Insulin-associated changes in carnitine palmitoyltransferase activity are mediated through the insulin-like growth factor I pathway in the cultured neonatal rat cardiac myocyte

The mitochondrial carnitine palmitoyltransferase (CPT) system is composed of two proteins, CPT-I and CPT-II, involved in the transport of fatty acids into the mitochondrial matrix to undergo $\beta$-oxidation. CPT-I is located outside the inner membrane and CPT-II is located on the inner aspect of the inner membrane. The CPT proteins are distinct with different molecular weights and activities. The malonyl-CoA sensitivity of CPT-I has been proposed as a regulatory step in $\beta$-oxidation. Using the neonatal rat cardiac myocyte, assays were designed to discriminate between these activities in situ using digitonin and Triton X-100. With this methodology, we are able to determine the involvement of the IGF-I pathway in the insulin-mediated increase in CPT activities. Concentrations of digitonin up to 25 $\mu$M fail to release citrate synthase from the mitochondrial matrix or alter the malonyl-CoA sensitivity of CPT-I. If the mitochondrial matrix was exposed, malonyl-CoA insensitive CPT-II would reduce malonyl-CoA sensitivity. In contrast to digitonin, Triton X-100 (0.15%) releases citrate synthase from the matrix and exposes CPT-II. CPT-II activity is confirmed by the absence of malonyl-CoA sensitivity. To examine the effects of various agents on the expression and/or activity of CPT, it is necessary to use serum-free medium to eliminate mitogenic effects of serum proteins. Comparison of different media to optimize CPT activity and cell viability resulted in the decision to use Dulbecco's Modified Eagle medium supplemented with transferrin. In three established models of cardiac hypertrophy using the neonatal rat cardiac myocyte there is a significant increase in CPT-I and CPT-II activity in the treated cells. Analogous to the situation seen in the hypertrophy model, insulin also significantly increases the activity of the mitochondrial proteins CPT-I, CPT-II and cytochrome oxidase with a coinciding increase the expression of CPT-II and cytochrome oxidase mRNA. The removal of serum increases the I$\sb{50}$ (concentration of inhibitor that halves enzyme activity) of CPT-I for malonyl-CoA by four-fold. Incubation with insulin returns I$\sb{50}$ values to serum levels. Incubation with insulin significantly increases malonyl-CoA and ATP levels in the cells with a resulting reduction in palmitate oxidation. Once malonyl-CoA inhibition of CPT-I is removed by permeabilizing the cells, insulin significantly increases the oxidation of palmitoyl-CoA in a manner which parallels the increase in CPT-I activity. Interestingly, CPT-II activity increases significantly only at the tissue culture concentration (1.7 $\mu$M) of insulin suggesting that the IGF-I pathway may be involved. Supporting a role for the IGF-I pathway in the insulin-induced increase in CPT activity is the significant increase in the synthesis of both cellular and mitochondrial proteins as well as increased synthesis of CPT-II. Consistent with an IGF-mediated pathway for the effect of insulin, IGF-I (10 ng/ml) significantly increases the activities of both CPT-I and -II. An IGF-I analogue which inhibits the autophosphorylation of the IGF-I receptor blunts the insulin-mediated increase in CPT-I and -II activity by greater than 70% and virtually eliminates the IGF-I response by greater than 90%. This is the first study to demonstrate the involvement of the IGF-I pathway in the regulation of mitochondrial protein expression, e.g. CPT. ^

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Proton therapy versus intensity modulated x-ray therapy in the treatment of prostate cancer: Estimating secondary cancer risks

External beam radiation therapy is used to treat nearly half of the more than 200,000 new cases of prostate cancer diagnosed in the United States each year. During a radiation therapy treatment, healthy tissues in the path of the therapeutic beam are exposed to high doses. In addition, the whole body is exposed to a low-dose bath of unwanted scatter radiation from the pelvis and leakage radiation from the treatment unit. As a result, survivors of radiation therapy for prostate cancer face an elevated risk of developing a radiogenic second cancer. Recently, proton therapy has been shown to reduce the dose delivered by the therapeutic beam to normal tissues during treatment compared to intensity modulated x-ray therapy (IMXT, the current standard of care). However, the magnitude of stray radiation doses from proton therapy, and their impact on this incidence of radiogenic second cancers, was not known. ^ The risk of a radiogenic second cancer following proton therapy for prostate cancer relative to IMXT was determined for 3 patients of large, median, and small anatomical stature. Doses delivered to healthy tissues from the therapeutic beam were obtained from treatment planning system calculations. Stray doses from IMXT were taken from the literature, while stray doses from proton therapy were simulated using a Monte Carlo model of a passive scattering treatment unit and an anthropomorphic phantom. Baseline risk models were taken from the Biological Effects of Ionizing Radiation VII report. A sensitivity analysis was conducted to characterize the uncertainty of risk calculations to uncertainties in the risk model, the relative biological effectiveness (RBE) of neutrons for carcinogenesis, and inter-patient anatomical variations. ^ The risk projections revealed that proton therapy carries a lower risk for radiogenic second cancer incidence following prostate irradiation compared to IMXT. The sensitivity analysis revealed that the results of the risk analysis depended only weakly on uncertainties in the risk model and inter-patient variations. Second cancer risks were sensitive to changes in the RBE of neutrons. However, the findings of the study were qualitatively consistent for all patient sizes and risk models considered, and for all neutron RBE values less than 100. ^