Giri-nya-la-nha (talk together) to explore acceptability of targeted smoking cessation resources with Australian Aboriginal women.

OBJECTIVES: To engage with health providers and Aboriginal women to understand what educational resources they want and need to support quit smoking attempts during pregnancy in order to develop a comprehensive evidence-based intervention. STUDY DESIGN: Resources were developed in partnership with Aboriginal people, communities and academics with the aim to be inclusive of diverse communities. We then recruited Aboriginal women of various ages for yarning circles (focus groups) held in three Australian states to explore the acceptability of the resources and seeking further guidance as to the needs of Aboriginal women to support smoking cessation during pregnancy. METHODS: Yarning circles were recorded and transcribed, and data were analysed independently by two researchers. Responses were coded using predetermined themes and further general inductive analysis for emergent themes. RESULTS: Twenty-four Aboriginal women reflected on the resources they included: one pregnant woman, 15 mothers and eight elders. Predetermined themes of attraction, comprehension, cultural acceptability, graphics and layout, persuasion and self-efficacy were explored. Women suggested the following: resources need to be visually attractive and interactive to enhance self-efficacy; additional scientific content on health consequences of smoking and combining with non-pharmacological approaches to quitting. CONCLUSION: Indigenous peoples prefer culturally targeted messages. However, developing effective Aboriginal health promotion requires more than a 'culturally appropriate' adaptation of mainstream resources. Consideration needs to be given to the diversity of Aboriginal communities when developing effective, evidence-based interventions. Aboriginal women are calling for innovative and interactive resources that enhance self-efficacy; the use of videos to explain medical and informational brochure content is well received. Requests for non-pharmacological cessation options were reported in New South Wales and Queensland and should be further explored.

Wula (Voices) of Aboriginal women on barriers to accepting smoking cessation support during pregnancy: Findings from a qualitative study.

AIM: To gather Aboriginal women's stories of smoking and becoming pregnant to identify the barriers in accepting smoking cessation support during pregnancy. METHODS: Qualitative data were collected through use of yarning methodology between August 2015 and January 2016 by an Aboriginal Researcher with experience in social and community services. A short on-line survey was used to collect quantitative data. Interviews only recorded the therapeutic yarning process, which ranged from 9 to 45min duration, averaging 30min. Audio-recorded interviews were transcribed and independently coded. A general inductive analysis was used to determine emergent themes. RESULTS: Twenty Aboriginal women between 17-38 years of age, who were pregnant or recently given birth, living in the Hunter New England (HNE) area took part. Eleven women were still smoking; nine had quit. Most were highly aware of the implications of smoking for their babies. Major themes identified for accepting support were: ambivalence towards a need for support, health professional advice, reduction in smoking, and attitudes to Nicotine Replacement Therapy (NRT). Women reported being advised to cut down, rather than to quit; reducing consumption may be a barrier to accepting NRT. Women recommended enhanced clinical support and Aboriginal community engagement in cessation care. DISCUSSION/CONCLUSIONS: Aboriginal women in the HNE area reported quitting or reducing their cigarette intake during pregnancy. Health Professionals working with Aboriginal women during pregnancy should give consistent messages to quit smoking completely, and offer increased, ongoing and extensive smoking cessation support to Aboriginal mothers. Clinical practices could partner with Aboriginal communities to support the delivery of smoking cessation services.

Isolation of a variant infectious bronchitis virus in Australia that further illustrates diversity among emerging strains.

Australian infectious bronchitis viruses (IBV) have undergone a separate evolution due to geographic isolation. Consequently, changes occurring in Australian IBV illustrate, independently from other countries, types of variability that could occur in emerging IBV strains. Previously, we have identified two distinct genetic groups of IBV, designated subgroups 1 and 2. IBV strains of subgroup 1 have S1 and N proteins that share a high degree of amino acid identity, 81 to 98% in S1 and 91 to 99% in N. Subgroup 2 strains possess S1 and N proteins that share a low level of identity with subgroup 1 strains: 54 to 62% in S1 and 60 to 62% in N. This paper describes the isolation and characterisation of a third, previously undetected genetic group of IBV in Australia. The subgroup 3 strains, represented by isolate chicken/Australia/N2/04, had an S1 protein that shared a low level of identity with both subgroups 1 and 2: 61 to 63% and 56 to 59%, respectively. However, the N protein and the 3' untranslated region were similar to subgroup 1: 90 to 97% identical with the N protein of subgroup 1 strains. This N4/02 subgroup 3 of IBV is reminiscent of two other strains, D1466 and DE072, isolated in the Netherlands and in the USA, respectively. The emergence of the subgroup 3 viruses in Australia, as well as the emergence of subgroup 2 in 1988, could not be explained by any of the mechanisms that are currently considered to be involved in generation of IBV variants.

