Skeletal muscle PI3K p110ß regulates expression of AMP-activated protein kinase.

Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110ß mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110ß inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110ß (TGX-221), siRNA against p110ß, or overexpression of kinase-dead p110ß. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110ß. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110ß siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110ß-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110ß (p110ß-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110ß-deficient myoblasts and in p110ß-mKO muscle. Loss of p110ß had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110ß-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110ß positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110ß-deficient myoblasts, loss of p110ß does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110ß in mediating skeletal muscle metabolic signaling.

Role of phosphoinositide 3-OH kinase p110ß in skeletal myogenesis.

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit ß (p110ß) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110ß delayed differentiation. We next generated mice with conditional deletion of p110ß in skeletal muscle (p110ß muscle knockout [p110ß-mKO] mice). While young p110ß-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110ß-mKO mice were less glucose tolerant than old control mice. Overexpression of p110ß accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110ß had the opposite effect. p110ß overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110ß inhibition. These findings reveal a role for p110ß during myogenesis and demonstrate that long-term reduction of skeletal muscle p110ß impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.

Improving glycemic control in the acute care setting through nurse education.

Patients with a primary or secondary diagnosis of diabetes present unique challenges during an inpatient hospital stay to treat an acute or chronic illness. Upon review of current hospital practice, an interprofessional team embarked on a performance improvement project to improve outcomes for the complex medical-surgical diabetic patient. The methods detailed herein--a comprehensive education plan, preceptorship and peer accountability, active engagement and support by the unit nursing leadership team, and interprofessional collaboration--offer strategies any organization can implement to positively impact diabetes care.

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110ß and PKCδ in myoblasts.

CONTEXT: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. OBJECTIVE: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. METHODS: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. RESULTS: Treatment of myoblasts with exogenous LPA revealed that PI3K p110ß mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. CONCLUSION: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110ß, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.

Enhanced Akt phosphorylation and myogenic differentiation in PI3K p110ß-deficient myoblasts is mediated by PI3K p110α and mTORC2.

Phosphoinositide 3-kinase (PI3K) is a principal regulator of Akt activation and myogenesis; however, the function of PI3K p110ß in these processes is not well defined. To address this, we investigated the role of p110ß in Akt activation and skeletal muscle cell differentiation. We found that Akt phosphorylation was enhanced in p110ß-deficient myoblasts in response to Insulin-like Growth Factor-I (IGF-I), epidermal growth factor, or p110α overexpression, as compared to p110ß-sufficient cells. This effect was associated with increased mammalian target of rapamycin complex 2 activation, even in myoblasts deficient in mSin1 and rictor. Conversely, in response to the G-protein-coupled receptor agonist lysophosphatidic acid, Akt phosphorylation was attenuated in p110ß-deficient myoblasts. Loss of p110ß also enhanced the expression of myogenic markers at the myoblast stage and during the first 48 h of differentiation. These data demonstrate that reductions in p110ß are associated with agonist-specific Akt hyperactivation and accelerated myogenesis, thus revealing a negative role for p110ß in Akt activation and during myoblast differentiation.

Osteoclasts direct bystander killing of bone cancer.

Primary and metastatic bone cancers are difficult to eradicate and novel approaches are needed to improve treatment and extend life. As bone cancer grows, osteoclasts, the principal bone-resorbing cells of the body, are recruited to and activated at sites of cancer. In this investigation, we determined if osteoclast lineage cells could function as a cell-based gene delivery system to bone cancers. We used the cytosine deaminase (CD) 5-fluorocytosine (5-FC) enzyme/prodrug system and studied bone marrow and bones from transgenic mice expressing a novel CD gene regulated by the osteoclast tartrate-resistant acid phosphatase (TRAP) gene promoter (Tg/NCD). DsRed2-labeled 2472 sarcoma cells were placed in Tg/NCD osteoclastogenic cultures and treated with 5-FC. 5-FC treatment resulted in profound bystander killing (90%; P < 0.05). The effect of 5-FC treatment on osteoclast lineage cells was most dramatic when administered at the beginning of the 7-day cultures, suggesting that mature osteoclasts are less sensitive to 5-FC. Evaluation of osteoclast-directed bystander killing in vivo revealed dramatic killing of bone cancer with only a modest effect on osteoclast number. Specifically, 5-FC treatment of tumor-bearing Tg/NCD mice or Tg/NCD bone marrow transplanted C3H mice (Tg/NCD-C3H) resulted in 92% and 44% reductions in tumor area, respectively (P < 0.05). Eight of ten 5-FC-treated Tg/NCD mice had complete bone tumor killing and five of six 5-FC-treated Tg/NCD-C3H mice had reduced tumor compared with controls. In addition, Tg/NCD osteoclasts were resistant to 5-FC treatment in vivo, a very important feature, as it identifies osteoclasts as an ideal CD gene delivery system.