The dwarf mutation decreases high dose insulin responses in skeletal muscle,the opposite of effects in liver

The in vivo status of the proximal components of the insulin signaling system was investigated in skeletal muscle of Ames (Prop1df/Prop1df) dwarf mice. The insulin-stimulated phosphorylation of the insulin receptor (IR) was reduced by 55% in Ames dwarf mice, while IR receptor protein content was not altered. Insulin-stimulated phosphorylation of IRS-1 and IRS-2 were decreased by 79 and 51%, respectively, while IRS-1 and IRS-2 protein levels were decreased by 66 and 43%. In addition, insulin-stimulated association of IRS-1 and IRS-2 with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase was significantly reduced (by 80 and 41%, respectively), whereas insulin-stimulated PI 3-kinase activity was reduced by 66%. However, insulin-stimulated phosphorylation of Akt was slightly reduced (by 20%), suggesting that the attenuation of insulin signaling downstream PI 3-kinase may involve other signaling molecules. Our current results demonstrate that the Prop1 mutation decreases high dose insulin responses in skeletal muscle. This alteration is remarkable because these animals are hypersensitive to insulin and display an augmented response to insulin in liver at the same signaling steps. Reduced response to insulin in skeletal muscle could be important for the control of glucose homeostasis in these animals and could have implications in their extended longevity.     

Age-associated alterations in cardiac and skeletal muscle glucose transporters, insulin and IGF-1 receptors, and PI3-kinase protein contents in the C57BL/6 mouse

We investigated potential age-related changes in cardiac and skeletal muscle protein contents of glut-4 and glut-1 transporters, insulin and insulin-like growth factor-1 (IGF-1) receptors, and phosphatidylinositol 3-kinase (PI3-kinase) in the C57B1/6 mouse. Myocardial glut-4 content increased four- to five-fold between mid- to late-adulthood with no further age-related changes. Increases in myocardial glut-1 preceded the increase in glut-4 and was of a much smaller magnitude (25-40%). Skeletal muscle glut-4 was also increased (38-49%) and no further changes were noted between adulthood and old age. Cardiac insulin receptor and the p85 alpha subunit of PI3-kinase both declined by about 40%, whereas the skeletal muscle content of these two proteins were unaffected by aging. Cardiac (-23 to -24%) and skeletal muscle (-40 to -62%) IGF-1 receptor levels were decreased in adult and old animals with senescence being associated with a further decrease in cardiac IGF-1 receptor levels to 20% of controls. A two- to three-fold increase in both basal and maximal in vitro autophosphorylation of the cardiac insulin and IGF-1 receptors by their respective ligands was observed with senescence. It appears that cardiac and skeletal muscle demonstrate differential responses in terms of the magnitude and temporal responses of age-associated alterations in glucose transport related protein contents in the C57B1/6 mouse.     

Insulin-inducible changes in the relative ratio of PTP1B splice variants

The skeletal muscle activity of protein tyrosine phosphates 1B (PTP1B), a modulator of insulin and IGF-1 signaling, is reduced in obese nondiabetic subjects and in subjects with type 2 diabetes in comparison with leaner, nondiabetic controls. PTP1B mRNA, like many other signaling molecules, including the insulin receptor, is alternatively spliced. Since we have shown that the ratio of the insulin receptor splice variants is modulated by insulin in vitro and is related to insulin levels in vivo, we hypothesized that the relative ratios of the alternatively spliced PTP1B mRNA might also vary in humans in proportion to the degree of hyperinsulinemia. This was tested in 21 nondiabetic Pima Indians, a population at increased risk for obesity and type 2 diabetes. The relative ratio of the PTP1B splice variants was quantified using RT-PCR of total RNA extracted from fractionated monocytes. The ratio of the splice variants was positively correlated with fasting plasma insulin concentration (r = 0.757; P = 0.0001), 2-h plasma insulin concentration following an oral glucose tolerance test (r = 0.614; P = 0.01, n = 16), and percentage of body fat (r = 0.746; P = 0.0001). These data indicate that variability in the ratio of the two splice variants is due, in part, to in vivo levels of chronic hyperinsulinemia. This simple, noninvasive assay is therefore a potential biomarker for chronic hyperinsulinemia, similar to the HbAlc assay in use to monitor glucose management in diabetic patients.     

