Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes

The binding of [3H]inositol hexakisphosphate ([3H] InsP6) to rat cerebellar membranes has been characterized with the objective of establishing the role, if any, of a membrane protein receptor. In the presence of EDTA, we have previously identified an InsP6-binding site with a capacity of approximately 20 pmol/mg protein (Hawkins, P. T., Reynolds, D. J. M., Poyner, D. R., and Hanley, M. R. (1990) Biochem. Biophys. Res. Commun. 167, 819-827). However, in the presence of 1 mM Mg2+, the capacity of [3H]InsP6 binding to membranes was increased approximately 9-fold. This enhancing effect of Mg2+ was reversed by addition of 10 microM of several cation chelators, suggesting that the increased binding required trace quantities of other metal cations. This is supported by experiments where it was possible to saturate binding by addition of excess membranes, despite not significantly depleting radioligand, pointing to removal of some other factor. Removal of endogenous cations from the binding assay by pretreatment with chelex resin also prevents the Mg(2+)-induced potentiation. Consideration of the specificity of the chelators able to abolish this potentiation suggested involvement of Fe3+ or Al3+. Both these ions (but not several others) were able to increase [3H]InsP6 binding to chelex-pretreated membranes at concentrations of 1 microM. It is possible to demonstrate synergy between Fe3+ and Mg2+ under these conditions. We propose that [3H]InsP6 may interact with membranes through non-protein recognition possibly via phospholipids, in a manner dependent upon trace metals. The implications of this for InsP6 biology are considered.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.

The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Effects of low-density lipoprotein receptor-related protein 4 and apolipoprotein E isoforms on bone metabolism in vivo

LRP4, member of the LDLR family, is a multifunctional membrane-bound receptor that is expressed in various tissues. The expression of LRP4 by osteoblasts, its novel interaction with Wnt-signaling inhibitors Dkk1 and SOST, and the lower levels of activated beta-catenin in different bone locations described here, adds another player to the long list of established factors that modulate canonical Wnt-signaling in bone. By demonstrating that in addition to Wise, LRP4 is able to interact with two additional important modulators of Wnt- and BMP-signaling, our perspective of the complexity of the integration of BMP and Wnt-signaling pathways on the osteoblast surface has expanded further. Nevertheless the recently described association of both the SOST and LRP4 genes with BMD in humans, together with our findings suggest that LRP4 plays a physiologically important role in the skeletal development and bone metabolism not only in rodents, but in humans as well. The efficiency with which LRP4 binds both SOST and Dkk1, presumably at the osteoblastic surface, LRP4 may act as a sink and competes with LRP5/6 for the binding of these Wnt antagonists, which then are no longer available for suppression of the signal through the LRP5/6 axis. rnApoE, a 299 amino acid glycoprotein, is a crucial regulator in the uptake of triglyceride, phospholipids, cholesteryl esters, and cholesterol into cells. ApoE has been linked to osteoporosis, and such a role is further strengthened by the present of a high bone mass phenotype in ApoE null mice. Until recently, the effects of respective ApoE isoforms E2, E3, and E4, and their impact on bone metabolism, have been unclear. Here we report that respective human ApoE knockin mice display diverse effects on bone metabolism. ApoE2 mice show decreased trabecular bone volume per total volume in femoral bone and lumbar spine in comparison to ApoE3 and E4 animals. In this context, urinary bone resorption marker DPD is increased in these animals, which is accompanied by a low ratio of osteoclastogenesis markers OPG/RANKL. Interestingly, serum bone formation markers ALP and OCN are diminished in ApoE4 mice. In contrast to this finding, ApoE2 mice show the lowest bone formation of all groups in vivo. These findings cannot be explained by the low receptor-affinity of ApoE2 and subsequent decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin. Thus, other crucial pathways relevant for bone metabolism, e. g. Wnt/beta-catenin-signaling pathways, must be, compared to the ApoE3/4 isoforms, more affected by the ApoE2 isoform.

Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

The heat shock protein 90 inhibitor, 17-allylamino-17- demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor κB ligand (BANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence causedby 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. © 2013 Moura et al; licensee BioMed Central Ltd.