Browsing by Subject "mTOR"
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Item Acute and chronic effects of [Beta]-hydroxy-[Beta]-methylbutyrate ([Beta]-HMB) on glucose tolerance, insulin sensitivity and muscle adaptation following chronic resistance training(2019-05-03) Solares, Geoffrey Josef; Farrar, Roger P.; Suggs, Laura J.; Bray, Molly S.; Wilcox, Richard E.; Todd, Janice S.Maximizing protein accretion and mitigating protein degradation is a major goal for resistance training regimens and intervention therapies. The branch chain amino acid leucine has historically demonstrated a significant role in the activation of protein synthesis via the activation of the mammalian target of rapamycin complex 1 (mTORC1). A derivative of leucine metabolism, beta-hydroxy-beta-methylbutyrate (HMB) has shown similar effects on mTORC1 with recent literature suggesting adverse effects of HMB on glucose homeostasis and regulation. Herein, we used animal models to test the effects of varying doses of HMB on glucose homeostasis and during a novel chronic resistance whole body training model. These data suggest that HMB effects possess acute modulation to the Akt/mTOR signaling pathway proteins, with minimal contributions to strength gains during chronic resistance whole body exercise. Our novel whole body exercise technique revealed a significant increase in strength gains with no differences in in situ force production in quadriceps and triceps surae muscle groups, but did show increased force per unit mass in both the triceps surae and quadriceps muscle groups. These data suggest an increase in whole body muscular coordination and/or synchronicity in force production that promotes increases in overall total strength. Furthermore, our data suggests that the overload placed on each individual contributing muscle to force output was not significant enough to induce a hypertrophic response. We conclude that HMB ingestion provides minimal benefit during prolonged exercise regimens and the effects of HMB on blood glucose and insulin sensitivity are not adverse. Finally, our whole body resistance model presents a novel paradigm for increasing work output that can be a model of whole body resistance training.Item Bioinformatic approaches to screening the molecular framework underlying local dendritic mRNA translation(2017-05) Namjoshi, Sanjeev Vinayak; Hofmann, Hans (Hans A.); Raab-Graham, Kimberly F.; Atkinson, Nigel S.; Marcotte, Edward M.; Sullivan, Christopher S.Learning and memory systems within the brain are believed to be the result of molecular events that occur at the synapse. These molecular events control synaptic efficacy – the modulation of the underlying molecular architecture of the synapse to influence the strength or weakness of connections between pre- and postsynaptic neurons. Deficits in any of these molecular processes results in neuronal dysfunction. The Mammalian/Mechanistic Target of Rapamycin Complex (mTORC1) is a protein complex within the brain composed of the serine/threonine kinase mTOR and other interacting proteins. Dysregulation of mTOR disrupts the many processes under its control such as local dendritic translation at the synapse. Since mTOR is at the core of many important signaling pathways, aberrant mTOR activity results in neuronal disease. The network of interactions between these molecular components is vast, forming an interconnected system that is dynamic and directed. In order to better understand the mechanistic nature of these interactions, the application of high-throughput technologies must be employed. Here we utilize multiple bioinformatics approaches combined with high-throughput technologies to clarify the role of mTOR in local dendritic translation. Using mass spectrometry, we provide the first evidence that mTOR bidirectionally controls the expression of over 700 proteins in the cortex, many of which are known to be associated with diseases in which mTOR is overactive (Chapter 2). Our investigations reveal a novel role PARK7 in tuberous sclerosis complex. Furthermore, we use and develop multiple bioinformatics tools to further delineate the nature of the RNA-binding properties of PARK7 and alternative avenues for drug discovery (Chapter 3). Finally, we provide evidence that the Fragile-X Mental Retardation protein sequesters a population of mRNAs involved in trans-synaptic signaling that mTOR translates to remodel the synapse, providing a mechanistic basis for the action of rapidly-acting antidepressants (Chapter 4). Collectively, our work stresses the importance of applying high-throughput technologies to answer long-standing questions in the field of local dendritic translation. Our findings provide new avenues of investigation and research to better understand neuronal disease and synaptic plasticityItem Dietary energy balance modulates growth factor signaling during multistage epithelial carcinogenesis in mouse skin(2010-12) Moore, Tricia Wallace; DiGiovanni, John; Hursting, Stephen; Kline, Kimberly; deGraffenried, Linda; Fischer, Susan; Vasquez, KarenEnergy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation.Item The effects of [beta]-hydroxy-[beta]-methylbutyrate (HMB) and leucine on cellular signaling pathways controlling protein synthesis and degradation during sedentary and post-exercise recovery in skeletal muscle(2012-08) Liao, Yi-Hung; Ivy, John, 1945-Recent research suggests that [beta]-hydroxy-[beta]-methylbutyrate (HMB), a metabolite of leucine (Leu), increases muscle mass and attenuates muscle damage during resistance training. Although Leu acts as a potent stimulator of protein synthesis, HMB, but not Leu, has been reported to be effective in