Hydrogen peroxide-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 microM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) inhibited this activation. An increase in [Ca2+], was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85alpha, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85alpha and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+]i was a regulation factor for phosphorylation of IRS-1.     

Molecular screening of the human glutamine-fructose-6-phosphate amidotransferase 1 (GFPT1) gene and association studies with diabetes and diabetic nephropathy

Increased glucose metabolism through the hexosamine pathway may result in insulin resistance, impaired insulin secretion, and diabetic nephropathy. We hypothesized that variants of GFPT1 encoding glutamine-fructose-6-phosphate amidotransferase, the rate limiting enzyme in this pathway, could increase GFPT1 gene expression and thus susceptibility to diabetes and diabetic nephropathy. To test this hypothesis, we screened for variation in the GFPT1 and flanking regions in Caucasian and African-American individuals. We tested each variant with over 5% allele frequency for an association with type 2 diabetes in Caucasian and African-American populations, and for an association with diabetic nephropathy in African-American subjects. We measured allele specific levels of GFPT1 mRNA and we compared mRNA levels across diagnostic categories for each ethnic group using RNA derived from transformed lymphocytes. None of the 8 variants detected altered the coding sequence or was present in a known regulatory region. We found a marginal association (p = 0.044) of 1/6 variants with diabetes in Caucasian subjects, and marginal associations of 2/7 variants with diabetic nephropathy among African-American subjects (p = 0.025, p = 0.041). Alleles marked by a variant in the 3' untranslated region were equally expressed, but in a small sample, GFPT1 mRNA levels were increased by 60% in Caucasians with diabetic nephropathy compared to diabetic individuals without nephropathy. Variants in the GFPT1 gene show suggestive evidence of an association with diabetic nephropathy among African-American individuals, and increased GFPT1 gene expression may characterize Caucasian subjects with diabetic nephropathy. Both findings need to be confirmed in other populations.     

不同波动水平的高糖对大鼠骨骼肌细胞PI-3K表达的影响

目的:探讨不同程度血糖升高及血糖波动对原代培养大鼠骨骼肌细胞的葡萄糖转运活动及磷脂酰肌醇3激酶蛋白表达的影响。方法: 原代培养大鼠骨骼肌细胞在5、25 mmol/L及间断的细胞外高浓度的血糖(5-25 mmol/L)波动(波动频者为B、波动小者A)中孵育48 h, 然后测定:葡萄糖转运活动;磷脂酰肌醇3激酶85亚单位(p85)的蛋白表达及其mRNA水平。结果: 高糖抑制了这些细胞的葡萄糖转运活动,削弱了磷脂酰肌醇3激酶85亚单位(p85)的蛋白表达及下调其mRNA水平。且血糖波动范围大者更为明显。结论: 高糖能抑制骨骼肌细胞的糖转运活动,诱导胰岛素抵抗。但是如果将培养基中葡萄糖浓度按高低循环的方式给予,即刺激血糖波动,那么血糖波动范围大者p85水平降低得更明显。     

Functional folding of a cytoplasmic single-chain variable fragment and its use as elutable protein purification tag

The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85alpha) of phosphatidylinositol-3-kinase (PI3K) (isc-p85alpha), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85alpha fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85alpha. This indicates that the iscFv part of isc-p85alpha does not negatively influence p85alpha folding and interaction with p110. Moreover, successful incorporation of the p85alpha-moiety of isc-p85alpha into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.     

