Gq-Coupled Receptors Inhibit PI 3-Kinase/Akt Signaling Pathway

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The goal of this proposal is to understand the role of class IA phosphatidylinositol 3‐kinase (PI3K) p110alpha and p110beta in regulating pathological and physiological cell growth in the pancreas and skeletal muscle. These two class IA PI3Ks are postulated to control protein synthesis and cell growth and survival. However, it has not been possible to investigate the biological roles of these enzymes using whole‐body gene deletion due to embryonic lethality. To overcome this experimental problem, mouse strains in which the two PI3K genes can be selectively deleted in specific tissues were generated. Using these animals, this proposal addresses four research questions. Aim 1 determines if pancreas‐specific ablation of p110alpha or p110beta blocks the formation of pancreatic tumors induced by constitutively active KrasG12D. Mice with pancreas‐specific expression of KrasG12D develop the full spectrum of malignant intraepithelial lesions commonly found in human pancreatic cancer. In addition, a fluorescence spectroscopy technique is used to measure the binding affinity between activated Kras and PI3K complexes containing p110alpha or p110beta, which might provide mechanistic insight into the phenotypes seen in the two knockout strains. Aim 2 investigates how insulin‐like growth factor‐1 (IGF‐1) activates mammalian target of rapamycin (mTOR) signaling in myotubes prepared from muscle‐specific p110alpha knockout mice. IGF‐1 activation of PI3K and then mTOR is thought to be a central regulatory signal for stimulating muscle growth. These studies pursue the molecular mechanisms that explain the unexpected finding of enhanced mTOR signaling in response to IGF‐1 in p110alpha‐null muscle, even though Akt activation is greatly reduced. Aim 3 investigates if ablation of p110alpha or p110beta affects skeletal muscle atrophy caused by hindlimb unloading or muscle regrowth following reambulation. This animal model mimics the process of muscle unloading and reloading that can occur during hospitalization. The degree of muscle atrophy and subsequent regrowth is monitored by microCT scans of muscle mass in the same animal. Aim 4 also uses microCT scans to determine if clenbuterol, a beta2 adrenergic receptor agonist known to promote muscle growth in humans and rodents, can still stimulate muscle hypertrophy in muscle‐specific p110alpha or p110beta knockout mice. Clenbuterol signaling to mTOR is also investigated in myotubes prepared from knockout mice. Knowledge gained from these experiments is important because drugs that inhibit PI3K are already being tested in clinical trials for the treatment of cancer. The identification of cancer patients who will respond to this targeted signal transduction therapy remains a major challenge. Moreover, systemic inhibition of PI3K runs the risk of adverse side effects if these enzymes play important roles in regulating organ function, including maintenance of muscle mass.