转化生长因子β1(Transforming growth factors-β1, TGF-β1)通过TGF-β1型受体即活化素受体样激酶5(the type Ⅰ activin receptor-like kinase (ALK)5, ALK5)进行信号转导,进而进一步激活ECM的主要成分纤连蛋白(fibronectin, FN)和可以诱导ECM降解的纤溶酶原激活物抑制剂1(plasminogen activator inhibitor type1, PAI-1)的表达,导致ECM的积聚和降解的减少。研究表明选择性抑制ALK5对与肾小球纤维化的这种慢性肾脏疾病是一种潜在治疗方法。盐诱导激酶1(Salt Inducible Kinase1, SIK1)是一种丝氨酸/苏氨酸激酶,SIK1可以与抑制型Smad7形成复合体并下调ALK5的表达而被认为是TGF-β的信号通路中的一个负向调控因子。虽然动物实验发现SIK1在大鼠肾脏高表达,但SIK1在糖尿病肾脏中表达和肾小球纤维化病变之间的关系还未完全阐明,而高糖是否影响SIK1表达也鲜有报道。
因此,本研究应用大鼠糖尿病模型和体外培养的大鼠肾系膜细胞,构建鼠SIK1基因的真核表达载体,从整体、细胞和分子水平,系统研究了糖尿病肾脏及高糖环境中系膜细胞SIK1蛋白表达情况及对ALK5信号通路的调控。
[方法]
1.SIK1在糖尿病肾病大鼠肾脏的表达和意义
将SPF级8周龄wistar大鼠随机分为正常对照组(normal组,10只)和糖尿病组(diabetic组,20只)。糖尿病模型采用腹腔注射链脲佐菌素(streptozotocin, STZ,溶于pH值4.5的0.1mol/L枸橼酸盐缓冲液中,60mg/kg),对照组只注射相当体积的枸橼酸盐缓冲液,72h后尾尖取血,血糖≥16.7mmol/L者确定为1型糖尿病模型。于12和16周龄糖尿病模型组取5只大鼠,乙醚麻醉大鼠,眼球内眦取血,离心分离血清,用于测定血糖；称重后于腹腔注射65mg/kg戊巴比妥钠麻醉,分离肾脏。于20周龄,将各组大鼠于代谢笼中,监测24h尿量,尿白蛋白及尿肌酐(Ucr)水平,并计算内生肌酐清除率(Ccr)和尿白蛋白排泄率(UAER)。麻醉取血,分离血清测定血糖,血肌酐及尿素氮水平。摘取肾脏,称重,计算肾脏指数。取部分肾组织置于4%多聚甲醛固定,用于HE, PAS染色观察其病理变化情况。
取肾组织制备切片进行免疫组化。分别提取肾组织及肾小球组织蛋白,应用免疫沉淀及Western blot方法以及观察糖尿病大鼠肾脏SIK1蛋白表达及磷酸化水平的影响。并应用Western blot方法观察20周龄大鼠肾小球ALK、FN. PAI-1表达情况,Masson染色观察20周龄大鼠肾小球胶原纤维及ECM聚集情况。
2.高糖刺激对系膜细胞SIK1的表达、活性、细胞内分布及ALK5信号通路的影响,SIK1表达质粒对高糖刺激下的系膜细胞ALK5信号通路及系膜细胞增殖的影响
大鼠肾系膜细胞HBZY-1细胞以含有10%胎牛血清、100KU/L青霉素及100mg/L链霉素的DMEM培养基培养,用高糖(30mmol/L, High glucose, HG)制备模型,于刺激后0、12、24和48h收集细胞,免疫沉淀、Western blot及半定量RT-PCR分别检测SIK1表达及磷酸化水平。构建pEGFP-SIK1表达质粒,激光共聚焦观察高糖刺激下细胞内生性及转染pEGFP空质粒及pEGFP-SIK1表达质粒后SIKl细胞内分布及核转运情况。Western blot及免疫细胞化学观察高糖对HBZY-1细胞ALK5信号通路的影响。
设计和构建针对SIK1基因的表达质粒,将对数生长期细胞接种于6孔培养板中,同步化后将细胞随机分为3组,空白细胞对照组、pCDF1空质粒对照组和pCDF1-SIK1质粒转染组,24h后细胞长至70-80%时开始转染,转染采用阳离子脂质体法。48h后荧光显微镜观察转染效率,终止培养进行蛋白、RNA提取。Western blot及半定量RT-PCR检测细胞内SIK-1蛋白和mRNA的表达。对各组转染成功细胞以高糖继续培养48h,终止培养后以Western blot及细胞免疫组化检测SIK1、ALK5、FN及PAI-1表达,MTT法检测细胞增殖情况。
[结果]
1.SIK1在糖尿病肾病大鼠肾脏的表达和意义
(1) wistar大鼠腹腔注射STZ成功建立1型糖尿病模型。与正常对照组比较,模型组表现出多饮、多食及体重减轻的特点。20周龄时,模型组餐后血糖水平升高,但血甘油三酯及胆固醇水平正常。与正常对照组相比,模型组出现糖尿病肾病的明显特征,各肾功能指标均表现出显著性差异。糖尿病模型组24h尿量、肾体重比、尿白蛋白分泌率、血肌酐,肌酐清除率,血尿素氮指标水平均明显升高,并出现肾小球肥大、纤维化,基底膜增厚等病理改变,肾小球面积及系膜增殖指数明显增大。
(2)免疫组织化学检测发现,SIK1在正常大鼠肾脏的肾小球及远曲小管表达明显,在近曲小管表达较少,且在各细胞的胞浆无着色,胞核无着色。在糖尿病模型组大鼠中,SIKl在肾小管的表达与正常对照组一致。而在肾小球的随病程月数的增加,SIK1的表达依次减少,呈时间依赖性。Western blot检测发现,在糖尿病大鼠肾组织SIK1的表达在三个时间点无明显变化,与正常对照组大鼠20周龄一致。而肾组织SIK1磷酸化水平在三个时间点却依次减少,且均明显低于正常对照组大鼠20周龄肾组织SIK1磷酸化水平。分离肾小球提取蛋白发现,糖尿病大鼠肾小球SIK1的表达在三个时间点表达却依次减少,均明显低于正常对照组大鼠20周龄肾小球SIK1的表达。
(3)与正常对照组大鼠相比,Western blot检测发现20周龄糖尿病大鼠肾小球ALK5、FN、PAI-1表达明显增高,Masson染色观察发现糖尿病大鼠肾小球肾小球胶原纤维及ECM明显聚集。
2.高糖刺激对系膜细胞SIKl的表达、活性、细胞内分布及ALK5信号通路的影响,SIK1表达质粒对高糖刺激下的系膜细胞ALK5信号通路及系膜细胞增殖的影响
(1)高糖刺激后0、12、24、48h,经免疫沉淀及Western blot检测发现,SIK1的表达及磷酸化水平依次递减,呈时间依赖性,有统计学意义。细胞免疫荧光后激光共聚焦观察发现SIK1在HBZY-1细胞的核内外均匀表达,高糖刺激下表达减少并向细胞内转运。转染pEGFP-SIKl表达质粒后,激光共聚焦观察发现GFP荧光信号在高糖刺激下由核外向核内转移。
(2)高糖刺激后0、12、24、48h,经Western blot检测发现,ALK5、FN、PAI-1表达水平依次递增,呈时间依赖性,有统计学意义。相对于高糖刺激后0h,细胞免疫组化发现在高糖刺激48h后FN、PAI-1表达水平明显增高。
(3)经酶切鉴定和测序分析,质粒符合设计要求,命名为pCDF1-SIK1。转染HBZY-1细胞24h后荧光倒置显微镜可见绿色荧光蛋白表达,48h表达量达60%以上。相对于正常对照组及pCDFl空质粒对照组,半定量RT-PCR及Western blot检测提示pCDFl-SIK组mRNA水平分别增加1.81和1.86倍,蛋白水平分别增加1.81和2.06,继续高糖刺激48h后,Western blot检测提示pCDF1-SIK组ALK5蛋白水平降低37.5%和39%,FN、PAI-1表达水平亦明显下降,并且MTT检测提示pCDFl-SIK可明显抑制高糖对HBZY-1细胞的增殖作用。
[结论]
(1) Wistar鼠腹腔注射STZ建立的1型糖尿病模型,在20周龄时出现糖尿病肾病的明显特征有,明显的ECM聚集；(2)SIK1主要在正常大鼠肾脏的远曲小管和肾小球表达明显,在近曲小管表达较少。随着糖尿病病程进展,肾脏SIK1表达无明显变化,但肾脏SIK1磷酸化水平及肾小球SIK1的表达逐渐减少。而在20周龄时肾小球ALK5,FN及PAI-1表达明显,提示SIK1在肾小球表达减少可能参与肾小球纤维化早期病变的发生和发展；(3)在细胞模型上,高糖可抑制SIK1的182位点磷酸化水平及表达水平,导致SIK1向细胞核内转运。同时高糖激活ALK5信号通路,提示SIK1的182位点磷酸化的抑制是高糖下调SIK1的主要原因,受抑制的SIK1可能导致ALK5信号通路的激活,进而导致细胞内的ECM聚集和细胞增殖。(4)重组质粒pCDF1-SIK能够激活体外培养的大鼠系膜细胞SIK1蛋白表达,下调ALK5信号通路,减少细胞内ECM聚集,抑制高糖诱导的细胞增殖。
(Objective]
Diabetic nephropathy is one of the most important causes of end-stage renal disease. Increasing evidence has shown that diabetic nephropathy in experimental animals, as well as in diabetic patients, develop fibrotic changes such as extracellular matrix (ECM) accumulation in glomeruli, which manifest as overt nephropathy. Transforming growth factors-β (TGF-β) plays a central role in the progression of glomerular fibrosis and transduces its signal through the type I activin receptor-like kinase (ALK)5, and then specifically induces expression of fibronectin (FN), the plasminogen activator inhibitor type1(PAI-1), and ECM accumulations. Several studies have shown that selective inhibit ALK5could be a potential therapy for chronic renal disease. Salt-inducible kinase1(SIKl), a serine/threonine protein kinase, is originally found in the adrenal glands of rats fed a high salt diet. SIK1belongs to a family of AMP-activated protein kinase (AMPK) due to the acid sequence closely related to catalytic a subunit of AMPK. Topical study also has shown that SIKl is identified as an inducible gene target of TGF-β/Smad signaling, and that SIK1forms complexes with the ALK5, the inhibitory Smad7and Smurf2to down-regulate ALK5as well as negative regulation of TGF-β signaling pathway. Although animal experiment reported that SIK mRNA is found in the highest levels in rat kidney, the specific role of SIK1in the kidney, especially in the glomeruli, of diabetic rats have not been evaluated yet, and there was limited knowledge concerning the specific rule of SIK1which involved in glucose metabolism at cellular level.
