Subcellular localization of IRS-1 in cell proliferation and differentiation.

The type 1 insulin-like growth factor receptor (IGF-IR) and its docking protein, insulin receptor substrate-1 (IRS-1), play important roles in cell transformation, cell differentiation and aging. IRS-1 and other IRS proteins can, under certain conditions, localize to the nuclei of cells, where they undergo interactions with nuclear and nucleolar proteins. In this study, we confirm and extend these observations, demonstrating that IRS-1 is preferentially nuclear in growing cells. Differentiation and inhibition of ribosomal RNA synthesis cause subcellular redistribution of IRS-1 and other nuclear proteins to the cytoplasm.     

Changes in tissue protein pattern in relation to auxin induction of DNA synthesis

Abstract. When artichoke tuber tissue was cultured in mineral salts, the induction of DNA synthesis and subsequent cell division was dependent upon the presence of auxin in the incubation medium. Evidence is provided that an obligatory set of metabolic events precedes auxin-induced DNA synthesis, and previous work has associated these with protein synthesis. As a consequence the auxin-induced changes in nuclear and cytoplasmic proteins have been investigated. Using 2D gel electrophoresis, qualitative alterations in nuclear non-histone proteins have been detected from the earliest treatment times with auxin. These changes were progressive, starting with four novel proteins after 3 h auxin treatment and ending with about forty when DNA synthesis commences some 18–21 h later. Qualitative alterations in phosphorylated nuclear proteins due to auxin treatment were only detectable when DNA synthesis commenced. In contrast, few qualitative alterations in cytoplasmic proteins were detectable, with the major change being in phosphorylated proteins at the onset of DNA synthesis.
A possible model of auxin action is outlined which involves sequential and progressive changes in the synthesis of nuclear proteins and the control of gene expression eventually leading to DNA synthesis.     

Insulin signaling: mechanisms altered in insulin resistance

Insulin has a major anabolic function leading to storage of lipidic and glucidic substrates. All its effects result from insulin binding to a specific membrane receptor which is expressed at a high level on the 3 insulin target tissues: liver, adipose tissue and muscles. The insulin receptor exhibits a tyrosine-kinase activity which leads, first, to receptor autophosphorylation and then to tyrosine phosphorylation of substrates proteins, IRS proteins in priority. This leads to the formation of macromolecular complexes close to the receptor. The two main transduction pathways are the phosphatidylinositol 3 kinase pathway activating protein kinase B which is involved in priority in metabolic effects, and the MAP kinase pathway involved in nuclear effects, proliferation and differentiation. However, in most cases, a specific effect of insulin requires the participation of the two pathways in a complex interplay which could explain the pleiotropy and the specificity of the insulin signal. The negative control of the insulin signal can result from hormone degradation or receptor dephosphorylation. However, the major negative control results from phosphorylation of serine/threonine residues on the receptor and/or IRS proteins. This phosphorylation is activated in response to different signals involved in insulin resistance, hyperinsulinism, TNFalpha or increased free fatty acids from adipose tissue, which are transformed inside the cell in acyl-CoA. A deleterious role for molecules issued from the adipose tissue is postulated in the resistance to insulin of the liver and muscles present in type 2 diabetes, obesity and metabolic syndrome.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

PPARγ变异与复杂疾病

过氧化物酶体增殖激活受体γ（PPARγ）是核激素受体超家族成员。PPARγ基因主要表达于脂肪组织,促进脂肪细胞分化,调控多种脂肪细胞分泌的蛋白质因子基因的表达。它也是糖尿病治疗药物噻唑烷二酮类化合物(TZDs)作用的靶分子。PPARγ的常见多态性影响胰腺β细胞功能,导致胰岛素分泌及外周组织对胰岛素敏感性的改变。它与2型糖尿病、肥胖、心血管疾病、癌症的发病风险相关联,阐明PPARγ的作用机制对复杂疾病的诊断、预防和治疗具有重要意义。     

Changes in the nuclear triiodothyronine receptor during the differentiation of preadipocytic cell lines of mice

During the differentiation process of ob 17 preadipocytes in the presence of insulin, a progressive increase in the nuclear T3 receptor concentration per cell (1.5 to 3 fold) was observed. This increase was not found related to the addition of insulin to the culture medium and was concomitant to the expression of several phenotypes of the mature adipocyte. Triiodothyronine (T3) added to the culture medium of these cells increases the lipogenic pathway and several enzymes involved in fatty acid synthesis and esterification. After T3 addition at the same optimal physiologic concentration of 1.5 nM, the cellular concentration of its nuclear receptor was clearly reduced, without any significant alteration in the apparent affinity constant. A similar behavior was observed in 3T3-L1 preadipocytes. The molecular mechanism of action of the T3 nuclear receptor within the chromatin is not known. The biological relevance of its depletion by T3 itself remains to be determined.     

Current theories on the mechanism of the effect of glucagon on carbohydrate metabolism

Glucagon is considered for mechanisms of its action on the carbon metabolism, its significance in the complex polyhormonal regulation of the glucose synthesis and metabolism being studied. Glucagon exerts its effect on cells through specific receptors arranged on the plasma membrane, while its effect on the carbohydrate metabolism is mediated, mainly, by cAMP-dependent phosphorylation os some proteins participating in regulation of carbohydrate synthesis and metabolism including the proteins controlling expression of many genes. The glucagon effect on the level and efficiency of the action of insulin and some other hormones is an important link in the glucagon action on the carbohydrate metabolism.     

羟基喜树碱诱导肝癌细胞凋亡前后细胞核蛋白质组变化的定量分析

对羟基喜树碱诱导肝癌细胞凋亡前后的细胞核蛋白质组进行定量研究,获得凋亡前后细胞核蛋白质组中差异蛋白表达量相对变化的信息,为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供实验依据。分离提取羟基喜树碱诱导肝癌细胞凋亡前后的细胞核并鉴定其纯度,用含稳定同位素的化学试剂c-ICAT标记细胞凋亡前后的细胞核蛋白,对细胞核标记蛋白进行消化和纯化,利用基于多维色谱-线性离子阱/静电场轨道阱质谱联用技术的鸟枪（Shotgun）法策略及c-ICAT定量策略分析鉴定蛋白质,获得同一种肝癌细胞核蛋白质在凋亡前后细胞中的表达量变化比值。分析鉴定了在细胞凋亡前后的表达量差异有显著统计学意义（P〈0.05）的肝癌细胞核蛋白42个,其中,12个蛋白的表达量在羟基喜树碱诱导细胞凋亡后下调,30个蛋白的表达量在凋亡细胞中上调。这些差异蛋白的分子功能主要与细胞增殖、凋亡和分化、能量代谢、核酸代谢以及细胞骨架相关。     

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.     

Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice

Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.     

The dynamic properties of neuronal chromatin are modulated by triiodothyronine

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.