Inhibition of phosphatidylinositide 3-kinase in OK-cells reduces Na/Pi-cotransport but does not interfere with its regulation by parathyroid hormone

The importance of phosphatidylinositide 3- kinase(s) [PI 3-kinase(s)] in membrane trafficking processes led us to examine its/their possible role in parathyroid-hormone- (PTH-) induced endocytosis and lysosomal degradation of the type IIa Na/P_i-cotransporter in opossum kidney cells (OK-cells). We used wortmannin, a potent inhibitor of several mammalian PI 3-kinase isoforms, and measured Na/P_i-cotransporter activity and type IIa Na/P_i-cotransporter protein expression; also the induction of a negative dominant subunit (Δp85) was used to reduce PI 3-kinase activity. Wortmannin and Δp85 led to a reduction of Na/P_i-cotransport activity but were unable to prevent its inhibition by PTH. Wortmannin led in a dose- and time-dependent manner to a reduction of Na/P_i-cotransport activity and transporter protein expression, and retarded their recovery from PTH-induced inhibition/degradation. The data suggest that a PI 3-kinase ”controlled” mechanism is involved in the synthesis (and/or routing) of the apical type IIa Na/P_i-cotransporter in OK-cells. Received: 19 February 1999 / Received after revision and accepted: 21 April 1999     

Characterization of GLUT4 and calpain expression in healthy human skeletal muscle during fasting and refeeding

Aims: Calpain‐10 and calpain‐3 and the diabetes ankyrin repeat protein (DARP) have all been linked to insulin resistance and type 2 diabetes. We set out to measure the expression of these genes in human skeletal muscle and relate them to functional measurements of insulin action during fasting (which induces insulin resistance) and refeeding (which reverses it). Methods: Ten healthy male volunteers underwent 48 h of starvation followed by 24 h of high carbohydrate refeeding. On three occasions, before and after starvation and after refeeding, subjects underwent a 16 min insulin tolerance test to quantify insulin sensitivity. Muscle biopsies were obtained before and after fasting and after refeeding for the analysis of calpain‐10 and calpain‐3, GLUT4 and DARP expression by Western blotting and real‐time PCR. Results: Fasting led to a marked reduction in whole body insulin sensitivity by approx. 45% (P < 0.01) and skeletal muscle GLUT4 gene expression by approx. 40% (P < 0.05). However, fasting had no effect on calpain‐10 and calpain‐3 mRNA or protein levels, or DARP mRNA expression. Refeeding only partly restored insulin sensitivity and GLUT4 gene expression to their pre‐fast values, but did not effect the expression of calpain‐10, calpain‐3 or DARP. Conclusions: These findings demonstrate that in healthy non‐diabetic humans induction of insulin resistance by fasting and its reversal by refeeding with a high CHO diet is mirrored by changes in skeletal muscle GLUT4 but not calpain‐10 and calpain‐3 expression.     

PI3K is a negative regulator of IgE production

The production of IgE, a main player in allergic disorders such as asthma and atopic dermatitis, is strictly regulated and the serum concentrations of IgE are normally kept at a much lower level than other isotypes. We found that mice deficient for the p85alpha regulatory subunit of class IA phosphoinositide 3-kinase (PI3K) produced increasing amounts of serum IgE. Purified p85alpha-/- B cells produced more IgE than wild-type B cells in vitro in response to anti-CD40 mAb and IL-4. PI3K inhibitors wortmannin and IC87114 enhanced IgE production by wild-type B cells stimulated with anti-CD40 mAb and IL-4. Under the same condition, antigen receptor cross-linking induced the expression of inhibitor of differentiation-2 and suppressed the expression of activation-induced cytidine deaminase and class switch recombination (CSR) in a PI3K-dependent manner. IgE production was also suppressed in a concentrated cell culture condition, which was completely reversed by PI3K inhibition. The selective suppression of IgE production by PI3K was also observed at a protein level after CSR. Our results indicate that PI3K negatively regulates IgE production at both CSR and protein levels.     

Deletion of PI3K-p85alpha gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells

Class IA phosphotidylinositol-3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that are important in regulating signaling events in B and T cells. However, their role in natural killer (NK) cells is not understood. Here, using mice that lack the regulatory p85alpha subunit and its alternatively spliced variants p55alpha/p50alpha (collectively termed as p85alpha(-/-)), we defined the role of PI3K in NK cell development and function. p85alpha(-/-) mice had impaired lineage commitment leading to reduced NK cellularity in the bone marrow and liver. p85alpha(-/-) NK cells showed a defective Ly49 subset specification and a decreased expression of CD43. Lack of p85alpha severely reduced the NK-mediated cytotoxicity against tumor cells representing 'induced-self' and 'missing-self'. More importantly, NKG2D and NK1.1 receptor-mediated cytokine and chemokine generation was significantly compromised in p85alpha(-/-) NK cells. These results reveal a previously unrecognized role of p85alpha in the development, terminal maturation, cytokine/chemokine generation and tumor clearance of NK cells.     