Therefore, in the current study, we assessed the possible role of SIK1in glomeruli of Streptozotocin (STZ)-induced type1diabetic rat. We were also using rat glomerular mesangial cells line (HBZY-1cells) to examine the modulation of SIK1protein under the condition of high glucose. Furthermore, we have constructed two specific SIK1expression plasmid and transfected them into HBZY-1cells respectively to explore whether SIK1play a beneficial role during mesangial cell fibrogenesis by regulating ALK5signaling pathway.
[Methods]
1.The expression of SIK1in the kidney of STZ-induced diabetic rats.
Thirty male Specific-pathogen-free (SPF) Wistar rats were housed in temperature (23±3℃)and humidity-controlled (60±5%) room with a12:12-hour light-dark cycle and fed with the standard diet. After1week adaptation, rats aged8weeks were randomized into2groups, ten rats were chosen randomly as normal group and the rest twenty rats as a model of diabetic mellitus. The STZ-induced rats were injected intraperitoneally with60mg/kg body weight STZ in50mM sodium citrate solution (pH4.5) to make type1diabetic model, meanwhile, rats in the normal group were injected with50mM sodium citrate solution. Type1diabetic were defined by fasting serum glucose level>16.7Mm after72h of STZ injection. All animals were given with equal volume distilled water every day. At the age of12,16weeks, diabetic rats were fasted for12h and anesthetized with intraperitoneal sodium pentobarbital (65mg/kg), its blood was then collected and its kidneys excised. After12weeks of administration, each last rat aged20was placed in a metabolic cage for24-h urine collection, then rats were weighed and sacrificed, the left kidney was removed and weighed. Glomeruli were isolated from rat kidneys by a modified procedure. Serum glucose was measured by an automatic biochemical analyzer. Urea and creatinine (Cr) were analyzed using enzymatic kits. Urinary microalbumin was measured by immunoturbidimetric assay using mALB kit. Albumin excretion rate (AER) was assessed as an index for kidney injury.
Renal tissue samples for light microscopy were fixed in4%buffered paraformaldehyde,4-um-thick paraffin-embedded samples were used and Routine Hematoxylin-eosin (HE) staining and periodic acid Schiff (PAS) staining was performed, the glomerular tuft area and mesangial matrix index were measured. The lysates extracted from kidney and glomeruli tissues were used for Western blot and immunoprecipitation to examine the expression of SIK1and pT186-SIKl. The expression ALK5, FN and PAI-1expression were also examined of rats at the age of20weeks. Meanwhile, Kidney glomerular fibrosis is predominantly featured as the production of extracellular matrix (ECM) and Masson staining was used to observe the accumulation of ECM.
2. High glucose significantly suppresses the expression and activity of SIK1, regulates the nuclear redistribution of SIK1and induces activation of the ALK5signaling pathway in HBZY-1cells, Overexpression of SIK1in HBZY-1cells resulted in downregulation of ALK5and its target gene FN and PAI-1, and reversed the high glucose-induced HBZY-1cellular overproliferation.
HBZY-1cells, a rat glomerular mesangial cells line, were maintained at37℃in a humidified atmosphere of5%CO2and cultured in Dulbecco's modified Eagle's medium supplemented with10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin. Cells were grown to-80%confluence and then incubated in serum-free medium for24h. To investigate the effects of glucose on protein expression of SIK1and pT186-SIK1, serum-deprived HBZY-1cells were cultured in DMEM containing30mM glucose (high glucose) for0,12,24and48h. At the end of each time, SIK1, pT186-SIK1, ALK5, FN and PAI-1expression were examined. In order to examine the direct effects of high glucose on the specific cellular localization of SIK1, HBZY-1cells were transfected with expression vectors for GFP-fused full-length SIK1(pEGFP-SIK1) and cultured with high glucose in glass-bottomed culture dishes for0and48h, and then observed and imaged by a Olympus confocal microscope. To examine SIK1negatively regulate ALK5signaling pathway, HBZY-1cells were randomly divided into three groups:untransfection normal group, empty vector control group and pCDFl-SIKl group. At48h after transfection, HBZY-1cells were collected and analyzed for SIK1expression, and were continuing cultured with high glucose for further48h to analyze SIK1, ALK5, FN and PAI-1expression by Western blot and immunocytochemistry analyses, MTT assays for HBZY-1cellular overproliferation analysis. Transient transfection of HBZY-1cells was carried out using Lipofectamine2000according to the manufacturer's instruction. At48h after transfection, fluorescent microscopy was used to examine GFP expression and then the cells were collected for the extraction of protein.
[Results]
Part1
(1). Twelve weeks after STZ injection, serum blood glucose level and systolic blood pressure were significantly higher in the type1diabetic group than in the normal group at the age of20weeks. The body weight of the rats in the diabetic groups was reduced in comparison with the normal group significantly. On the contrary, the ratio of kidney-to-body weight (KW/BW) was significantly higher in diabetic groups than in the normal group. The urine volume, AER, Cr, Ccr and Urea in the diabetic group were significantly increased compared with the normal group, indicating a damaged renal function in diabetic rats. Twelve wks after STZ injection, based on the results of HE and PAS staining, diabetic rats exhibited larger glomerular tuft area and an increased mesangial matrix index by two-fold compared with rats in normal group. Kidneys of the type1diabetic rats exhibited profound extracellular matrix deposition inside glomeruli. These indicating the pathological abnormalities were noteworthy in kidney of diabetic rats.
(2). The expression of SIK1protein was investigated by immunohistochemistry and result showed that expression of the endogenous SIK1was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. To our surprise, gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. To further prove this finding and to investigate the activity changes of SIK1during diabetic nephropathy, we explored kidney SIK1protein expression and SIK1activity (Thr-182phosphorylation, pT182) by immunoprecipitation and Western blot. We found that there was no significant difference of the kidney SIK1expression among diabetic rats at the age of12,16and20weeks and normal rats at the age of20weeks. Nevertheless, SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Activity of SIK1(pT182), with the diabetic state extending, was decrease rapidly in kidney from diabetic rats. Without any other process, Glomeruli-enriched tissues isolated from rat kidneys were lysed for Western blot analysis immediately. We found that the level of SIK1protein expression in the glomeruli isolated from kidneys of diabetic rat at the age of12,16and20weeks was significantly decreased than that of normal rats at the age of20weeks, glomerular SIK1expression of diabetic rat at the age of16and20weeks also markedly decreased than that of diabetic rat at the age of12weeks, but there was a negligible difference between diabetic rat at the age of16and20weeks.
(3). To clarify the possible relationship between SIK1and ALK5signaling pathway under the state of diabetic glomerulopathy, Western blot analysis of ALK5, FN and PAI-1in glomeruli was performed. Of diabetes rats at the age of20weeks, as expected, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks, in agreement with this, our further Masson staining showing the increased accumulation of glomerular ECM of diabetic rats compared with normal rats at the age of20weeks.
Part2
(1). HBZY-1cells were stimulated with high glucose, and the activity resulting from Thr-182phosphorylation of the SIK1gene was monitored. The HBZY-1cells were cultured in media containing30mM glucose for0,12,24, or48h. Immunoprecipitation followed by Western blot analysis showed that SIK1activity decreased after glucose treatment in a time-dependent manner. The levels of Thr-182phosphorylation at12,24, and48h were24.8%,58%, and58.4%, respectively, compared to0h. The level of phospho-Thr-182is important to maintain the SIK1protein level, which was also true in the HBZY-1cells treated with high glucose. The density ratios of SIKl/β-actin at12,24, and48h were18.2%,61.2%, and72%, respectively, of that at0h. Further semi-quantitative RT-PCR analysis also demonstrated equivalent results at the transcriptional level under high glucose conditions at each time point. These results showed that high glucose concentrations induced the decreased activity and expression of SIK1. Because the intracellular distribution of SIK1reflects its functional activity we decided to examine the localization of SIK1protein in HBZY-1cells, immuno-positive signals for SIK1were detected in the cytoplasm and nucleus of unstimulated HBZY-1cells. After stimulation with high glucose, the SIK1signals gradually redistributed into the nucleus with decreased intensity.
To confirm our results from immunocytochemical analysis, green fluorescence protein (GFP)-tagged SIK1protein was expressed in HBZY-1cells and the subcellular translocation of GFP signals was monitored before and after the addition of high glucose. In unstimulated cells, the GFP-SIK1signal was observed in both the nuclear and cytoplasmic compartments. When the cells were treated with high glucose, the green fluorescence signal was subsequently translocated into the nuclei from the cytoplasm. These results indicated that high glucose induced the nuclear import of SIK1, which might be a result from the reduction in SIK1kinase activity.
(2). Western blot analysis using HBZY-1cells also showed that the level of ALK5was upregulated by high glucose with1.82-and2.92-fold inductions at24and48h, respectively (normalized with β-actin), whereas no significant difference in the ALK5level was observed between0and12h. In contrast to ALK5, protein levels for FN and PAI-1, downstream targets of ALK5, were also increased by high glucose in a time-dependent manner. Immunocytochemical analysis of FN and PAI-1protein in HBZY-1cells also provided consistent results at48h, suggesting that high glucose induces the activation of the ALK5signaling pathway, which may be attributed to the downregulation of SIK1.
(3). To examine the involvement of the reduced levels of SIK1in the upregulation of ALK5signaling in glomerular mesangial cells, we decided to perform paradoxical assays through forced expression of SIK1in HBZY-1cells. The transformation efficiency, monitored by the control copGFP-expression, was approximately60%. When the SIK1expression plasmid was transformed into HBZY-1cells, the levels of SIK1protein and mRNA increased approximately1.8-and1.9-fold, respectively, compared to non-or mock-transfected cells. After stimulation of the cells with high glucose for48h, a negative correlation between the levels of SIK1protein and ALK5, FN, and PAI-1proteins was observed. When the SIK1expression plasmid was transformed into HBZY-1cells, the ALK5protein expression decreased to approximately38%in pCDFl-SIK1transfection cells compared to control cells. Immunocytochemical analysis of FN and PAI-I also supported the negative correlation between SIK1and ALK5signaling. A significant reduction of proliferation of HBZY-1cells from overexpression of SIK1was also observed.
[Conclusions]
(1).STZ-induced type1diabetes rat model to demonstrate the possible role of SIK1in early diabetic nephropathy. Twelve weeks later, diabetic rats developed diabetic nephropathy showing typical disorder features which developed severe hyperglycemia, albuminuria and renal pathological changes such as the expansion of mesangial matrix and the accumulation of ECM, all of those are characteristic of early diabetic nephropathy, and demonstrated that an animal model of diabetic nephropathy was successfully established。
(2). Endogenous SIKl was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. Gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Of diabetes rats at the age of20weeks, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks. Our results indicated that decreasing expression and activity of SIK1in glomerular cells of diabetic rats may play an important role associated with abnormal fibrosis processing.
