Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Al-(L-丙氨酸)胰岛素晶体学研究

应用微量过饱和静置法在含柠檬酸钠和氯化钠的晶体生长体系中得到适合Ｘ射线衍射分析用的Ａｌ－（Ｌ－丙氯酸）胰岛素单晶体。晶体属于三方晶系,空间群为Ｒ３晶体衍射分辨率可达１．８Ａ以上。晶胞参数：ａＨ＝８０．８９Ａ,ｂＨ＝８０．８９Ａ,ｃＨ＝３７＼６４Ａ,α＝β＝９０°,γ＝１２０°。每个结晶学不对称单位含有两个Ａｌ－（Ｌ－丙氨酸）胰岛素分子。     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.     

The 3-Phosphorylated Phosphoinositide Response of 3T3-L1 Preadipose Cells Exposed to Insulin,Insulin-Like Growth Factor-1, or Platelet-Derived Growth Factor

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P₂ either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P₃ peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P₃ response. Insulin failed to increase PI(3,4)P₂ at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P₂ and PI(3,4,5)P₃ rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P₃ with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P₂.     

糖尿病大鼠肝细胞膜胰岛素受体的变化及机制探讨

本文采用受体的放射配体分析法,观察链佐霉素（ＳＴＺ）糖尿病大鼠肝细胞膜胰岛素受体的变化,并用核酸杂交方法对ＳＴＺ大鼠肝组织胰岛素受体变化的机制进行探讨。主要结果如下,Ｗｉｓｔａｒ大鼠在腹腔注射ＳＴＺ后２周,血糖升高,血清胰岛素水平降低,肝细胞膜胰岛素受体数目增加,与正常对照组相比增加了１５５．６％,而受体结合的亲和力下降。用斑点杂交方法观察到,ＳＴＺ注射２周后,大鼠肝组织胰岛素受体ｍＲＮＡ含量明显增加,而补充胰岛素后,受体ｍＲＮＡ含量基本恢复正常。提示ＳＴＺ大鼠肝细胞膜胰岛素受体数目增加的原因,可能主要是低胰岛素血症引起肝组织胰岛素受体ｍＲＮＡ表达水平增加,而补充胰岛素可使其恢复正常。从而进一步证明,血清胰岛素水平在ＳＴＺ糖尿病大鼠肝细胞膜胰岛素受体的变化中具有重要作用。     

Antihypertensive effects of captopril without adverse effects on glucose tolerance in hyperinsulinemic rhesus monkeys

The rhesus monkey (Macaca mulatta), which has been found to develop spontaneous obesity, non-insulin dependent diabetes mellitus (NIDDM; Type 2), and hypertension, was used to evaluate the potential blood pressure-lowering effects of captopril as well as the specific effects, if any, on the prediabetic state. Intravenous and oral glucose tolerance testing was carried out with oral captopril dosing. Results showed that captopril significantly decreased both systolic and diastolic blood pressure in all monkeys and significantly decreased fasting plasma glucose levels. Based on these preliminary studies in monkeys, we conclude that captopril exerted antihypertensive effects without adverse effects on glucose metabolism.     

Immunoneutralization of insulin partially modifies dorsoventral patterning in developing frog embryos

Determination of anteroposterior and dorsoventral axes is an important early event in the development of vertebrates involving extensive cellular interactions including inductive events. Recently we showed that insulin plays an essential role in prepancreatic development of the frogMicrohyla ornata. In the present study we have investigated the effects of immunoneutralization of endogenous insulin on the process of pattern formation. Treatment of neurulating embryos with antiserum to insulin caused abnormal pattern formation. The defects included loss of normal architecture of the neural tube, reduction in the size of the neural tube and, most conspicuously, rotation of the dorsoventral axis of the neural tube, notochord and adjoining mesodermal elements. The effects could be alleviated partially by pretreatment of embryos with exogenous insulin. This supports our belief that insulin plays an important role in induction and pattern formation of the amphibian nervous system. In addition, using 2-deoxy-α-D-glucose, an inhibitor of glucose metabolism, it is shown that the stimulatory effects of exogenous insulin on developing frog embryos are, at least partially, through the glucose metabolism pathway. Preliminary results of this study were presented at the National Symposium on Genes and Human Environment, held at Hyderabad, February 1994 and DAE Symposium on Stress and Adaptive Responses in Biological Systems, held at Vadodara, March 1994.     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.