Calpain inhibition and insulin action in cultured human muscle cells

Variation in the calpain 10 gene has been reported to increase susceptibility to type 2 diabetes. Part of this susceptibility appears to be mediated by a decrease in whole body insulin sensitivity. As skeletal muscle is the primary tissue site of the peripheral insulin resistance in type 2 diabetes, the aim of this study was to use a human skeletal muscle cell culture system to explore the effects of calpain inhibition on insulin action. Calpain 10 mRNA and protein expression was examined in cultured myoblasts, myotubes, and whole skeletal muscle from non-diabetic subjects using RT-PCR and Western blotting. Changes in insulin-stimulated glucose uptake and glycogen synthesis in response to the calpain inhibitors ALLN and ALLM were measured. Calpain 10 expression was confirmed in cultured human myoblasts, myotubes, and native skeletal muscle. Insulin-stimulated glucose uptake was significantly decreased following preincubation with ALLN [404+/-40 vs 505+/-55 (mean+/-SEM)pmol/mg/min; with vs without ALLN: p = 0.04] and ALLM [455+/-38 vs 550+/-50 pmol/mg/min; with vs without ALLM: p = 0.025] in day 7 fused myotubes, but not in myoblasts. Neither ALLN nor ALLM affected insulin-stimulated glycogen synthesis in myoblasts or myotubes. These studies confirm calpain 10 expression in cultured human muscle cells and support a role for calpains in insulin-stimulated glucose uptake in human skeletal muscle cells that may be relevant to the pathogenesis of the peripheral insulin resistance in type 2 diabetes.     

AMPK/PGC-1α在有氧运动改善2型糖尿病大鼠骨骼肌萎缩中的作用

文题释义：腺苷酸活化蛋白激酶(AMPK)：是生物能量代谢调节的关键分子,AMPK在低氧、缺血、运动和营养缺乏等条件下易被激活,是研究糖尿病及其他代谢相关疾病的核心因子。 肌萎缩：宏观上表现为肌肉体积和质量的降低,微观上表现为肌纤维数目或直径减少。骨骼肌是摄取和利用葡萄糖的重要组织,肌萎缩的发生将增加2型糖尿病等代谢性疾病的发病风险。 背景：腺苷酸活化蛋白激酶(AMP-activated kinase,AMPK)对线粒体能量代谢功能的调节障碍是导致肥胖和2型糖尿病患者脂肪堆积的重要原因,长期慢性炎症反应还将进一步诱导骨骼肌萎缩的发生,有氧运动可以提高AMPK的活性并调节能量代谢,但是通过有氧运动提高AMPK改善2型糖尿病骨骼肌萎缩的作用机制尚不明确。 目的：探究有氧运动对2型糖尿病大鼠骨骼肌萎缩的影响,以及AMPK在其中的作用机制。 方法：采用高脂饲养联合链脲佐菌素注射建立2型糖尿病大鼠模型20只,建模后将大鼠分为糖尿病组(n=8)和糖尿病运动组(n=12),同时将正常大鼠15只分为安静对照组(n=6)和运动组(n=9),其中安静对照组和糖尿病组继续饲养4周,运动组和糖尿病运动组进行有4周有氧运动干预(跑速16 m/min,60 min/d,5 d/周),运动干预后取大鼠比目鱼肌免疫组织化学染色观察各组肌萎缩情况,Western blot检测AMPK、PGC-1α、MAFbx和MuRF1蛋白表达情况。实验已于2016-06-25通过北京体育大学运动科学实验伦理委员会批准(批准号：2016014)。 结果与结论：①高脂饲养联合链脲佐菌素注射建立的2型糖尿病模型大鼠血糖显著升高、体质量和胰岛素水平显著下降(P < 0.01);②糖尿病组大鼠比目鱼肌肌纤维平均横截面积较安静对照组显著降低(P < 0.01),糖尿病运动组大鼠比目鱼肌肌纤维平均横截面积较糖尿病组显著升高(P < 0.01);③糖尿病组大鼠比目鱼肌中AMPK和PGC-1α表达水平较安静对照组显著降低,MAFbx和MuRF1表达水平较安静对照组显著升高(P < 0.01);糖尿病运动组大鼠AMPK表达水平较糖尿病组显著升高,MAFbx和MuRF1的水平较糖尿病组显著降低(P < 0.01);④上述结果说明,有氧运动通过激活AMPK/PGC-1α信号通路,提高线粒体功能,抑制MAFbx和MuRF1表达水平,改善骨骼肌萎缩,在一定程度上恢复了2型糖尿病的代谢平衡。 ORCID: 0000-0003-0979-7741(王继) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Activation of phosphoinositide 3-kinase by the NBS1 DNA repair protein through a novel activation motif