(3). High glucose induces the depression of the activity and expression of SIK, the activation of the ALK5signaling pathway in HBZY-1cells and regulates the specific localization and distribution of SIKl in HBZY-1cells. But, forced expression of SIK1induces the degradation of ALK5and reduces the ECM production and reversed the high glucose-induced overproliferation in HBZY-1cells.
引文
[1]A.I. Adler, R.J. Stevens, S.E. Manley, R.W. Bilous, C.A. Cull, R.R. Holman, Development and progression of nephropathy in type 2 diabetes:the United Kingdom Prospective Diabetes Study (UKPDS 64), Kidney international 63 (2003) 225-232.
[2]B. Stengel, S. Billon, P.C. van Dijk, K.J. Jager, F.W. Dekker, K. Simpson, J.D. Briggs, Trends in the incidence of renal replacement therapy for end-stage renal disease in Europe,1990-1999, Nephrology Dialysis Transplantation 18 (2003) 1824-1833.
[3]V. Sakhuja, K. Sud, End-stage renal disease in India and Pakistan:Burden of disease and management issues, Kidney international 63 (2003) S115-S118.
[4]W.A. Border, N.A. Noble, Evidence that TGF-β should be a therapeutic target in diabetic nephropathy, Kidney international 54 (1998) 1390-1391.
[5]D.J. Leehey, A.K. Singh, N. Alavi, R. Singh, Role of angiotensin Ⅱ in diabetic nephropathy, Kidney international 58 (2000) S93-S98.
[6]C.M. Zimmerman, R.W. Padgett, Transforming growth factor β signaling mediators and modulators, Gene 249 (2000) 17-30.
[7]H.W. Schnaper, T. Hayashida, S.C. Hubchak, A.C. Poncelet, TGF-β signal transduction and mesangial cell fibrogenesis, American Journal of Physiology-Renal Physiology 284 (2003) F243-F252.
[8]F. Lebrin, M. Deckers, P. Bertolino, P. Ten Dijke, TGF-β receptor function in the endothelium, Cardiovascular research 65 (2005) 599-608.
[9]J. Moon, H. Kim, I. Cho, Y. Sheen, D. Kim, IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy, Kidney international 70 (2006) 1234-1243.
[10]N.J. Laping, ALK5 inhibition in renal disease, Current opinion in pharmacology 3 (2003) 204-208.
[11]N. Laping, E. Grygielko, A. Mathur, S. Butter, J. Bomberger, C. Tweed, W. Martin, J. Fornwald, R. Lehr, J. Harling, Inhibition of transforming growth factor (TGF)-βi-nduced extracellular matrix with a novel inhibitor of the TGF-P type Ⅰ receptor kinase activity:SB-431542, Molecular pharmacology 62 (2002) 58-64.
[12]M. Petersen, M. Thorikay, M. Deckers, M. Van Dinther, E. Grygielko, F. Gellibert, A. De Gouville, S. Huet, P. Ten Dijke, N. Laping, Oral administration of GW788388, an inhibitor of TGF-β type Ⅰ and Ⅱ receptor kinases, decreases renal fibrosis, Kidney international 73 (2007) 705-715.
[13]A.C. Gouville, V. Boullay, G. Krysa, J. Pilot, J.M. Brusq, F. Loriolle, J.M. Gauthier, S.A. Papworth, A. Laroze, F. Gellibert, Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis, British journal of pharmacology 145 (2005) 166-177.
[14]Z. Wang, H. Takemori, S.K. Halder, Y. Nonaka, M. Okamoto, Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal, FEBS letters 453 (1999) 135-139.
[15]G.R. Steinberg, B.E. Kemp, AMPK in health and disease, Science Signalling 89 (2009) 1025.
[16]N. Bright, C. Thornton, D. Carling, The regulation and function of mammalian AMPK-related kinases, Acta physiologica 196 (2009) 15-26.
[17]H. Takemori, Y. Katoh, N. Horike, J. Doi, M. Okamoto, ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase, Journal of Biological Chemistry 277 (2002) 42334-42343.
[18]Y. Katoh, H. Takemori, L. Min, M. Muraoka, N. Horike, M. Okamoto, Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm, European Journal of Biochemistry 271 (2004) 4307-4319.
[19]Y.K. Hashimoto, T. Satoh, M. Okamoto, H. Takemori, Importance of autophosphorylation at Serl86 in the A-loop of salt inducible kinase 1 for its sustained kinase activity, Journal of cellular biochemistry 104 (2008) 1724-1739.
[20]A.K. Al-Hakim, O. Goransson, M. Deak, R. Toth, D.G. Campbell, N.A. Morrice, A.R. Prescott, D.R. Alessi,14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK, Science Signalling 118 (2005) 5661.
[21]A. Romito, E. Lonardo, G Roma, G. Minchiotti, A. Ballabio, G Cobellis, Lack of sikl in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2, PloS one 5 (2010)e9029.
[22]R. Berdeaux, N. Goebel, L. Banaszynski, H. Takemori, T. Wandless, G.D. Shelton, M. Montminy, SIK1 is a class Ⅱ HDAC kinase that promotes survival of skeletal myocytes, Nature medicine 13 (2007) 597-603.
[23]H. Cheng, P. Liu, Z.C. Wang, L. Zou, S. Santiago, V. Garbitt, O.V. Gjoerup, J.D. Iglehart, A. Miron, A.L. Richardson, SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis, Science Signalling 2 (2009) ra35.
[24]S.H. Koo, L. Flechner, L. Qi, X. Zhang, R.A. Screaton, S. Jeffries, S. Hedrick, W. Xu, F. Boussouar, P. Brindle, The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism, Nature 437 (2005) 1109-1111.
[25]Y.S. Yoon, W.Y. Seo, M.W. Lee, S.T. Kim, S.H. Koo, Salt-inducible kinase regulates hepatic lipogenesis by controlling SREBP-lc phosphorylation, Journal of Biological Chemistry 284 (2009) 10446-10452.
[26]R. Berdeaux, Metabolic regulation by salt inducible kinases, Frontiers in Biology 6 (2011) 231-241.
[27]M. Kowanetz, P. Lonn, M. Vanlandewijck, K. Kowanetz, C.H. Heldin, A. Moustakas, TGFβ induces SIK to negatively regulate type I receptor kinase signaling, The Journal of cell biology 182 (2008) 655-662.
[28]P. Lonn, M. Vanlandewijck, E. Raja, M. Kowanetz, Y. Watanabe, K. Kowanetz, E. Vasilaki, C.H. Heldin, A. Moustakas, Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase, Journal of Biological Chemistry 287 (2012) 12867-12878.
[29]J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, The Salt-Inducible Kinase, SIK, Is Induced by Depolarization in Brain, Journal of neurochemistry 74 (2002) 2227-2238.
[30]C.X. Liu, Q. Hu, Y. Wang, W. Zhang, Z.Y. Ma, J.B. Feng, R. Wang, X.P. Wang, B. Dong, F. Gao, Angiotensin-converting enzyme (ACE) 2 overexpression ameliorates glomerular injury in a rat model of diabetic nephropathy:a comparison with ACE inhibition, Molecular Medicine 17 (2011) 59.
[31]G Wolf, S. Chen, F.N. Ziyadeh, From the periphery of the glomerular capillary wall toward the center of disease podocyte injury comes of age in diabetic nephropathy, Diabetes 54 (2005) 1626-1634.
[32]N. Horike, H. Takemori, Y. Katoh, J. Doi, L. Min, T. Asano, X.J. Sun, H. Yamamoto, S. Kasayama, M. Muraoka, Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2, Journal of Biological Chemistry 278(2003)18440-18447.
[33]D.F. Ding, N. You, X.M. Wu, J.R. Xu, A.P. Hu, X.L. Ye, Q. Zhu, X.Q. Jiang, H. Miao, C. Liu, Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK, American journal of nephrology 31 (2010) 363-374.
[34]P.G. Cammisotto, I. Londono, D. Gingras, M. Bendayan, Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats, American Journal of Physiology-Renal Physiology 294 (2008)F881-F889.
[35]T. Sasaki, H. Takemori, Y. Yagita, Y. Terasaki, T. Uebi, N. Horike, H. Takagi, T. Susumu, H. Teraoka, K. Kusano, SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB, Neuron 69 (2011) 106-119.
[36]R. Zatz, B.R. Dunn, T.W. Meyer, S. Anderson, H.G. Rennke, B.M. Brenner, Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension, Journal of Clinical Investigation 77 (1986) 1925.
[37]M. Toyoda, B. Najafian, Y. Kim, M.L. Caramori, M. Mauer, Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy, Diabetes 56 (2007) 2155-2160.
[38]M.R. Rudnick, A. Kesselheim, S. Goldfarb, Contrast-induced nephropathy:how it develops, how to prevent it, Cleveland Clinic journal of medicine 73 (2006) 75-80.
[39]M. Ries, F. Basseau, B. Tyndal, R. Jones, C. Deminiere, B. Catargi, C. Combe, C.W. Moonen, N. Grenier, Renal diffusion and BOLD MRI in experimental diabetic nephropathy, Journal of Magnetic Resonance Imaging 17 (2003) 104-113.
[40]M. Nangaku, Chronic hypoxia and tubulointerstitial injury:a final common pathway to end-stage renal failure, Journal of the American Society of Nephrology 17 (2006) 17-25.
[41]C. Kusmic, A. L'Abbate, G. Sambuceti, G. Drummond, C. Barsanti, M. Matteucci, J. Cao, F. Piccolomini, J. Cheng, N.G. Abraham, Improved myocardial perfusion in chronic diabetic mice by the up-regulation of pLKB1 and AMPK signaling, Journal of cellular biochemistry 109 (2010) 1033-1044.
[42]D.B. Shackelford, R.J. Shaw, The LKB1-AMPK pathway: metabolism and growth control in tumour suppression, Nature Reviews Cancer 9 (2009) 563-575.
[43]L. Wu, R. Derynck, Essential role of TGF-β signaling in glucose-induced cell hypertrophy, Developmental cell 17 (2009) 35-48.
[44]R. Mishra, B.L. Cool, K.R. Laderoute, M. Foretz, B. Viollet, M.S. Simonson, AMP-activated protein kinase inhibits transforming growth factor-β-induced Smad3-dependent transcription and myofibroblast transdifferentiation, Journal of Biological Chemistry 283 (2008) 10461-10469.
[45]A. Barthel, D. Schmoll, K.-D. Kruger, R.A. Roth, H.-G. Joost, Regulation of the forkhead transcription factor FKHR (FOXOla) by glucose starvation and AICAR, an activator of AMP-activated protein kinase, Endocrinology 143 (2002)3183-3183.
[46]H. Esumi, K. Izuishi, K. Kato, K. Hashimoto, Y. Kurashima, A. Kishimoto, T. Ogura, T. Ozawa, Hypoxia and nitric oxide treatment confer tolerance to glucose starvation in a 5'-AMP-activated protein kinase-dependent manner, Journal of Biological Chemistry 277 (2002) 32791-32798.
