Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling. Am J Physiol Endocrinol Metab 290: E154-E162, 2006. First published August 23, 2005; doi:10.1152/ajpendo. 00330.2005.-Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser645, Ser649, Ser653, Ser657) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. Insulin resistance is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.

Promoter studies of a polydnavirus gene from Cotesia rubecula (Hym : Braconidae)

The Cotesia rubecula polydnavirus gene, CrV1, is expressed in a highly transient fashion. Within four hours after egg deposition and virus infection, tissues of the host caterpillar, Pieris rapae, express high levels of the transcript. Twelve hours after infection no transcripts are visible. We have previously shown that the CrV1 secreted protein is mainly produced in host haemocytes. In haemocytes, immune functions such as phagocytosis and cell spreading are abolished by destabilization of the cell cytoskeleton. To test whether the observed down-regulation of CrV1 transcripts is mediated by transcriptional control or by other factors, such as the disruption of cytoskeleton in CrV1-inactivated cells, we cloned the promoter and the 3' untranslated region of the CrV1 gene to study CrV1 expression. The promoter region of the CrV1 gene was cloned into baculovirus expression systems along with the CAT reporter gene. Molecular analyses showed that the CAT gene under the control of CrV1 promoter is expressed as early as 2 h post infection and continues until late phase of infection suggesting that down-regulation of CrV1 expression in host haemocytes is perhaps mediated by post-transcriptional mechanisms.

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappa B

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.

Vitamin D receptor expression in the embryonic rat brain

We are interested in determining whether low maternal vitamin D-3 affects brain development in utero. Whilst the vitamin D receptor (VDR) has been identified in embryonic rat brains, the timing and magnitude of its expression across the brain remains unclear. In this study we have quantitated VDR expression during development as well correlated the timing of its appearance with two vital developmental events, apoptosis and mitosis. Brains from embryonic rats (embryonic days 15-23) were examined. We show that the well-described increase in apoptotic cells and decrease in mitotic cells during development correlates with the appearance of the VDR in brain tissue. Given that vitamin D-3 regulates mitosis and apoptosis in non-neuronal tissue we speculate that the timing of VDR expression in embryonic brain may directly or indirectly mediate features of neuronal apoptosis and mitosis.

Patterns of variation within self-incompatibility loci

Diverse self-incompatibility (SI) mechanisms permit flowering plants to inhibit fertilization by pollen that express specificities in common with the pistil. Characteristic of at least two model systems is greatly reduced recombination across large genomic tracts surrounding the S-locus, which regulates SI. In three angiosperm families, including the Solanaceae, the gene that controls the expression of gametophytic SI in the pistil encodes a ribonuclease (S-RNase). The gene that controls pollen SI expression is currently unknown, although several candidates have recently been proposed. Although each candidate shows a high level of polymorphism and complete allelic disequilibrium with the S-RNase gene, such properties may merely reflect tight linkage to the S-locus, irrespective of any functional role in SI. We analyzed the magnitude and nature of nucleotide variation, with the objective of distinguishing likely candidates for regulators of SI from other genes embedded in the S-locus region. We studied the S-RNase gene of the Solanaceae and 48A, a candidate for the pollen gene in this system, and we also conducted a parallel analysis of the regulators of sporophytic SI in Brassica, a system in which both the pistil and pollen genes are known. Although the pattern of variation shown by the pollen gene of the Brassica system is consistent with its role as a determinant of pollen specificity, that of 48A departs from expectation. Our analysis further suggests that recombination between 48A and S-RNase may have occurred during the interval spanned by the gene genealogy, another indication that 48A may not regulate SI expression in pollen.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.