Class IA phosphoinositide 3-kinases (PI 3-kinases) are key signaling components downstream of tyrosine kinases and Ras, regulating many different cellular functions and contributing to tumorigenesis. Class IA PI 3-kinases are heterodimers comprised of a p85 regulatory and a p110 catalytic subunit. Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product p95 (also known as NBS1) is part of the Mre11-Rad50-Nbs1 complex, a central player associated with double-strand break repair. We previously demonstrated that NBS1 overexpression induces transformation through activation of PI 3-kinase/Akt. In this study, we show that NBS1 directly interacts, through a highly conserved C-terminal motif (aa 653-669) of NBS1, with the N-terminal domain (aa 1-108) of the p110alpha catalytic subunit of PI 3-kinase, and stimulates PI 3-kinase activity. Mutations of different regions of the conserved motif abolish the ability of NBS1 to activate PI 3-kinase in vitro and in vivo. Co-expression of NBS1/p110alpha/p-Akt is observed in certain percentage of head and neck cancer patient samples. These results demonstrate that NBS1 can function as an adaptor/activator of p110alpha PI 3-kinase through a novel activation motif, consistent with its possible role in cell transformation and tumorigenesis.     

Frontline: The p85alpha isoform of phosphoinositide 3-kinase is essential for a subset of B cell receptor-initiated signaling responses

Phosphoinositide 3-kinase (PI3K) is a ubiquitously expressed signaling enzyme that plays an integral role in development and activation of B cells. B cell receptor (BCR)-driven proliferation is completely blocked either in cells lacking the p85alpha regulatory isoform of PI3K or in wild-type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85alpha to early signaling events has not been fully investigated. Here we show that B cells lacking p85alpha have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85alpha results in partial reductions in Ca2+ mobilization and IkappaB phosphorylation, whereas ERK phosphorylation is not diminished. Moreover, although Akt phosphorylation is partially reduced, phosphorylation of several proteins downstream of Akt is preserved. These partial impairments suggest that there are other routes to PI3K activation in B cells apart from p85alpha-associated catalytic subunits. Notably, addition of phorbol ester restores BCR-mediated proliferation in p85alpha-deficient cells but not wild-type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85alpha is a failure to activate diacylglycerol-regulated signaling enzymes, most likely protein kinase C.     

The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers

MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR-126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR-126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR-126 gene targets revealed p85beta, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal, as one of the potential substrates. Restoration of miR-126 in cancer cells induced a > or =3-fold reduction in p85beta protein levels, with no concomitant change in p85alpha, a gene that is functionally related to p85beta but not a supposed target of miR-126. Additionally, using reporter constructs, we show that the p85beta-3' untranslated region is directly targeted by miR-126. Furthermore, this miR-126 mediated reduction of p85beta was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR-126 down-regulation together with an increase in the p85beta protein level. Taken together, we propose that miR-126 regulates PI3K signaling partly by targeting p85beta, and that the loss of miR-126 may provide a selective growth advantage during colon carcinogenesis.