[47]J. Du, Q. Chen, H. Takemori, H. Xu, SIK2 can be activated by deprivation of nutrition and it inhibits expression of lipogenic genes in adipocytes, Obesity 16(2008)531-538.
[48]A. Suzuki, G.-i. Kusakai, A. Kishimoto, Y. Minegichi, T. Ogura, H. Esumi, Induction of cell-cell detachment during glucose starvation through F-actin conversion by SNARK, the fourth member of the AMP-activated protein kinase catalytic subunit family, Biochemical and biophysical research communications 311 (2003) 156-161.
[49]D.L. Lefebvre, Y. Bai, N. Shahmolky, M. Sharma, R. Poon, D.J. Drucker, C.F. Rosen, Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK, Biochemical Journal 355 (2001) 297.
[50]D.L. Lefebvre, C.F. Rosen, Regulation of SNARK activity in response to cellular stresses, Biochimica et Biophysica Acta (BBA)-General Subjects 1724 (2005) 71-85.
[51]M.J. Lee, D. Feliers, K. Sataranatarajan, M.M. Mariappan, M. Li, J.L. Barnes, G.G. Choudhury, B.S. Kasinath, Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells, Cellular signalling 22 (2010) 65-70.
[52]X. Lin, H. Takemori, Y. Katoh, J. Doi, N. Horike, A. Makino, Y. Nonaka, M. Okamoto, Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells, Molecular Endocrinology 15 (2001) 1264-1276.
[53]Y. Katoh, H. Takemori, X. Lin, M. Tamura, M. Muraoka, T. Satoh, Y. Tsuchiya, L. Min, A. Miyauchi, L.A. Witters, Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade, FEBS Journal 273 (2006) 2730-2748.
[54]J.M. Lizcano, O. Goransson, R. Toth, M. Deak, N.A. Morrice, J. Boudeau, S.A. Hawley, L. Udd, T.P. Makela, D.G. Hardie, LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1, The EMBO journal 23 (2004) 833-843.
[55]J. Xu, Y. Wu, P. Song, M. Zhang, S. Wang, M.H. Zou, Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus, Circulation 116 (2007) 944-953.
[56]A. Trumper, K. Trumper, D. Horsch, Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta (INS-1)-cells, Journal of endocrinology 174 (2002) 233-246.
[57]S. Wang, R. Hirschberg, Diabetes-relevant regulation of cultured blood outgrowth endothelial cells, Microvascular research 78 (2009) 174-179.
[58]A.C. Gouville, V. Boullay, G Krysa, J. Pilot, J.M. Brusq, F. Loriolle, J.M. Gauthier, S.A. Papworth, A. Laroze, F. Gellibert, Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethyInitrosamine-induced liver fibrosis, British journal of pharmacology 145 (2009) 166-177.
[59]Y. Yata, P. Gotwals, V. Koteliansky, D.C. Rockey, Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-β soluble receptor:Implications for antifibrotic therapy, Hepatology 35 (2002) 1022-1030.
[1]A. Andersen, J.S. Christiansen, J. Andersen, S. Kreiner, T. Deckert, Diabetic nephropathy in type 1 (insulin-dependent) diabetes:an epidemiological study, Diabetologia 25 (1983) 496-501.
[2]A. Scheen, Thiazolidinediones and liver toxicity, Diabetes & metabolism 27 (2001).
[3]D.E. Moller, New drug targets for type 2 diabetes and the metabolic syndrome, Nature 414 (2001) 821-827.
[4]M. Okamoto, H. Takemori, Y. Katoh, Salt-inducible kinase in steroidogenesis and adipogenesis, Trends in Endocrinology & Metabolism 15 (2004) 21-26.
[5]J.C. Ruiz, F.L. Conlon, E.J. Robertson, Identification of novel protein kinases expressed in the myocardium of the developing mouse heart, Mechanisms of development 48 (1994) 153-164.
[6]Z.-n. Wang, H. Takemori, S.K. Halder, Y. Nonaka, M. Okamoto, Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal, FEBS letters 453 (1999) 135-139.
[7]Y. Katoh, H. Takemori, N. Horike, J. Doi, M. Muraoka, L. Min, M. Okamoto, Salt-inducible kinase (SIK) isoforms:their involvement in steroidogenesis and adipogenesis, Molecular and cellular endocrinology 217 (2004) 109-112.
[8]A. Lanjuin, P. Sengupta, Regulation of chemosensory receptor expression and sensory signaling by the KIN-29 Ser/Thr kinase, Neuron 33 (2002) 369-381.
[9]B. Wang, J. Goode, J. Best, J. Meltzer, P.E. Schilman, J. Chen, D. Garza, J.B. Thomas, M. Montminy, The Insulin-Regulated CREB Coactivator TORC Promotes Stress Resistance in< i> Drosophila, Cell metabolism 7 (2008) 434-444.
[10]S. Choi, W. Kim, J. Chung, Drosophila salt-inducible kinase (SIK) regulates starvation resistance through CREB-regulated transcription coactivator (CRTC), Journal of Biological Chemistry (2010).
[11]N. Horike, H. Takemori, Y. Katoh, J. Doi, L. Min, T. Asano, X.J. Sun, H. Yamamoto, S. Kasayama, M. Muraoka, Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2, Journal of Biological Chemistry 278(2003)18440-18447.
[12]Y. Katoh, H. Takemori, L. Min, M. Muraoka, J. Doi, N. Horike, M. Okamoto, Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm, European Journal of Biochemistry 271 (2004) 4307-4319.
[13]S.-H. Koo, L. Flechner, L. Qi, X. Zhang, R.A. Screaton, S. Jeffries, S. Hedrick, W. Xu, F. Boussouar, P. Brindle, The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism, Nature 437 (2005) 1109-1111.
[14]R. Berdeaux, N. Goebel, L. Banaszynski, H. Takemori, T. Wandless, G.D. Shelton, M. Montminy, SIK1 is a class Ⅱ HDAC kinase that promotes survival of skeletal myocytes, Nature medicine 13 (2007) 597-603.
[15]N. Bright, C. Thornton, D. Carling, The regulation and function of mammalian AMPK-related kinases, Acta physiologica 196 (2009) 15-26.
[16]D.G. Hardie, The AMP-activated protein kinase pathway-new players upstream and downstream, Journal of cell science 117 (2004) 5479-5487.
[17]J.M. Lizcano, O. Goransson, R. Toth, M. Deak, N.A. Morrice, J. Boudeau, S.A. Hawley, L. Udd, T.P. Makela, D.G. Hardie, LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1, The EMBO journal 23 (2004) 833-843.
[18]R.J. Shaw, M. Kosmatka, N. Bardeesy, R.L. Hurley, L.A. Witters, R.A. DePinho, L.C. Cantley, The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress, Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 3329-3335.
[19]S.A. Hawley, M.A. Selbert, E.G. Goldstein, A.M. Edelman, D. Carling, D.G. Hardie,5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms, Journal of Biological Chemistry 270(1995)27186-27191.
[20]S.A. Hawley, D.A. Pan, K.J. Mustard, L. Ross, J. Bain, A.M. Edelman, B.G. Frenguelli, D.G. Hardie, Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase, Cell metabolism 2 (2005) 9-19.
[21]C.A. Witczak, N. Fujii, M.F. Hirshman, L.J. Goodyear, Ca2+/calmodulin-dependent protein kinase kinase-a regulates skeletal muscle glucose uptake independent of AMP-activated protein kinase and Akt activation, Diabetes 56 (2007) 1403-1409.
[22]M. Momcilovic, S.-P. Hong, M. Carlson, Mammalian TAK1 activates Snfl protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro, Journal of Biological Chemistry 281 (2006) 25336-25343.
[23]K. Sakamoto, A. McCarthy, D. Smith, K.A. Green, D.G. Hardie, A. Ashworth, D.R. Alessi, Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction, The EMBO journal 24 (2005) 1810-1820.
[24]R.J. Shaw, K.A. Lamia, D. Vasquez, S.-H. Koo, N. Bardeesy, R.A. DePinho, M. Montminy, L.C. Cantley, The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin, Science Signalling 310 (2005) 1642.
[25]M. Jaleel, F. Villa, M. Deak, R. Toth, A.R. Prescott, D.M. Van Aalten, D.R. Alessi, The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation, Biochemical Journal 394 (2006) 545.
[26]Y. Katoh, H. Takemori, J. Doi, M. Okamoto, Identification of the nuclear localization domain of salt-inducible kinase, Endocrine research 28 (2002) 315-318.
[27]H. Takemori, Y. Katoh, N. Horike, J. Doi, M. Okamoto, ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase, Journal of Biological Chemistry 277 (2002) 42334-42343.
[28]J. Liu, E. Park, A. Gurney, W. Roesler, R. Hanson, Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements, Journal of Biological Chemistry 266 (1991) 19095-19102.
[29]M.D. Conkright, G. Canettieri, R. Screaton, E. Guzman, L. Miraglia, J.B. Hogenesch, M. Montminy, TORCs:transducers of regulated CREB activity, Molecular cell 12 (2003) 413-423.
[30]B. Mayr, M. Montminy, Transcriptional regulation by the phosphorylation-dependent factor CREB, Nature Reviews Molecular Cell Biology 2 (2001) 599-609.
[31]W.A. Carlezon, R.S. Duman, E.J. Nestler, The many faces of CREB, Trends in neurosciences 28 (2005) 436-445.
[32]M. Johannessen, M.P. Delghandi, U. Moens, What turns CREB on?, Cellular signalling 16 (2004) 1211.
[33]H.A. Giebler, I. Lemasson, J.K. Nyborg, p53 recruitment of CREB binding protein mediated through phosphorylated CREB:a novel pathway of tumor suppressor regulation, Molecular and cellular biology 20 (2000) 4849-4858.
[34]R.A. Screaton, M.D. Conkright, Y. Katoh, J.L. Best, G. Canettieri, S. Jeffries, E. Guzman, S. Niessen, J.R. Yates Ⅲ, H. Takemori, The CREB coactivator TORC2 functions as a calcium-and cAMP-sensitive coincidence detector, Cell 119 (2004)61-74.
[35]R. Dentin, Y. Liu, S.-H. Koo, S. Hedrick, T. Vargas, J. Heredia, J. Yates, M. Montminy, Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2, Nature 449 (2007) 366-369.
[36]M. Muraoka, A. Fukushima, S. Viengchareun, M. Lombes, F. Kishi, A. Miyauchi, M. Kanematsu, J. Doi, J. Kajimura, R. Nakai, Involvement of SIK2/TORC2 signaling cascade in the regulation of insulin-induced PGC-1α and UCP-1 gene expression in brown adipocytes, American Journal of Physiology-Endocrinology And Metabolism 296 (2009) E1430-E1439.
