We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110␣ and p110␤, and do not contain p110␦. Injection of specific inhibitory antibodies to p110␣ induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110␤ antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110␣ antibodies. In summary, the p110␣ isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.PI 3-kinases 1 are important signaling intermediates in a variety of regulated cellular processes (1). They are classified based on their regulation and substrate specificity (2). Class I enzymes produce PI[3]P, PI[3,4]P 2 , and PI[3,4,5]P 3 , whereas class II and III enzymes produce PI[3]P and PI[3,4]P 2 , or only PI[3]P, respectively. Class Ia enzymes exhibit the greatest diversity of the known PI 3-kinases, with multiple isoforms of both the regulatory (p85) and catalytic (p110) subunits (2). Differential phosphorylation of p85␣ and p85 and differential activation of p85␣-and p85␤-associated PI 3-kinase have been reported (3, 4). Knockouts of p85␣ further suggest that the p85␣ and p85␤ are not redundant (5, 6). Distinct class Ia catalytic subunit isoforms also have different functions. Both p110␣ and p110␤ play a role in mitogenesis, although p110␣ is required for responses to a broader range of growth factors (7,8). Recently, distinct signaling properties for p110 isoforms have been demonstrated in macrophages (9).We examined the specific functions of p110␣ and p110␤ in MTLn3 cells, a metastatic variant of the 13762NF rat mammary adenocarcinoma. MTLn3 cells undergo chemotaxis in an EGF gradient (10,11). This response involves the actin-dependent extension of a lamellipod in the direction of increasing EGF concentrations, with a zone of newly polymerized F-actin at the leading edge (12). Using isoform-specific inhibitory antibodies against p110␣ and p110␤, we now show that EGFstimulated lamellipod extension requires p110␣ but not p110␤. Significantly, anti-p110␣ antibodies blocked the formation of new barbed ends during an in situ actin nucleation assay. These studies provide direct evidence that p110␣ is required for the regulation of actin nucleation by EGF.

BackgroundWhey protein isolates (WPI) supplementation is known to improve resistance training adaptations. However, limited information is available on the effects of WPI plus carbohydrate (CHO) supplementation on endurance training adaptations.MethodSix endurance trained male cyclists and triathletes (age 29 ± 4 years, weight 74 ± 2 kg, VO2 max 63 ± 3 ml oxygen. kg-1. Min-1, height 183 ± 5 cm; mean ± SEM) were randomly assigned to one of two dietary interventions in a single blind cross over design; CHO or CHO + WPI. Each dietary intervention was followed for 16 days which included the last 2 days having increased CHO content, representing a CHO loading phase. The dietary interventions were iso-caloric and carbohydrate content matched. On completion of the dietary intervention, participants performed an exercise bout, consisting of cycling for 60 min at 70% VO2 max, followed by time trial to exhaustion at 90% VO2 max and recovered in the laboratory for 6 hours. Blood samples and muscle biopsies were taken at various time points at rest and through the exercise trial and recovery.ResultsCompared to CHO, CHO + WPI increased plasma insulin during recovery at 180 mins (P < 0.05) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) mRNA expression at the end of 6 hours of recovery (P < 0.05). Muscle glycogen did not differ between the two trials.ConclusionThis study showed co-ingestion of CHO + WPI may have beneficial effects on recovery and adaptations to endurance exercise via, increased insulin response and up regulation of PGC-1α mRNA expression.

Background: Acute exercise reduces postprandial oxidative stress and glycemia; however, the effects of exercise intensity are unclear. We investigated the effect of acute low-volume high-intensity interval-exercise (LV-HIIE) and continuous moderate-intensity exercise (CMIE) on glycemic control and oxidative stress in overweight and obese, inactive adults.Methods: Twenty-seven adults were randomly allocated to perform a single session of LV-HIIE (9 females, 5 males; age: 30 ± 1 years; BMI: 29 ± 1 kg·m−2; mean ± SEM) or CMIE (8 females, 5 males; age: 30 ± 2.0; BMI: 30 ± 2.0) 1 h after consumption of a standard breakfast. Plasma redox status, glucose and insulin were measured. Continuous glucose monitoring (CGM) was conducted during the 24-h period before (rest day) and after exercise (exercise day).Results: Plasma thiobarbituric acid reactive substances (TBARS; 29 ±13%, p < 0.01; mean percent change ±90% confidence limit), hydrogen peroxide (44 ± 16%, p < 0.01), catalase activity (50 ± 16%, p < 0.01), and superoxide dismutase activity (21 ± 6%, p < 0.01) significantly increased 1 h after breakfast (prior to exercise) compared to baseline. Exercise significantly decreased postprandial glycaemia in whole blood (−6 ± 5%, p < 0.01), irrespective of the exercise protocol. Only CMIE significantly decreased postprandial TBARS (CMIE: −33 ± 8%, p < 0.01; LV-HIIE: 11 ± 22%, p = 0.34) and hydrogen peroxide (CMIE: −25 ± 15%, p = 0.04; LV-HIIE: 7 ± 26%; p = 0.37). Acute exercise provided a similar significant improvement in 24-h average glucose levels (−5 ± 2%, p < 0.01), hyperglycemic excursions (−37 ± 60%, p < 0.01), peak glucose concentrations (−8 ± 4%, p < 0.01), and the 2-h postprandial glucose response to dinner (−9 ± 4%, p < 0.01), irrespective of the exercise protocol.Conclusion: Despite elevated postprandial oxidative stress compared to CMIE, LV-HIIE is an equally effective exercise mode for improving 24-h glycemic control in overweight and obese adults.

TLR9 is critical for the recognition of unmethylated CpG DNA in innate immunity. Accumulating evidence suggests distinct patterns of TLR9 expression in various types of cells. However, the molecular mechanism of TLR9 expression has received little attention. In the present study, we demonstrate that transcription of murine TLR9 is induced by IFN-β in peritoneal macrophages and a murine macrophage cell line RAW264.7. TLR9 is regulated through two cis-acting regions, a distal regulatory region (DRR) and a proximal promoter region (PPR), which are separated by ∼2.3 kbp of DNA. Two IFN-stimulated response element/IFN regulatory factor-element (ISRE/IRF-E) sites, ISRE/IRF-E1 and ISRE/IRF-E2, at the DRR and one AP-1 site at the PPR are required for constitutive expression of TLR9, while only the ISRE/IRF-E1 motif is essential for IFN-β induction. In vivo genomic footprint assays revealed constitutive factor occupancy at the DRR and the PPR and an IFN-β-induced occupancy only at the DRR. IRF-2 constitutively binds to the two ISRE/IRF-E sites at the DRR, while IRF-1 and STAT1 are induced to bind to the two ISRE/IRF-E sites and the ISRE/IRF-E1, respectively, only after IFN-β treatment. AP-1 subunits, c-Jun and c-Fos, were responsible for the constitutive occupancy at the proximal region. Induction of TLR9 by IFN-β was absent in STAT1−/− macrophages, while the level of TLR9 induction was decreased in IRF-1−/− cells. This study illustrates the crucial roles for AP-1, IRF-1, IRF-2, and STAT1 in the regulation of murine TLR9 expression.

Our results support the concept that p110beta appears to be the predominant functional class I PI 3-kinase isoform in prostate cancer cells.