Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.     

“Fluorescent glycogen” formation with sensibility for <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis> detection

There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a D-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.     

Modeling of α(1–4) chain arrangements in α(1–4)(1–6) glucans: The action and outcome of β-amylase and Pseudomonas stutzeri amylase on an α(1–4)(1–6) glucan model

Simulated enzymic debranching of a β-limit dextrin model, prepared from a computed construct made by random extension and branching, and given the CCL value of w-maize amylopectin (and equal amounts of external chains with ECL values of 2 and 3) has been related to experimental chromatograms of the debranched β-limit dextrin of the amylopectin. The profile was similar to those from gel chromatograms and IEC-PAD chromatography.The equivalent lengths in glucosyl units of grid-links (g-links) of internal and external chains in constructs were calculated from the ICL and ECL values of amylopectin and models produced from the constructs with the appropriate lengths for internal and external chains. These derived models were subjected to simulated hydrolysis by Pseudomonas stutzeri amylase and the products compared with those of the experimental distribution from w-maize amylopectin. With the model the amounts of maltotetraose and maltodextrins released were similar to the experimental values but the distribution of branched maltodextrins was quite different. Unlike w-maize amylopectin – a polymer with the cluster structure – which has given a profile of molecular sizes of maltodextrins with low amounts of single and small numbers of internal chains and with a peak at a M_W of about 14,000 (13 chains), in the model the proportion of maltodextrin with one internal chain was high and as d.p. increased the amounts decreased exponentially. This would be expected if the distribution of internal chains in the core was random. It is suggested that in the core of a model prepared from a construct made with alternating probabilities of extension – one in which this probability is high relative to branching, and a second in which it is low – may give clusters of branched maltodextrins with short internal chains which are joined by longer chains; more closely approximating the distribution of internal chains of different lengths in amylopectin.An arrangement for amylopectin molecules in the starch granule has been proposed. In this, they have a wafer-like, discoidal shape, composed of the amorphous zone overlain with the double helical, crystalline region. The flat macromolecules are concentrically layered with the former on the inside and the latter oriented to the outside of the granule.     

Immunocytochemical Analysis of Glycogen Phosphorylase Isozymes in the Developing and Adult Retina of the Domestic Chicken (<Emphasis Type="Italic">Gallus domesticus</Emphasis>)

Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses. Special issue article in honor of Dr. Frode Fonnum.     

Externalization of phosphatidylserine during apoptosis does not specifically require either isoform of phosphatidylserine synthase

Phosphatidylserine (PtdSer) is made in mammalian cells by two PtdSer synthases, PSS1 and PSS2. In the plasma membrane PtdSer is normally localized on the inner leaflet but undergoes transbilayer movement during apoptosis and becomes exposed on the cell surface. We induced apoptosis with staurosporine in four Chinese hamster ovary (CHO) cell lines that are deficient in PSS1 and/or PSS2 to determine if PtdSer generated by either of these enzymes is required for externalization on the cell surface during apoptosis. The onset of apoptosis was confirmed by the appearance of morphological changes and DNA fragmentation while the plasma membrane remained largely intact. In all cell lines, regardless of their content of PSS1 and/or PSS2, apoptosis occurred to approximately the same extent, and within approximately the same time frame, as in parental CHO-K1 cells. The exposure of PtdSer on the cell surface was assessed by annexin V labeling and flow cytometry. Cells that were deficient in either PSS1 or PSS2, as well as cells that were deficient in both PSS1 and PSS2, externalized normal amounts of PtdSer. Our study demonstrates, that reduction of in vitro serine-exchange activity, even by 97%, does not restrict the externalization of PtdSer during apoptosis. Moreover, a normal level of expression of PSS1 and/or PSS2 is not required for generating the pool of PtdSer externalized during apoptosis.     

不同时辰电针对大鼠杏仁核一氧化氮合酶表达的影响

目的观察不同时辰电针对大鼠杏仁核一氧化氮合酶(NOS)表达的影响.方法采用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)法,观察不同时辰电针大鼠一侧"足三里"穴对杏仁核NOS表达的影响.结果电针对大鼠杏仁皮质内侧核群、基底外侧核群NOS的表达有上调作用,并存在时辰差异(P<0.05);电针对大鼠杏仁中央核NOS表达无明显作用(P>0.05).结论电针对大鼠杏仁核NOS表达的影响有时辰差异.     

Inhibition of GSK-3 reduces infarct volume and improves neurobehavioral functions

In the present study, we have investigated the effects of glycogen synthase kinase-3 (GSK-3) inhibition on infarct volume and neurobehavioral functions in a focal cerebral ischemia model. To achieve our goals, GSK-3 inhibitor II or VIII was injected at several time points and in varing dosages. GSK-3 inhibitor VIII was more effective than inhibitor II, and infarct volume and water content in the VIII group were significantly decreased 24 h after the onset of ischemic stroke, as compared with the control group. These protective effects were associated with reductions of TUNEL-positive cells, neutrophil infiltration, glucose levels after ischemia, and GSK-3 enzyme activity. In addition, expressions of death and inflammation-related signals decreased and those of survival-related signals increased. Lastly, neurobehavioral functions were restored to a greater extent in the VIII group than in the control group. Together, these results suggest that GSK-3 inhibition reduces infarct volume and restores neurobehavioral functions.     

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP₃) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [³H]inositol display a significant increase in InsP₃ synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [³H] content of the InsP₃ fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP₃ from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP₃. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.     

Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.