[37]Y. Katoh, H. Takemori, X.z. Lin, M. Tamura, M. Muraoka, T. Satoh, Y. Tsuchiya, L. Min, A. Miyauchi, L.A. Witters, Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade, FEBS Journal 273 (2006) 2730-2748.
[38]Y.-S. Yoon, W.-Y. Seo, M.-W. Lee, S.-T. Kim, S.-H. Koo, Salt-inducible kinase regulates hepatic lipogenesis by controlling SREBP-lc phosphorylation, Journal of Biological Chemistry 284 (2009) 10446-10452.
[39]J. Bricambert, J. Miranda, F. Benhamed, J. Girard, C. Postic, R. Dentin, Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice, The Journal of clinical investigation 120 (2010) 4316.
[40]S.N. Jakobsen, D.G. Hardie, N. Morrice, H.E. Tornqvist,5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside, Journal of Biological Chemistry 276 (2001) 46912-46916.
[41]S. Herzig, F. Long, U.S. Jhala, S. Hedrick, R. Quinn, A. Bauer, D. Rudolph, G. Schutz, C. Yoon, P. Puigserver, CREB regulates hepatic gluconeogenesis through the coactivator PGC-1, Nature 413 (2001) 179-183.
[42]J. Le Lay, G. Tuteja, P. White, R. Dhir, R. Ahima, K.H. Kaestner, CRTC2 (T0RC2) contributes to the transcriptional response to fasting in the liver but is not required for the maintenance of glucose homeostasis, Cell metabolism 10(2009)55-62.
[43]Y. Wang, H. Inoue, K. Ravnskjaer, K. Viste, N. Miller, Y. Liu, S. Hedrick, L. Vera, M. Montminy, Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity, Proceedings of the National Academy of Sciences 107 (2010) 3087-3092.
[44]M. Saberi, D. Bjelica, S. Schenk, T. Imamura, G. Bandyopadhyay, P. Li, V. Jadhar, C. Vargeese, W. Wang, K. Bowman, Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes, American Journal of Physiology-Endocrinology And Metabolism 297 (2009) E1137-E1146.
[45]X.-z. Lin, H. Takemori, Y. Katoh, J. Doi, N. Horike, A. Makino, Y. Nonaka, M. Okamoto, Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Yl mouse adrenocortical tumor cells, Molecular Endocrinology 15 (2001) 1264-1276.
[46]T. Sasaki, H. Takemori, Y. Yagita, Y. Terasaki, T. Uebi, N. Horike, H. Takagi, T. Susumu, H. Teraoka, K.-i. Kusano, SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB, Neuron 69 (2011) 106-119.
[47]R. Dentin, S. Hedrick, J. Xie, J. Yates III, M. Montminy, Hepatic glucose sensing via the CREB coactivator CRTC2, Science Signalling 319 (2008) 1402.
[48]L. He, A. Sabet, S. Djedjos, R. Miller, X. Sun, M.A. Hussain, S. Radovick, F.E. Wondisford, Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein, Cell 137 (2009) 635-646.
[49]D.M. Erion, I.D. Ignatova, S. Yonemitsu, Y. Nagai, P. Chatterjee, D. Weismann, J.J. Hsiao, D. Zhang, T. Iwasaki, R. Stark, Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP response element-binding protein, Cell metabolism 10 (2009) 499-506.
[50]J.G. Salway, Metabolism at a Glance, Blackwell Scientific Publications Ltd, 1994.
[51]R. Raghow, C. Yellaturu, X. Deng, E.A. Park, M.B. Elam, SREBPs:the crossroads of physiological and pathological lipid homeostasis, Trends in Endocrinology & Metabolism 19 (2008) 65-73.
[52]H. Yamashita, M. Takenoshita, M. Sakurai, R.K. Bruick, W.J. Henzel, W. Shillinglaw, D. Arnot, K. Uyeda, A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver, Proceedings of the National Academy of Sciences 98 (2001) 9116-9121.
[53]R. Dentin, J.-P. Pegorier, F. Benhamed, F. Foufelle, P. Ferre, V. Fauveau, M.A. Magnuson, J. Girard, C. Postic, Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression, Journal of Biological Chemistry 279 (2004) 20314-20326.
[54]J. Du, Q. Chen, H. Takemori, H. Xu, SIK2 can be activated by deprivation of nutrition and it inhibits expression of lipogenic genes in adipocytes, Obesity 16(2008)531-538.
[55]Y. Song, J. Altarejos, M.O. Goodarzi, H. Inoue, X. Guo, R. Berdeaux, J.-H. Kim, J. Goode, M. Igata, J.C. Paz, CRTC3 links catecholamine signalling to energy balance, Nature 468 (2010) 933-939.
[56]S. Kajimura, P. Seale, B.M. Spiegelman, Transcriptional control of brown fat development, Cell metabolism 11 (2010) 257-262.
[57]P. Vague, T. Coste, M. Jannot, D. Raccah, M. Tsimaratos, C-peptide, Na, Experimental Diabetes Research 5 (2004) 37-50.
[58]H. Poulsen, P. Morth, J. Egebjerg, P. Nissen, Phosphorylation of the Na< sup>+, K< sup>+-ATPase and the H< sup>+, K< sup>+-ATPase, FEBS letters 584 (2010) 2589-2595.
[59]S. Sackmann, U. Lichtenauer, I. Shapiro, M. Reincke, F. Beuschlein, Aldosterone producing adrenal adenomas are characterized by activation of calcium/calmodulin-dependent protein kinase (CaMK) dependent pathways, Hormone and metabolic research 43 (2011) 106.
[60]R.L. Hurley, K.A. Anderson, J.M. Franzone, B.E. Kemp, A.R. Means, L.A. Witters, The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases, Journal of Biological Chemistry 280 (2005) 29060-29066.
[61]H.S. Jun, K. Hwang, Y. Kim, T. Park, High-fat Diet Alters PP2A, TC10, and CIP4 Expression in Visceral Adipose Tissue of Rats, Obesity 16 (2012) 1226-1231.
[62]E. Lecuona, L.A. Dada, H. Sun, M.L. Butti, G. Zhou, T.-L. Chew, J.I. Sznajder, Na, K-ATPase al-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane, The FASEB journal 20 (2006) 2618-2620.
[63]S. Longin, J. Jordens, E. Martens, I. Stevens, V. Janssens, E. Rondelez, I. De Baere, R. Derua, E. Waelkens, J. Goris, An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator, Biochemical Journal 380 (2004) 111.
[64]M. Sjostrom, K. Stenstrom, K. Eneling, J. Zwiller, A.I. Katz, H. Takemori, A.M. Bertorello, SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process, Proceedings of the National Academy of Sciences 104 (2007) 16922-16927.
[65]K. Stenstrom, H. Takemori, G. Bianchi, A.I. Katz, A.M. Bertorello, Blocking the salt-inducible kinase 1 network prevents the increases in cell sodium transport caused by a hypertension-linked mutation in human [alpha]-adducin, Journal of hypertension 27 (2009) 2452.
[66]Y. Lee, H. Han, Regulatory mechanisms of Na+/glucose cotransporters in renal proximal tubule cells, Kidney International 72 (2007) S27-S35.
[67]A. Fekete, K. Rosta, L. Wagner, A. Prokai, P. Degrell, E. Ruzicska, E. Vegh, M. Toth, K. Ronai, K. Rusai, Na+, K+- ATPase is modulated by angiotensin Ⅱ in diabetic rat kidney-another reason for diabetic nephropathy?, The Journal of Physiology 586 (2008) 5337-5348.
[68]A. Jaitovich, A.M. Bertorello, Intracellular sodium sensing:SIK1 network, hormone action and high blood pressure, Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1802 (2010) 1140-1149.
[69]K. Stenstrom, Salt-inducible kinase 1:a novel regulator within a cell sodium-sensing network, Karolinska institutet,2010.
[70]N. Horike, A. Kumagai, Y. Shimono, T. Onishi, Y. Itoh, T. Sasaki, K. Kitagawa, O. Hatano, H. Takagi, T. Susumu, Downregulation of SIK2 expression promotes the melanogenic program in mice, Pigment cell & melanoma research 23 (2010) 809-819.
[71]C. Hyman, M. Hofer, Y.-A. Barde, M. Juhasz, G.D. Yancopoulos, S.P. Squinto, R.M. Lindsay, BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra, Nature 350 (1991) 230-232.
[72]J. Cheng, M. Uchida, W. Zhang, M.R. Grafe, P.S. Herson, P.D. Hum, Role of salt-induced kinase 1 in androgen neuroprotection against cerebral ischemia, Journal of Cerebral Blood Flow & Metabolism 31 (2010) 339-350.
[73]J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, The Salt-Inducible Kinase, SIK, Is Induced by Depolarization in Brain, Journal of neurochemistry 74 (2002) 2227-2238.
[74]H. Cheng, P. Liu, Z.C. Wang, L. Zou, S. Santiago, V. Garbitt, O.V. Gjoerup, J.D. Iglehart, A. Miron, A.L. Richardson, SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis, Science Signalling 2 (2009) ra35.
[75]A. Romito, E. Lonardo, G. Roma, G. Minchiotti, A. Ballabio, G. Cobellis, Lack of sikl in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2, PloS one 5 (2010)e9029.
[76]A.M. van der Linden, K.M. Nolan, P. Sengupta, KIN-29 SIK regulates chemoreceptor gene expression via an MEF2 transcription factor and a class Ⅱ HDAC, The EMBO journal 26 (2006) 358-370.
[77]E. Kozhemyakina, T. Cohen, T.-P. Yao, A.B. Lassar, Parathyroid hormone-related peptide represses chondrocyte hypertrophy through a protein phosphatase 2A/histone deacetylase 4/MEF2 pathway, Molecular and cellular biology 29 (2009) 5751-5762.
[78]M. Kowanetz, P. Lonn, M. Vanlandewijck, K. Kowanetz, C.H. Heldin, A. Moustakas, TGFβ induces SIK to negatively regulate type I receptor kinase signaling, The Journal of cell biology 182 (2008) 655-662.
[79]P. Lonn, M. Vanlandewijck, E. Raja, M. Kowanetz, Y. Watanabe, K. Kowanetz, E. Vasilaki, C.H. Heldin, A. Moustakas, Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type Ⅰ receptor signaling in cooperation with the Smurf2 ubiquitin ligase, Journal of Biological Chemistry 287 (2012) 12867-12878.
[80]E. Raja, Cross-regulation between TGFβ/BMP Signalling and the metabolic LKB1 pathway, Uppsala University,2012.
[81]M. Vanlandewijck, Diversification of TGF-β Signaling in Homeostasis and Disease, Uppsala University,2011.
[82]I. Magnusson, D. Rothman, L. Katz, R. Shulman, G. Shulman, Increased rate of gluconeogenesis in type II diabetes mellitus. A 13C nuclear magnetic resonance study, Journal of Clinical Investigation 90 (1992) 1323.
