降糖增效方对糖尿病大鼠肝脏胰岛素信号通路IRS1位点磷酸化的影响

目的通过观察降糖增效方对糖尿病大鼠肝脏胰岛素受体底物1(insulin receptor substrates 1,IRS1)磷酸化的影响来探讨该方发挥降糖增效作用的机制。方法将40只6~8周龄雄性ZDF(fa/fa)大鼠随机分为模型组、二甲双胍组(0.134 g/kg)、降糖增效方组(0.64 g/kg)、联合组(降糖增效方0.64 g/kg+二甲双胍0.134 g/kg),另选ZDF(fa/+)大鼠10只为正常组,连续干预6周;实验结束检测体空腹血糖(fasting blood glucose,FBG)、血清胰岛素水平(fasting insulin,FINS)、胰岛素抵抗指数(homeostatic model assessment of insulin resistance,HOMA-IR)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、血肌酐(serum creatinine,Scr)、尿素氮(blood urea nitrogen,BUN)、口服葡萄糖耐量试验(oral glucose tolerance test,OGTT);蛋白免疫印迹法(Western bloting)检测IRS1丝氨酸磷酸化位点ser307、ser612、ser1101表达。结果与模型组相比较,各治疗组大鼠FBG、FIns、HOMA-IR、OGTT2 h血糖水平、AUC、SCr、BUN显著降低(P<0.05,P<0.01);肝组织p-IRS1 Ser307、p-IRS1 ser612、p-IRS1 ser1101蛋白表达显著降低(P<0.05,P<0.01);与二甲双胍、降糖增效方组比较,联合组大鼠FBG、FIns、HOMA-IR显著降低(P<0.05,P<0.01);OGTT2 h血糖水平以及AUC显著降低(P<0.05,P<0.01);肝组织p-IRS1 Ser307、p-IRS1 ser612、p-IRS1 Ser1101蛋白表达显著降低(P<0.05,P<0.01)。结论降糖增效方降低糖尿病大鼠肝脏IRS1 ser307/612/1101位点磷酸化水平,这可能是其增加降糖疗效的机制。     

The docking protein FRS2α is a critical regulator of VEGF receptors signaling

Vascular endothelial growth factors (VEGFs) signal via their cognate receptor tyrosine kinases designated VEGFR1-3. We report that the docking protein fibroblast growth factor receptor substrate 2 (FRS2α) plays a critical role in cell signaling via these receptors. In vitro FRS2α regulates VEGF-A and VEGF-C–dependent activation of extracellular signal-regulated receptor kinase signaling and blood and lymphatic endothelial cells migration and proliferation. In vivo endothelial-specific deletion of FRS2α results in the profound impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis. We conclude that FRS2α is a previously unidentified component of VEGF receptors signaling.Vascular endothelial growth factors (VEGFs) are key regulators of blood and lymphatic vessel development and homeostasis. The absence of VEGF-A during development results in a complete failure of blood vasculature formation (1), whereas VEGF-C knockout abolishes lymphangiogenesis (2). Both VEGF-A and VEGF-C play equally critical roles in postnatal formation and maintenance of various blood and lymphatic vessel beds (3–6). The two VEGFs signal via, respectively, the two receptor tyrosine kinases (RTK) VEGFR2 and VEGFR3 with the former primarily expressed in the arterial and venous vasculature and the latter in the lymphatic vasculature in adult tissues (7). The other VEGF receptor, VEGFR1, is thought to function largely as a “decoy” receptor in endothelial cells but can transmit signaling in mononuclear cells (7).All VEGF receptors share a number of structural similarities including an extracellular ligand binding domain, a single transmembrane region, and a cytoplasmic domain containing a tyrosine kinase domain with an insert region. Receptor activation requires ligand-induced dimerization that results in autophosphorylation of cytoplasmic tyrosines that serve as binding sites for various signaling proteins. In the case of VEGF-A receptor, VEGFR2 phosphorylation of Y¹⁰⁵⁴/Y¹⁰⁵⁹ is required for maximal VEGFR2 kinase activity that leads to phosphorylation of Y¹¹⁷⁵ (a phospholipase Cγ1 binding site also required for ERK activation) and Y⁹⁵¹ (a TSAd binding site leading to Src activation) among others (7). VEGF-C signals via VEGFR3 in a similar manner.In the course of studying fibroblast growth factor (FGF) signaling, we noticed that an endothelial knockdown or deletion of a scaffold protein FRS2α, known to be involved in FGF and neural growth factor (NGF) receptor signaling (8–10), also affects VEGF signaling. FRS2α is a docking protein that contains an N-terminal myristylation site, a PTB domain, and a large C-terminal tail that contains four binding sites for the SH2 domain of the adaptor protein Grb2 and two binding sites for the SH2 domain of the tyrosine phosphatase Shp2 (11). Tyrosine phosphorylation of FRS2α at these binding sites leads to activation of MAPK signaling (12).In this study, we found that FRS2α plays a central role in regulation of VEGF signaling in blood and lymphatic endothelial cells. Its deletion profoundly reduced VEGF signaling in vitro and in vivo and resulted in impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis.     