Recurrent chylothorax in a patient with non-Hodgkins lymphoma: case report.

Spontaneous chylothorax could arise as a complication of Iymphoma. There are no reports on the frequency of it's occurrence. It is associated with a high mortality rate. This is mainly due to severe nutritional deficiencies and wasting. This case describes a patient with non-Hodgkins Iymphoma who developed recurrent bilateral chylothorax requiring repeated pleural aspirations and eventually talc pleurodesis which failed.

The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias.

Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.

Low grade chronic inflammation in women with polycystic ovarian syndrome.

Low grade chronic inflammation as reflected by increased C-reactive protein (CRP) concentrations independently predicts those at risk for coronary heart disease (CHD) and type 2 diabetes. Women with polycystic ovarian syndrome (PCOS) are insulin resistant and have increased risk for CHD and type 2 diabetes, but currently there are no data on markers of inflammation in women with PCOS. Seventeen women with PCOS (defined on the basis of elevated testosterone and oligomenorrhea) and 15 healthy women matched as a group for body mass index were recruited. Measurement of CRP concentrations was made using a highly sensitive assay. Insulin resistance was assessed using the hyperinsulinemic euglycemic clamp technique. The women with PCOS had significantly elevated CRP concentrations relative to controls (geometric means, 2.12 and 0.67 mg/L, respectively; P = 0.016). Log CRP correlated with body mass index in both PCOS and controls (r = 0.58; P < 0.05 and r = 0.78; P < 0.01, respectively) and inversely with insulin sensitivity (r = -0.57; P < 0.05 and r = -0.69; P < 0.01). Total testosterone did not correlate with log CRP in either group. On adjustment for body mass index and age, there remained a significant difference in log CRP between PCOS and controls (t = 2.13; P < 0.05). On further adjustment for insulin sensitivity, log CRP was no longer significantly different between groups (t = 1.51; P = 0.14). We conclude that women with PCOS have significantly increased CRP concentrations relative to women with normal menstrual rhythm and normal androgen levels. We propose low grade chronic inflammation as a novel mechanism contributing to increased risk of CHD and type 2 diabetes in these women.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis.

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts ( approximately 25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Regulation of glucose transport in aortic smooth muscle cells by cAMP and cGMP.

We have studied the ability of cGMP and cAMP to modulate platelet-derived growth factor (PDGF)-stimulated 2-deoxy-D-glucose (deGlc) transport in primary cultures of vascular smooth muscle cells (VMSC) from rat aorta. PDGF stimulated deGlc transport in a time- and concentration-dependent manner. 8-Bromo-cGMP and atrial natriuretic peptide(1-28) [ANP(1-28)] were found to reduce PDGF-stimulated deGlc transport without affecting basal (unstimulated) transport activity. In contrast, 8-bromo-cAMP and dibutyryl-cAMP stimulated basal deGlc transport 2-fold and were without effect on PDGF-stimulated deGlc transport. 8-Bromo-cGMP also inhibited 8-bromo-cAMP-stimulated deGlc transport. The stimulation of deGlc transport by PDGF was sensitive to the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059, and we show that ERK1/2 was activated by PDGF. Neither 8-bromo-cGMP nor ANP(1-28) inhibited PDGF-stimulated ERK activation, suggesting that the effects of cGMP and ANP(1-28) were not mediated by inhibition of this kinase. Our data also argue against a role for cGMP-dependent protein kinase in mediating the effects of cGMP or ANP(1-28). Collectively, our data suggest that in VSMC: (i) cGMP and cAMP have opposing effects on deGlc transport; (ii) PDGF and cAMP have common elements in the pathways by which they activate deGlc transport; and (iii) a common element may be the target of the cGMP-mediated inhibition of deGlc transport.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4.

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

The long term health consequences of polycystic ovary syndrome.

Women with polycystic ovary syndrome have both insulin resistance and beta cell dysfunction. Consequently, they are at increased risk of developing diabetes and cardiovascular disease. Women with polycystic ovary syndrome present to clinicians at a young age and as such offer a unique opportunity to identify insulin resistant patients at an early stage. This enables the modification of risk factors and diagnosis of diabetes before the onset of macro- and micro-vascular symptoms. Increased emphasis should thus be placed on long term risk management and diabetic screening with advice on smoking, exercise and, if appropriate, weight loss. Where possible drugs that exacerbate insulin resistance should be avoided and consideration should be given to the use of insulin sensitising agents, particularly in the obese.

Sex hormones induce insulin resistance in 3T3-L1 adipocytes by reducing cellular content of IRS proteins.