[83]J.C. Yoon, P. Puigserver, G. Chen, J. Donovan, Z. Wu, J. Rhee, G. Adelmant, J. Stafford, C.R. Kahn, D.K. Granner, Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1, Nature 413 (2001) 131-138.
[2]B. Stengel, S. Billon, P.C. van Dijk, K.J. Jager, F.W. Dekker, K. Simpson, J.D. Briggs, Trends in the incidence of renal replacement therapy for end-stage renal disease in Europe,1990-1999, Nephrology Dialysis Transplantation 18 (2003) 1824-1833.
[3]V. Sakhuja, K. Sud, End-stage renal disease in India and Pakistan:Burden of disease and management issues, Kidney international 63 (2003) S115-S118.
[4]W.A. Border, N.A. Noble, Evidence that TGF-β should be a therapeutic target in diabetic nephropathy, Kidney international 54 (1998) 1390-1391.
[5]D.J. Leehey, A.K. Singh, N. Alavi, R. Singh, Role of angiotensin Ⅱ in diabetic nephropathy, Kidney international 58 (2000) S93-S98.
[6]C.M. Zimmerman, R.W. Padgett, Transforming growth factor β signaling mediators and modulators, Gene 249 (2000) 17-30.
[7]H.W. Schnaper, T. Hayashida, S.C. Hubchak, A.C. Poncelet, TGF-β signal transduction and mesangial cell fibrogenesis, American Journal of Physiology-Renal Physiology 284 (2003) F243-F252.
[8]F. Lebrin, M. Deckers, P. Bertolino, P. Ten Dijke, TGF-β receptor function in the endothelium, Cardiovascular research 65 (2005) 599-608.
[9]J. Moon, H. Kim, I. Cho, Y. Sheen, D. Kim, IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy, Kidney international 70 (2006) 1234-1243.
[10]N.J. Laping, ALK5 inhibition in renal disease, Current opinion in pharmacology 3 (2003) 204-208.
[11]N. Laping, E. Grygielko, A. Mathur, S. Butter, J. Bomberger, C. Tweed, W. Martin, J. Fornwald, R. Lehr, J. Harling, Inhibition of transforming growth factor (TGF)-βi-nduced extracellular matrix with a novel inhibitor of the TGF-P type Ⅰ receptor kinase activity:SB-431542, Molecular pharmacology 62 (2002) 58-64.
[12]M. Petersen, M. Thorikay, M. Deckers, M. Van Dinther, E. Grygielko, F. Gellibert, A. De Gouville, S. Huet, P. Ten Dijke, N. Laping, Oral administration of GW788388, an inhibitor of TGF-β type Ⅰ and Ⅱ receptor kinases, decreases renal fibrosis, Kidney international 73 (2007) 705-715.
[13]A.C. Gouville, V. Boullay, G. Krysa, J. Pilot, J.M. Brusq, F. Loriolle, J.M. Gauthier, S.A. Papworth, A. Laroze, F. Gellibert, Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis, British journal of pharmacology 145 (2005) 166-177.
[14]Z. Wang, H. Takemori, S.K. Halder, Y. Nonaka, M. Okamoto, Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal, FEBS letters 453 (1999) 135-139.
[15]G.R. Steinberg, B.E. Kemp, AMPK in health and disease, Science Signalling 89 (2009) 1025.
[16]N. Bright, C. Thornton, D. Carling, The regulation and function of mammalian AMPK-related kinases, Acta physiologica 196 (2009) 15-26.
[17]H. Takemori, Y. Katoh, N. Horike, J. Doi, M. Okamoto, ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase, Journal of Biological Chemistry 277 (2002) 42334-42343.
[18]Y. Katoh, H. Takemori, L. Min, M. Muraoka, N. Horike, M. Okamoto, Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm, European Journal of Biochemistry 271 (2004) 4307-4319.
[19]Y.K. Hashimoto, T. Satoh, M. Okamoto, H. Takemori, Importance of autophosphorylation at Serl86 in the A-loop of salt inducible kinase 1 for its sustained kinase activity, Journal of cellular biochemistry 104 (2008) 1724-1739.
[20]A.K. Al-Hakim, O. Goransson, M. Deak, R. Toth, D.G. Campbell, N.A. Morrice, A.R. Prescott, D.R. Alessi,14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK, Science Signalling 118 (2005) 5661.
[21]A. Romito, E. Lonardo, G Roma, G. Minchiotti, A. Ballabio, G Cobellis, Lack of sikl in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2, PloS one 5 (2010)e9029.
[22]R. Berdeaux, N. Goebel, L. Banaszynski, H. Takemori, T. Wandless, G.D. Shelton, M. Montminy, SIK1 is a class Ⅱ HDAC kinase that promotes survival of skeletal myocytes, Nature medicine 13 (2007) 597-603.
[23]H. Cheng, P. Liu, Z.C. Wang, L. Zou, S. Santiago, V. Garbitt, O.V. Gjoerup, J.D. Iglehart, A. Miron, A.L. Richardson, SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis, Science Signalling 2 (2009) ra35.
[24]S.H. Koo, L. Flechner, L. Qi, X. Zhang, R.A. Screaton, S. Jeffries, S. Hedrick, W. Xu, F. Boussouar, P. Brindle, The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism, Nature 437 (2005) 1109-1111.
[25]Y.S. Yoon, W.Y. Seo, M.W. Lee, S.T. Kim, S.H. Koo, Salt-inducible kinase regulates hepatic lipogenesis by controlling SREBP-lc phosphorylation, Journal of Biological Chemistry 284 (2009) 10446-10452.
[26]R. Berdeaux, Metabolic regulation by salt inducible kinases, Frontiers in Biology 6 (2011) 231-241.
[27]M. Kowanetz, P. Lonn, M. Vanlandewijck, K. Kowanetz, C.H. Heldin, A. Moustakas, TGFβ induces SIK to negatively regulate type I receptor kinase signaling, The Journal of cell biology 182 (2008) 655-662.
[28]P. Lonn, M. Vanlandewijck, E. Raja, M. Kowanetz, Y. Watanabe, K. Kowanetz, E. Vasilaki, C.H. Heldin, A. Moustakas, Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase, Journal of Biological Chemistry 287 (2012) 12867-12878.
[29]J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, The Salt-Inducible Kinase, SIK, Is Induced by Depolarization in Brain, Journal of neurochemistry 74 (2002) 2227-2238.
[30]C.X. Liu, Q. Hu, Y. Wang, W. Zhang, Z.Y. Ma, J.B. Feng, R. Wang, X.P. Wang, B. Dong, F. Gao, Angiotensin-converting enzyme (ACE) 2 overexpression ameliorates glomerular injury in a rat model of diabetic nephropathy:a comparison with ACE inhibition, Molecular Medicine 17 (2011) 59.
[31]G Wolf, S. Chen, F.N. Ziyadeh, From the periphery of the glomerular capillary wall toward the center of disease podocyte injury comes of age in diabetic nephropathy, Diabetes 54 (2005) 1626-1634.
[32]N. Horike, H. Takemori, Y. Katoh, J. Doi, L. Min, T. Asano, X.J. Sun, H. Yamamoto, S. Kasayama, M. Muraoka, Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2, Journal of Biological Chemistry 278(2003)18440-18447.
[33]D.F. Ding, N. You, X.M. Wu, J.R. Xu, A.P. Hu, X.L. Ye, Q. Zhu, X.Q. Jiang, H. Miao, C. Liu, Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK, American journal of nephrology 31 (2010) 363-374.
[34]P.G. Cammisotto, I. Londono, D. Gingras, M. Bendayan, Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats, American Journal of Physiology-Renal Physiology 294 (2008)F881-F889.
[35]T. Sasaki, H. Takemori, Y. Yagita, Y. Terasaki, T. Uebi, N. Horike, H. Takagi, T. Susumu, H. Teraoka, K. Kusano, SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB, Neuron 69 (2011) 106-119.
[36]R. Zatz, B.R. Dunn, T.W. Meyer, S. Anderson, H.G. Rennke, B.M. Brenner, Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension, Journal of Clinical Investigation 77 (1986) 1925.
[37]M. Toyoda, B. Najafian, Y. Kim, M.L. Caramori, M. Mauer, Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy, Diabetes 56 (2007) 2155-2160.
[38]M.R. Rudnick, A. Kesselheim, S. Goldfarb, Contrast-induced nephropathy:how it develops, how to prevent it, Cleveland Clinic journal of medicine 73 (2006) 75-80.
[39]M. Ries, F. Basseau, B. Tyndal, R. Jones, C. Deminiere, B. Catargi, C. Combe, C.W. Moonen, N. Grenier, Renal diffusion and BOLD MRI in experimental diabetic nephropathy, Journal of Magnetic Resonance Imaging 17 (2003) 104-113.
[40]M. Nangaku, Chronic hypoxia and tubulointerstitial injury:a final common pathway to end-stage renal failure, Journal of the American Society of Nephrology 17 (2006) 17-25.
[41]C. Kusmic, A. L'Abbate, G. Sambuceti, G. Drummond, C. Barsanti, M. Matteucci, J. Cao, F. Piccolomini, J. Cheng, N.G. Abraham, Improved myocardial perfusion in chronic diabetic mice by the up-regulation of pLKB1 and AMPK signaling, Journal of cellular biochemistry 109 (2010) 1033-1044.
[42]D.B. Shackelford, R.J. Shaw, The LKB1-AMPK pathway: metabolism and growth control in tumour suppression, Nature Reviews Cancer 9 (2009) 563-575.
[43]L. Wu, R. Derynck, Essential role of TGF-β signaling in glucose-induced cell hypertrophy, Developmental cell 17 (2009) 35-48.
[44]R. Mishra, B.L. Cool, K.R. Laderoute, M. Foretz, B. Viollet, M.S. Simonson, AMP-activated protein kinase inhibits transforming growth factor-β-induced Smad3-dependent transcription and myofibroblast transdifferentiation, Journal of Biological Chemistry 283 (2008) 10461-10469.
[45]A. Barthel, D. Schmoll, K.-D. Kruger, R.A. Roth, H.-G. Joost, Regulation of the forkhead transcription factor FKHR (FOXOla) by glucose starvation and AICAR, an activator of AMP-activated protein kinase, Endocrinology 143 (2002)3183-3183.
[46]H. Esumi, K. Izuishi, K. Kato, K. Hashimoto, Y. Kurashima, A. Kishimoto, T. Ogura, T. Ozawa, Hypoxia and nitric oxide treatment confer tolerance to glucose starvation in a 5'-AMP-activated protein kinase-dependent manner, Journal of Biological Chemistry 277 (2002) 32791-32798.
[47]J. Du, Q. Chen, H. Takemori, H. Xu, SIK2 can be activated by deprivation of nutrition and it inhibits expression of lipogenic genes in adipocytes, Obesity 16(2008)531-538.