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1_S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1_S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1_L, Thr²⁹. Furthermore, the association of HVCN1_S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1_S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1_S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.The voltage-gated proton channel HVCN1 (or H_V1 or VSOP) is a small protein that conducts protons across membranes selectively (1, 2) and in a regulated manner. Previously, we described its function in B lymphocytes, where proton channels sustain B-cell receptor (BCR) signaling via regulation of reactive oxygen species production by the NADPH oxidase enzyme complex (3). In addition, we found HVCN1 to be directly associated with the BCR. Upon receptor stimulation, the BCR and HVCN1 were cointernalized to late endosomal/lysosomal organelles called “MIICs,” or MHC class II-containing compartments, where antigens bound to the BCR are digested into small peptides and loaded onto MHC class II molecules for presentation to T cells (3).HVCN1 is expressed not only by normal but also by malignant B cells, such as those in chronic lymphocytic leukemia (CLL) (3). CLL cells are characterized by their reliance on BCR signaling for survival and growth (4), so it is possible that they maintain or upregulate HVCN1 expression to sustain their growth. Other tumor cells, such as those in breast (5) and colorectal cancer (6), have been found to rely on HVCN1 for survival. In these tumor cells, proton channels prevent excessive acidification of the cytoplasm and allow increased cell migration. In malignant B cells, HVCN1 may regulate intracellular pH and at the same time sustain BCR signaling. However, its precise roles remain to be elucidated.We show here that CLL cells and other B-cell lines specifically express higher levels of a shorter isoform of HVCN1, HVCN1_S. We identified the existence of two distinct isoforms of relatively similar size when immunoblotting B-cell lysates with an HVCN1-specific antibody (3). HVCN1_S is only weakly expressed in normal B cells, and in light of its apparent upregulation in tumor cells, we set out to characterize its function. We show that HVCN1_S responds more strongly to phosphorylation by protein kinase C (PKC) and identify the phosphorylation site. We provide evidence that HVCN1_S in B cells is preferentially expressed at the plasma membrane, even upon BCR stimulation and subsequent internalization, due to a weaker association with the BCR. Finally, we show that HVCN1_S expression results in stronger BCR signaling, increased proliferation, and augmented chemokine-dependent migration. Overall, our data indicate that HVCN1_S is an alternative protein isoform that mediates stronger currents upon PKC phosphorylation, is more highly expressed at the plasma membrane, and can confer a growth advantage to malignant B cells.     

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.     

Modulation of cardiac contractility by serine/threonine protein phosphatase type 5

Background

Protein phosphatase 5 (PP5) a serine/threonine phosphatase is ubiquitously expressed in mammalian tissues including the heart, but its physiological role in the heart is still unknown. Therefore, we used a transgenic mouse model to get a first insight into the cardiac role of PP5.

Methods and results

We generated transgenic mice with cardiac myocyte specific overexpression of PP5. Successful overexpression of PP5 was demonstrated by Western blotting, immunohistochemistry and enhanced arachidonic acid-stimulated protein phosphatase activity in transgenic hearts. Cardiac function was examined on the level of isolated cardiac myocytes, isolated organs and in intact animals. Whereas Ca²⁺ transients and cell shortening remained unchanged, L-type Ca²⁺ currents were decreased in isolated cardiac myocytes from transgenic mice. Ventricular contractility was reduced in isolated perfused hearts under basal conditions and after β-adrenergic stimulation. In intact animals, echocardiography revealed increased left ventricular diameters and decreased contractility and invasively measured hemodynamic performance by left ventricular catheterization demonstrated a reduced response to β-adrenergic stimulation in transgenic mice compared to wild type.

Conclusions

Overexpression of PP5 affected contractility and β-adrenergic signaling in the hearts of transgenic mice. Taken together, these findings are indicative of a regulatory role of PP5 in cardiac function.     