AIM/HYPOTHESIS: Numerous studies have suggested a relation between sex hormones and insulin sensitivity but the ability of sex hormones to directly influence insulin action in peripheral tissues has not been investigated. METHODS: We have examined the effects of estriol, estradiol and estrone on insulin action in cultured 3T3-L1 adipocytes, a useful model of adipocytes. RESULTS: Treatment of these cells with each of these sex hormones resulted in a statistically significant reduction in the ability of insulin to stimulate glucose transport independently of a reduction in total cellular GLUT-4 content. This diminished ability of insulin to stimulate glucose transport was accompanied by a reduction in the total cellular content of insulin receptor substrates -1 and -2 and the p85alpha subunit of phosphatidylinositol 3'-kinase. By contrast, cellular content of protein kinase B was unchanged by hormone treatment but the magnitude of insulin-stimulated kinase activity was statistically significantly reduced after incubation with each of the sex hormones tested. We have further shown that treatment of 3T3-L1 adipocytes with these hormones alters the subcellular distribution of insulin receptor substrate proteins such that the particulate and soluble pools of these proteins were differentially affected by hormone treatment. CONCLUSION/INTERPRETATION: These data show that sex hormones can directly induce a state of insulin resistance in 3T3-L1 adipocytes in culture. The mechanism of this defect seems to be at least in part due to decreased cellular content and altered subcellular distribution of insulin receptor substrate proteins which in turn results in a reduction in proximal insulin-stimulated signalling cascades.

5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes.

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a compound that activates 5'-AMP-activated protein kinase (AMPK), has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study, we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes, in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently, closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation, assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may, therefore, give insight into the mechanism of insulin action.

Preservation: past, present and future.

Foods deteriorate in quality due to a wide range of reactions including some that are physical, some that are chemical, some enzymic and some microbiological. The various forms of spoilage and food poisoning caused by micro-organisms are preventable to a large degree by a number of preservation techniques, most of which act by preventing or slowing microbial growth. These include freezing, chilling, drying, curing, conserving, vacuum packing, modified atmosphere packing, acidifying, fermenting, and adding preservatives. In contrast, a smaller number of techniques act by inactivating micro-organisms, predominantly heating (pasteurization and sterilization). Complementary techniques restrict access of micro-organisms to food products, e.g. aseptic processing and packaging. New and 'emerging' preservation techniques include more that act by inactivation. They include the application of ionizing radiation, high hydrostatic pressure, high voltage electric discharges, high intensity light, ultrasonication in combination with heat and slightly raised pressure ('manothermosonication'), and the addition to foods of bacteriolytic enzymes, bacteriocins, and other naturally-occurring antimicrobials. Major trends, reacting to consumers' needs, are towards the use of procedures that deliver food products that are less 'heavily' preserved, higher quality, more convenient, more 'natural', freer from additives, nutritionally healthier, and still with high assurance of microbiological safety.

P2Y receptor-mediated inhibition of tumor necrosis factor alpha -stimulated stress-activated protein kinase activity in EAhy926 endothelial cells.

In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor alpha (TNFalpha)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-2, the downstream target of p38 MAP kinase. In NCT2544 keratinocytes, UTP and a proteinase-activated receptor-2 agonist caused similar inhibition, but in 13121N1 cells, transfected with the human P2Y(2) or P2Y(4) receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P2Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca(2+) levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFalpha-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318220 and Go6983 reversed the inhibitory action of UTP, but GF109203X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(2+)-independent isoforms of protein kinase C.

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.

Tumour necrosis factor stimulates stress-activated protein kinases and the inhibition of DNA synthesis in cultures of bovine aortic endothelial cells.

In this study, we examined the ability of tumour necrosis factor-alpha (TNF) to stimulate the mitogen-activated protein (MAP) kinase homologues p42/44 MAP kinase, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase and its effect upon DNA synthesis in primary cultures of bovine aortic endothelial cells (BAECs). TNF strongly stimulated p38 MAP kinase and JNK activity in both a time- and concentration-dependent manner. By contrast, TNF was a very poor activator of p42/44 MAP kinase relative to the known activator of p42/44 MAP kinase in endothelial cells, adenosine triphosphate (ATP). TNF-stimulated activation of p38 MAP kinase, and MAPKAP kinase-2, a known downstream target of p38 MAP kinase, was strongly inhibited by pre-incubation with the p38 MAP kinase inhibitor SB203580, whereas the minor activation of p42/44 MAP kinase was abolished by pre-incubation of the cell with the novel MAP kinase kinase 1 inhibitor PD098059. Addition of TNF resulted in a 50-60% decrease in DNA synthesis in BAECs. Pre-incubation with PD098059 or co-incubation with ATP failed to modify the inhibitory effect of TNF upon DNA synthesis. SB203580 reduced basal DNA synthesis by approximately 50%; however, if failed to modify the inhibition mediated by TNF. These results indicate that TNF strongly activates both p38 MAP kinase, JNK and, to a minor extent, p42/p44 MAP kinase. It is likely that only one of these kinases, JNK, plays a role in the regulation of DNA synthesis in these cells.