[48]A. Suzuki, G.-i. Kusakai, A. Kishimoto, Y. Minegichi, T. Ogura, H. Esumi, Induction of cell-cell detachment during glucose starvation through F-actin conversion by SNARK, the fourth member of the AMP-activated protein kinase catalytic subunit family, Biochemical and biophysical research communications 311 (2003) 156-161.
[49]D.L. Lefebvre, Y. Bai, N. Shahmolky, M. Sharma, R. Poon, D.J. Drucker, C.F. Rosen, Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK, Biochemical Journal 355 (2001) 297.
[50]D.L. Lefebvre, C.F. Rosen, Regulation of SNARK activity in response to cellular stresses, Biochimica et Biophysica Acta (BBA)-General Subjects 1724 (2005) 71-85.
[51]M.J. Lee, D. Feliers, K. Sataranatarajan, M.M. Mariappan, M. Li, J.L. Barnes, G.G. Choudhury, B.S. Kasinath, Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells, Cellular signalling 22 (2010) 65-70.
[52]X. Lin, H. Takemori, Y. Katoh, J. Doi, N. Horike, A. Makino, Y. Nonaka, M. Okamoto, Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells, Molecular Endocrinology 15 (2001) 1264-1276.
[53]Y. Katoh, H. Takemori, X. Lin, M. Tamura, M. Muraoka, T. Satoh, Y. Tsuchiya, L. Min, A. Miyauchi, L.A. Witters, Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade, FEBS Journal 273 (2006) 2730-2748.
[54]J.M. Lizcano, O. Goransson, R. Toth, M. Deak, N.A. Morrice, J. Boudeau, S.A. Hawley, L. Udd, T.P. Makela, D.G. Hardie, LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1, The EMBO journal 23 (2004) 833-843.
[55]J. Xu, Y. Wu, P. Song, M. Zhang, S. Wang, M.H. Zou, Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus, Circulation 116 (2007) 944-953.
[56]A. Trumper, K. Trumper, D. Horsch, Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta (INS-1)-cells, Journal of endocrinology 174 (2002) 233-246.
[57]S. Wang, R. Hirschberg, Diabetes-relevant regulation of cultured blood outgrowth endothelial cells, Microvascular research 78 (2009) 174-179.
[58]A.C. Gouville, V. Boullay, G Krysa, J. Pilot, J.M. Brusq, F. Loriolle, J.M. Gauthier, S.A. Papworth, A. Laroze, F. Gellibert, Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethyInitrosamine-induced liver fibrosis, British journal of pharmacology 145 (2009) 166-177.
[59]Y. Yata, P. Gotwals, V. Koteliansky, D.C. Rockey, Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-β soluble receptor:Implications for antifibrotic therapy, Hepatology 35 (2002) 1022-1030.
[1]A. Andersen, J.S. Christiansen, J. Andersen, S. Kreiner, T. Deckert, Diabetic nephropathy in type 1 (insulin-dependent) diabetes:an epidemiological study, Diabetologia 25 (1983) 496-501.
[2]A. Scheen, Thiazolidinediones and liver toxicity, Diabetes & metabolism 27 (2001).
[3]D.E. Moller, New drug targets for type 2 diabetes and the metabolic syndrome, Nature 414 (2001) 821-827.
[4]M. Okamoto, H. Takemori, Y. Katoh, Salt-inducible kinase in steroidogenesis and adipogenesis, Trends in Endocrinology & Metabolism 15 (2004) 21-26.
[5]J.C. Ruiz, F.L. Conlon, E.J. Robertson, Identification of novel protein kinases expressed in the myocardium of the developing mouse heart, Mechanisms of development 48 (1994) 153-164.
[6]Z.-n. Wang, H. Takemori, S.K. Halder, Y. Nonaka, M. Okamoto, Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal, FEBS letters 453 (1999) 135-139.
[7]Y. Katoh, H. Takemori, N. Horike, J. Doi, M. Muraoka, L. Min, M. Okamoto, Salt-inducible kinase (SIK) isoforms:their involvement in steroidogenesis and adipogenesis, Molecular and cellular endocrinology 217 (2004) 109-112.
[8]A. Lanjuin, P. Sengupta, Regulation of chemosensory receptor expression and sensory signaling by the KIN-29 Ser/Thr kinase, Neuron 33 (2002) 369-381.
[9]B. Wang, J. Goode, J. Best, J. Meltzer, P.E. Schilman, J. Chen, D. Garza, J.B. Thomas, M. Montminy, The Insulin-Regulated CREB Coactivator TORC Promotes Stress Resistance in< i> Drosophila, Cell metabolism 7 (2008) 434-444.
[10]S. Choi, W. Kim, J. Chung, Drosophila salt-inducible kinase (SIK) regulates starvation resistance through CREB-regulated transcription coactivator (CRTC), Journal of Biological Chemistry (2010).
[11]N. Horike, H. Takemori, Y. Katoh, J. Doi, L. Min, T. Asano, X.J. Sun, H. Yamamoto, S. Kasayama, M. Muraoka, Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2, Journal of Biological Chemistry 278(2003)18440-18447.
[12]Y. Katoh, H. Takemori, L. Min, M. Muraoka, J. Doi, N. Horike, M. Okamoto, Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm, European Journal of Biochemistry 271 (2004) 4307-4319.
[13]S.-H. Koo, L. Flechner, L. Qi, X. Zhang, R.A. Screaton, S. Jeffries, S. Hedrick, W. Xu, F. Boussouar, P. Brindle, The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism, Nature 437 (2005) 1109-1111.
[14]R. Berdeaux, N. Goebel, L. Banaszynski, H. Takemori, T. Wandless, G.D. Shelton, M. Montminy, SIK1 is a class Ⅱ HDAC kinase that promotes survival of skeletal myocytes, Nature medicine 13 (2007) 597-603.
[15]N. Bright, C. Thornton, D. Carling, The regulation and function of mammalian AMPK-related kinases, Acta physiologica 196 (2009) 15-26.
[16]D.G. Hardie, The AMP-activated protein kinase pathway-new players upstream and downstream, Journal of cell science 117 (2004) 5479-5487.
[17]J.M. Lizcano, O. Goransson, R. Toth, M. Deak, N.A. Morrice, J. Boudeau, S.A. Hawley, L. Udd, T.P. Makela, D.G. Hardie, LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1, The EMBO journal 23 (2004) 833-843.
[18]R.J. Shaw, M. Kosmatka, N. Bardeesy, R.L. Hurley, L.A. Witters, R.A. DePinho, L.C. Cantley, The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress, Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 3329-3335.
[19]S.A. Hawley, M.A. Selbert, E.G. Goldstein, A.M. Edelman, D. Carling, D.G. Hardie,5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms, Journal of Biological Chemistry 270(1995)27186-27191.
[20]S.A. Hawley, D.A. Pan, K.J. Mustard, L. Ross, J. Bain, A.M. Edelman, B.G. Frenguelli, D.G. Hardie, Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase, Cell metabolism 2 (2005) 9-19.
[21]C.A. Witczak, N. Fujii, M.F. Hirshman, L.J. Goodyear, Ca2+/calmodulin-dependent protein kinase kinase-a regulates skeletal muscle glucose uptake independent of AMP-activated protein kinase and Akt activation, Diabetes 56 (2007) 1403-1409.
[22]M. Momcilovic, S.-P. Hong, M. Carlson, Mammalian TAK1 activates Snfl protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro, Journal of Biological Chemistry 281 (2006) 25336-25343.
[23]K. Sakamoto, A. McCarthy, D. Smith, K.A. Green, D.G. Hardie, A. Ashworth, D.R. Alessi, Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction, The EMBO journal 24 (2005) 1810-1820.
[24]R.J. Shaw, K.A. Lamia, D. Vasquez, S.-H. Koo, N. Bardeesy, R.A. DePinho, M. Montminy, L.C. Cantley, The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin, Science Signalling 310 (2005) 1642.
[25]M. Jaleel, F. Villa, M. Deak, R. Toth, A.R. Prescott, D.M. Van Aalten, D.R. Alessi, The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation, Biochemical Journal 394 (2006) 545.
[26]Y. Katoh, H. Takemori, J. Doi, M. Okamoto, Identification of the nuclear localization domain of salt-inducible kinase, Endocrine research 28 (2002) 315-318.
[27]H. Takemori, Y. Katoh, N. Horike, J. Doi, M. Okamoto, ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase, Journal of Biological Chemistry 277 (2002) 42334-42343.
[28]J. Liu, E. Park, A. Gurney, W. Roesler, R. Hanson, Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements, Journal of Biological Chemistry 266 (1991) 19095-19102.
[29]M.D. Conkright, G. Canettieri, R. Screaton, E. Guzman, L. Miraglia, J.B. Hogenesch, M. Montminy, TORCs:transducers of regulated CREB activity, Molecular cell 12 (2003) 413-423.
[30]B. Mayr, M. Montminy, Transcriptional regulation by the phosphorylation-dependent factor CREB, Nature Reviews Molecular Cell Biology 2 (2001) 599-609.
[31]W.A. Carlezon, R.S. Duman, E.J. Nestler, The many faces of CREB, Trends in neurosciences 28 (2005) 436-445.
[32]M. Johannessen, M.P. Delghandi, U. Moens, What turns CREB on?, Cellular signalling 16 (2004) 1211.
[33]H.A. Giebler, I. Lemasson, J.K. Nyborg, p53 recruitment of CREB binding protein mediated through phosphorylated CREB:a novel pathway of tumor suppressor regulation, Molecular and cellular biology 20 (2000) 4849-4858.
[34]R.A. Screaton, M.D. Conkright, Y. Katoh, J.L. Best, G. Canettieri, S. Jeffries, E. Guzman, S. Niessen, J.R. Yates Ⅲ, H. Takemori, The CREB coactivator TORC2 functions as a calcium-and cAMP-sensitive coincidence detector, Cell 119 (2004)61-74.
[35]R. Dentin, Y. Liu, S.-H. Koo, S. Hedrick, T. Vargas, J. Heredia, J. Yates, M. Montminy, Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2, Nature 449 (2007) 366-369.
[36]M. Muraoka, A. Fukushima, S. Viengchareun, M. Lombes, F. Kishi, A. Miyauchi, M. Kanematsu, J. Doi, J. Kajimura, R. Nakai, Involvement of SIK2/TORC2 signaling cascade in the regulation of insulin-induced PGC-1α and UCP-1 gene expression in brown adipocytes, American Journal of Physiology-Endocrinology And Metabolism 296 (2009) E1430-E1439.
[37]Y. Katoh, H. Takemori, X.z. Lin, M. Tamura, M. Muraoka, T. Satoh, Y. Tsuchiya, L. Min, A. Miyauchi, L.A. Witters, Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade, FEBS Journal 273 (2006) 2730-2748.