Calorie restriction in mice overexpressing UCP3: evidence that prior mitochondrial uncoupling alters response

Calorie restriction (CR) without malnutrition is the only intervention to consistently increase lifespan in all species tested, and lower age-related pathologies in mammals including humans. It has been suggested that uncoupling of mitochondrial oxidative phosphorylation, using chemical uncouplers, mimics CR, and that overlapping mechanisms underlie the phenotypic changes induced by uncoupling and CR. We aimed to critically assess this using a unique mouse model of skeletal muscle-targeted UCP3-induced uncoupling (UCP3Tg), and focused our studies mainly on skeletal muscle mitochondria. Compared to ad libitum fed Wt mice, skeletal muscle mitochondria from ad libitum fed UCP3Tg mice showed higher basal uncoupling and lower H(2)O(2) emission, with unchanged maximal oxidative phosphorylation, and mitochondrial content. UCP3Tg CR mice showed some tendency for differential adaptation to CR, with lowered H(+) leak conductance and evidence for higher H(2)O(2) emission from skeletal muscle mitochondria following 2 weeks CR, and failure to lower H(2)O(2) emission after 1 month CR. Differential adaptation was also apparent at the whole body level: while UCP3Tg CR mice lost as much weight as Wt CR mice, the proportion of muscle lost was higher in UCP3Tg mice. However, a striking outcome of our studies was the absence of change with CR in many of the parameters of mitochondrial function and content that we measured in mice of either genotype. Overall, our study raises the question of whether CR can consistently modify skeletal muscle mitochondria; alterations with CR may only be apparent under certain conditions such as during the 2 wk CR intervention in the UCP3Tg mice.     

阿尔茨海默病大鼠海马胆固醇水平升高对tau蛋白磷酸化程度增加的影响

目的观察海马胆固醇含量升高后tau蛋白磷酸化程度的变化,探讨其在阿尔茨海默病(AD)中可能的作用。方法水迷宫训练大鼠7 d筛选合格大鼠后,海马直接注射不同剂量胆固醇,72 h后水迷宫检测行为学变化,并以免疫印迹和免疫组织化学方法检测大鼠海马tau蛋白磷酸化水平。结果注射胆固醇后72 h,中高剂量组大鼠水迷宫潜伏期明显延长(P<0.01),tau蛋白PHF-1位点磷酸化水平明显增高(P<0.01),tau-1明显降低(P<0.01),总tau蛋白(R134d)无明显变化。结论海马胆固醇水平升高可导致tau蛋白磷酸化程度增加,可能参与AD的形成。     

Leber's Hereditary Optic Neuropathy Arising From the Synergy Between ND1 3635G>A Mutation and Mitochondrial YARS2 Mutations

PurposeTo investigate the mechanism underlying the synergic interaction between Leber''s hereditary optic neuropathy (LHON)-associated ND1 and mitochondrial tyrosyl-tRNA synthetase (YARS2) mutations.MethodsMolecular dynamics simulation and differential scanning fluorimetry were used to evaluate the structure and stability of proteins. The impact of ND1 3635G>A and YARS2 p.G191V mutations on the oxidative phosphorylation machinery was evaluated using blue native gel electrophoresis and enzymatic activities assays. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analysis of effect of mutations on autophagy was undertaken via flow cytometry for autophagic flux.ResultsMembers of one Chinese family bearing both the YARS2 p.191Gly>Val and m.3635G>A mutations exhibited much higher penetrance of optic neuropathy than those pedigrees carrying only the m.3635G>A mutation. The m.3635G>A (p.Ser110Asn) mutation altered the ND1 structure and function, whereas the p.191Gly>Val mutation affected the stability of YARS2. Lymphoblastoid cell lines harboring both m.3635G>A and p.191Gly>Val mutations revealed more reductions in the levels of mitochondrion-encoding ND1 and CO2 than cells bearing only the m.3635G>A mutation. Strikingly, both m.3635G>A and p.191Gly>Val mutations exhibited decreases in the nucleus-encoding subunits of complex I and IV. These deficiencies manifested greater defects in the stability and activities of complex I and complex IV and overproduction of ROS and promoted greater autophagy in cell lines harboring both m.3635G>A and p.191Gly>Val mutations compared with cells bearing only the m.3635G>A mutation.ConclusionsOur findings provide new insights into the pathophysiology of LHON arising from the synergy between ND1 3635G>A mutation and mitochondrial YARS2 mutations.