Stress-activated protein kinases: activation, regulation and function.

The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes. Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists. Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation. More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1). These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-alpha (TNF-alpha), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion. Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses. In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress.

The glucose transporter GLUT4 and the aminopeptidase vp165 colocalise in tubulo-vesicular elements in adipocytes and cardiomyocytes.

The aminopeptidase vp165 is one of the major polypeptides enriched in GLUT4-containing vesicles immuno-isolated from adipocytes. In the present study we have confirmed and quantified the high degree of colocalisation between GLUT4 and vp165 using double label immuno-electron microscopy on vesicles isolated from adipocytes and heart. The percentage of vp165-containing vesicles that also contained GLUT4 was 91%, 76%, and 86% in rat adipocytes, 3T3-L1 adipocytes, and rat heart, respectively. Internalisation of a transferrin/HRP (Tf/HRP) conjugate by 3T3-L1 adipocytes, followed by diaminobenzidine treatment in intact cells, resulted in ablation of only 41% and 45% of GLUT4 and vp165, respectively, whereas endosomal markers are almost quantitatively ablated. Using immuno-electron microscopy on cryosections it was determined that in atrial cardiomyocytes GLUT4 and vp165 colocalised in a population of tubulo-vesicular (T-V) elements that were often found close to the plasma membrane. Double label immunocytochemistry indicated a high degree of overlap in these T-V elements between GLUT4 and vp165. However, in atrial cardiomyocytes a large proportion of GLUT4 was also present in secretory granules containing atrial natriuretic factor (ANF). In contrast, very little vp165 was detected in ANF granules. These data indicate that GLUT4 and vp165 are colocalised in an intracellular, post-endocytic, tubulo-vesicular compartment in adipocytes and cardiomyocytes suggesting that both proteins are sorted in a similar manner in these cells. However, GLUT4 but not vp165 is additionally localised in the regulated secretory pathway in atrial cardiomyocytes.

Characterization of the intracellular signalling pathways that underlie growth-factor-stimulated glucose transport in Xenopus oocytes: evidence for ras- and rho-dependent pathways of phosphatidylinositol 3-kinase activation.

The stimulation of glucose transport is one of the early cellular responses to growth factors and is essential for cell proliferation, yet the molecular processes that underlie this response are poorly defined. The aim of this study was to characterize the role of the low-molecular-mass G-proteins, Ras and Rho, and their downstream targets, Raf protein kinase and phosphatidylinositol 3-kinase, in the regulation of glucose transport in Xenopus oocytes by two distinct growth-factor receptors: the insulin-like growth factor I (IGF-I) tyrosine kinase receptor and the heterotrimeric G-protein-coupled lysophosphatidic acid (LPA) receptor. Microinjection of a neutralizing anti-Ras antibody partially blocked IGF-I-stimulated deoxyglucose uptake but was without effect on LPA-stimulated deoxyglucose uptake. In contrast, microinjection of the C3 coenzyme of botulinum toxin, which selectively ADP-ribosylates and inactivates Rho, inhibited LPA-stimulated, but not IGF-I-stimulated, deoxyglucose uptake. Similarly, LPA- but not IGF-I-stimulated deoxyglucose uptake was attenuated in oocytes expressing a dominant negative rho construct. Cells expressing a dominant negative mutant of Raf protein kinase exhibited markedly reduced sensitivity to both LPA and IGF-I, consistent with a role for endogenous Raf in glucose uptake by both growth factors. Furthermore, expression of a constitutively activated form of raf-1 resulted in a growth-factor-independent increase in deoxyglucose uptake. Measurements of phosphatidylinositol 3-kinase activity in microinjected cells support the hypothesis that the IGF-I receptor stimulates glucose transport by a Ras-dependent activation of phosphatidylinositol 3-kinase, whereas the G-protein-coupled LPA receptor controls this response by a pathway that involves Rho-dependent activation of a distinct phosphatidylinositol 3-kinase. Thus we provide evidence for clear differences in the signalling pathways that control glucose transport by G-protein-coupled and tyrosine kinase growth-factor receptors. Furthermore this is the first demonstration that active Rho is involved in the signalling pathways that regulate glucose uptake in response to some growth factors.