[38]Y.-S. Yoon, W.-Y. Seo, M.-W. Lee, S.-T. Kim, S.-H. Koo, Salt-inducible kinase regulates hepatic lipogenesis by controlling SREBP-lc phosphorylation, Journal of Biological Chemistry 284 (2009) 10446-10452.
[39]J. Bricambert, J. Miranda, F. Benhamed, J. Girard, C. Postic, R. Dentin, Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice, The Journal of clinical investigation 120 (2010) 4316.
[40]S.N. Jakobsen, D.G. Hardie, N. Morrice, H.E. Tornqvist,5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside, Journal of Biological Chemistry 276 (2001) 46912-46916.
[41]S. Herzig, F. Long, U.S. Jhala, S. Hedrick, R. Quinn, A. Bauer, D. Rudolph, G. Schutz, C. Yoon, P. Puigserver, CREB regulates hepatic gluconeogenesis through the coactivator PGC-1, Nature 413 (2001) 179-183.
[42]J. Le Lay, G. Tuteja, P. White, R. Dhir, R. Ahima, K.H. Kaestner, CRTC2 (T0RC2) contributes to the transcriptional response to fasting in the liver but is not required for the maintenance of glucose homeostasis, Cell metabolism 10(2009)55-62.
[43]Y. Wang, H. Inoue, K. Ravnskjaer, K. Viste, N. Miller, Y. Liu, S. Hedrick, L. Vera, M. Montminy, Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity, Proceedings of the National Academy of Sciences 107 (2010) 3087-3092.
[44]M. Saberi, D. Bjelica, S. Schenk, T. Imamura, G. Bandyopadhyay, P. Li, V. Jadhar, C. Vargeese, W. Wang, K. Bowman, Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes, American Journal of Physiology-Endocrinology And Metabolism 297 (2009) E1137-E1146.
[45]X.-z. Lin, H. Takemori, Y. Katoh, J. Doi, N. Horike, A. Makino, Y. Nonaka, M. Okamoto, Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Yl mouse adrenocortical tumor cells, Molecular Endocrinology 15 (2001) 1264-1276.
[46]T. Sasaki, H. Takemori, Y. Yagita, Y. Terasaki, T. Uebi, N. Horike, H. Takagi, T. Susumu, H. Teraoka, K.-i. Kusano, SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB, Neuron 69 (2011) 106-119.
[47]R. Dentin, S. Hedrick, J. Xie, J. Yates III, M. Montminy, Hepatic glucose sensing via the CREB coactivator CRTC2, Science Signalling 319 (2008) 1402.
[48]L. He, A. Sabet, S. Djedjos, R. Miller, X. Sun, M.A. Hussain, S. Radovick, F.E. Wondisford, Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein, Cell 137 (2009) 635-646.
[49]D.M. Erion, I.D. Ignatova, S. Yonemitsu, Y. Nagai, P. Chatterjee, D. Weismann, J.J. Hsiao, D. Zhang, T. Iwasaki, R. Stark, Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP response element-binding protein, Cell metabolism 10 (2009) 499-506.
[50]J.G. Salway, Metabolism at a Glance, Blackwell Scientific Publications Ltd, 1994.
[51]R. Raghow, C. Yellaturu, X. Deng, E.A. Park, M.B. Elam, SREBPs:the crossroads of physiological and pathological lipid homeostasis, Trends in Endocrinology & Metabolism 19 (2008) 65-73.
[52]H. Yamashita, M. Takenoshita, M. Sakurai, R.K. Bruick, W.J. Henzel, W. Shillinglaw, D. Arnot, K. Uyeda, A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver, Proceedings of the National Academy of Sciences 98 (2001) 9116-9121.
[53]R. Dentin, J.-P. Pegorier, F. Benhamed, F. Foufelle, P. Ferre, V. Fauveau, M.A. Magnuson, J. Girard, C. Postic, Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression, Journal of Biological Chemistry 279 (2004) 20314-20326.
[54]J. Du, Q. Chen, H. Takemori, H. Xu, SIK2 can be activated by deprivation of nutrition and it inhibits expression of lipogenic genes in adipocytes, Obesity 16(2008)531-538.
[55]Y. Song, J. Altarejos, M.O. Goodarzi, H. Inoue, X. Guo, R. Berdeaux, J.-H. Kim, J. Goode, M. Igata, J.C. Paz, CRTC3 links catecholamine signalling to energy balance, Nature 468 (2010) 933-939.
[56]S. Kajimura, P. Seale, B.M. Spiegelman, Transcriptional control of brown fat development, Cell metabolism 11 (2010) 257-262.
[57]P. Vague, T. Coste, M. Jannot, D. Raccah, M. Tsimaratos, C-peptide, Na, Experimental Diabetes Research 5 (2004) 37-50.
[58]H. Poulsen, P. Morth, J. Egebjerg, P. Nissen, Phosphorylation of the Na< sup>+, K< sup>+-ATPase and the H< sup>+, K< sup>+-ATPase, FEBS letters 584 (2010) 2589-2595.
[59]S. Sackmann, U. Lichtenauer, I. Shapiro, M. Reincke, F. Beuschlein, Aldosterone producing adrenal adenomas are characterized by activation of calcium/calmodulin-dependent protein kinase (CaMK) dependent pathways, Hormone and metabolic research 43 (2011) 106.
[60]R.L. Hurley, K.A. Anderson, J.M. Franzone, B.E. Kemp, A.R. Means, L.A. Witters, The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases, Journal of Biological Chemistry 280 (2005) 29060-29066.
[61]H.S. Jun, K. Hwang, Y. Kim, T. Park, High-fat Diet Alters PP2A, TC10, and CIP4 Expression in Visceral Adipose Tissue of Rats, Obesity 16 (2012) 1226-1231.
[62]E. Lecuona, L.A. Dada, H. Sun, M.L. Butti, G. Zhou, T.-L. Chew, J.I. Sznajder, Na, K-ATPase al-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane, The FASEB journal 20 (2006) 2618-2620.
[63]S. Longin, J. Jordens, E. Martens, I. Stevens, V. Janssens, E. Rondelez, I. De Baere, R. Derua, E. Waelkens, J. Goris, An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator, Biochemical Journal 380 (2004) 111.
[64]M. Sjostrom, K. Stenstrom, K. Eneling, J. Zwiller, A.I. Katz, H. Takemori, A.M. Bertorello, SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process, Proceedings of the National Academy of Sciences 104 (2007) 16922-16927.
[65]K. Stenstrom, H. Takemori, G. Bianchi, A.I. Katz, A.M. Bertorello, Blocking the salt-inducible kinase 1 network prevents the increases in cell sodium transport caused by a hypertension-linked mutation in human [alpha]-adducin, Journal of hypertension 27 (2009) 2452.
[66]Y. Lee, H. Han, Regulatory mechanisms of Na+/glucose cotransporters in renal proximal tubule cells, Kidney International 72 (2007) S27-S35.
[67]A. Fekete, K. Rosta, L. Wagner, A. Prokai, P. Degrell, E. Ruzicska, E. Vegh, M. Toth, K. Ronai, K. Rusai, Na+, K+- ATPase is modulated by angiotensin Ⅱ in diabetic rat kidney-another reason for diabetic nephropathy?, The Journal of Physiology 586 (2008) 5337-5348.
[68]A. Jaitovich, A.M. Bertorello, Intracellular sodium sensing:SIK1 network, hormone action and high blood pressure, Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1802 (2010) 1140-1149.
[69]K. Stenstrom, Salt-inducible kinase 1:a novel regulator within a cell sodium-sensing network, Karolinska institutet,2010.
[70]N. Horike, A. Kumagai, Y. Shimono, T. Onishi, Y. Itoh, T. Sasaki, K. Kitagawa, O. Hatano, H. Takagi, T. Susumu, Downregulation of SIK2 expression promotes the melanogenic program in mice, Pigment cell & melanoma research 23 (2010) 809-819.
[71]C. Hyman, M. Hofer, Y.-A. Barde, M. Juhasz, G.D. Yancopoulos, S.P. Squinto, R.M. Lindsay, BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra, Nature 350 (1991) 230-232.
[72]J. Cheng, M. Uchida, W. Zhang, M.R. Grafe, P.S. Herson, P.D. Hum, Role of salt-induced kinase 1 in androgen neuroprotection against cerebral ischemia, Journal of Cerebral Blood Flow & Metabolism 31 (2010) 339-350.
[73]J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, The Salt-Inducible Kinase, SIK, Is Induced by Depolarization in Brain, Journal of neurochemistry 74 (2002) 2227-2238.
[74]H. Cheng, P. Liu, Z.C. Wang, L. Zou, S. Santiago, V. Garbitt, O.V. Gjoerup, J.D. Iglehart, A. Miron, A.L. Richardson, SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis, Science Signalling 2 (2009) ra35.
[75]A. Romito, E. Lonardo, G. Roma, G. Minchiotti, A. Ballabio, G. Cobellis, Lack of sikl in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2, PloS one 5 (2010)e9029.
[76]A.M. van der Linden, K.M. Nolan, P. Sengupta, KIN-29 SIK regulates chemoreceptor gene expression via an MEF2 transcription factor and a class Ⅱ HDAC, The EMBO journal 26 (2006) 358-370.
[77]E. Kozhemyakina, T. Cohen, T.-P. Yao, A.B. Lassar, Parathyroid hormone-related peptide represses chondrocyte hypertrophy through a protein phosphatase 2A/histone deacetylase 4/MEF2 pathway, Molecular and cellular biology 29 (2009) 5751-5762.
[78]M. Kowanetz, P. Lonn, M. Vanlandewijck, K. Kowanetz, C.H. Heldin, A. Moustakas, TGFβ induces SIK to negatively regulate type I receptor kinase signaling, The Journal of cell biology 182 (2008) 655-662.
[79]P. Lonn, M. Vanlandewijck, E. Raja, M. Kowanetz, Y. Watanabe, K. Kowanetz, E. Vasilaki, C.H. Heldin, A. Moustakas, Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type Ⅰ receptor signaling in cooperation with the Smurf2 ubiquitin ligase, Journal of Biological Chemistry 287 (2012) 12867-12878.
[80]E. Raja, Cross-regulation between TGFβ/BMP Signalling and the metabolic LKB1 pathway, Uppsala University,2012.
[81]M. Vanlandewijck, Diversification of TGF-β Signaling in Homeostasis and Disease, Uppsala University,2011.
[82]I. Magnusson, D. Rothman, L. Katz, R. Shulman, G. Shulman, Increased rate of gluconeogenesis in type II diabetes mellitus. A 13C nuclear magnetic resonance study, Journal of Clinical Investigation 90 (1992) 1323.
[83]J.C. Yoon, P. Puigserver, G. Chen, J. Donovan, Z. Wu, J. Rhee, G. Adelmant, J. Stafford, C.R. Kahn, D.K. Granner, Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1, Nature 413 (2001) 131-138.