suppressing proteolysis in skeletal muscle. However, mechanisms for the effects of HMB on cell signaling pathways controlling muscle protein turnover during rest and after endurance exercise are still poorly understood. Furthermore, the effects of HMB on cell signaling pathways controlling protein synthesis and degradation under normal in vivo conditions warrant further investigation. For optimal gains in muscle mass, the appropriate type and amount of protein (PRO) is required for positive protein balance to occur in skeletal muscle. Therefore, this dissertation was designed to determine the effect of HMB, PRO and Leu, individually and in combination, on the regulation of cellular signaling pathways controlling muscle protein turnover during sedentary and post-exercise conditions. Study 1 demonstrated that, compared with HMB and PRO alone, the combination of HMB and PRO was more effective in activating the mTOR signaling pathway, which controls protein synthesis, and inhibiting FOXO3A, a major regulator of the ubiquitin-proteasome proteolytic signaling pathway. Study 2 demonstrated that, compared with its individual components, a novel HMB/PRO/Leu supplement better activated protein-synthetic signals and inhibited proteolytic signals in skeletal muscle, and these effects were better sustained. Finally, Study 3 demonstrated that adding Leu to PRO-enriched mixtures after exercise additively activated protein-synthetic signals in a fiber type-specific manner, and adding HMB clearly inhibited proteolytic signaling proteins. Furthermore, provision of an HMB/PRO/Leu supplement after exercise was found to favorably modulate signaling pathways controlling both protein synthesis and degradation. Taken together, the results of these studies suggest that a novel nutrient supplement, composed of HMB, Leu and PRO, additively enhances the intracellular signaling proteins controlling protein synthesis and attenuates signaling proteins controlling proteolysis in skeletal muscle during sedentary and post-exercise recovery. Therefore, such a supplement may be beneficial for both athletic and therapeutic purposes.Item Leucine supplementation differentially enhances pancreatic cancer growth in lean and overweight mice(Cancer and Metabolism, 2014-03-18) Liu, Kristyn A.; Lashinger, Laura M.; Rasmussen, Audrey J.; Hursting, Stephend D.Background: The risk of pancreatic cancer, the 4th deadliest cancer for both men and women in the United States, is increased by obesity. Calorie restriction (CR) is a well-known dietary regimen that prevents or reverses obesity and suppresses tumorigenesis in a variety of animal models, at least in part via inhibition of mammalian target of rapamycin (mTOR) signaling. Branched-chain amino acids (BCAA), especially leucine, activate mTOR and enhance growth and proliferation of myocytes and epithelial cells, which is why leucine is a popular supplement among athletes. Leucine is also increasingly being used as a treatment for pancreatic cancer cachexia, but the effects of leucine supplementation on pancreatic tumor growth have not been elucidated. Results: Supplementation with leucine increased pancreatic tumor growth in both lean (104 ± 17 mm3 versus 46 ± 13 mm3; P <0.05) and overweight (367 ± 45 mm3 versus 230 ± 39 mm3; P <0.01) mice, but tumor enhancement was associated with different biological outcomes depending on the diet. In the lean mice, leucine increased phosphorylation of mTOR and downstream effector S6 ribosomal protein, but in the overweight mice, leucine reduced glucose clearance and thus increased the amount of circulating glucose available to the tumor. Conclusion: These findings show that leucine supplementation enhances tumor growth in both lean and overweight mice through diet-dependent effects in a murine model of pancreatic cancer, suggesting caution against the clinical use of leucine supplementation for the purposes of skeletal muscle enhancement in cachectic patients.Item mTOR dependent regulation of Kv1.1 in normal and disease states by the RNA binding factors, HuD and miR-129-5p(2014-05) Sosanya, Natasha Marie; Raab-Graham, Kimberly F.; Atkinson, Nigel; Golding, Nace; Haris, Adron; Iyer, VishwanathLittle is known about how a neuron undergoes site-specific changes in intrinsic excitability in normal and diseased conditions. We provide evidence for a novel mechanism for the mammalian Target of Rapamycin Complex 1 (mTORC1) kinase dependent translational regulation of the voltage-gated potassium channel Kv1.1 mRNA (Chapter 2). First, we identified a microRNA, miR-129-5p, that represses Kv1.1 mRNA translation when mTORC1 is active. When mTORC1 is inactive, we found that the RNA-binding protein, HuD, binds to Kv1.1 mRNA and promotes its translation. Surprisingly, mTORC1 activity does not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA but affects the degradation of high-affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Thus, high affinity HuD target mRNAs can serve two purposes under normal physiological conditions: 1) to provide functional proteins, such as CaMKIIα, that change the architecture of the synapse and 2) serve as a sponge sequestering HuD from translating mRNAs like Kv1.1. To determine if this mechanism for repression of Kv1.1 expression is conserved in a disease model where mTORC1 activity is overactive, we assessed the expression levels of active mTORC1, Kv1.1, and miR-129-5p in a rat model of temporal lobe epilepsy (TLE; Chapter 3). We found that when mTOR activity is low in TLE, Kv1.1 expression is high and behavioral seizure number is low. In contrast, when behavioral seizure activity starts to rise there is a corresponding increase in mTOR activity and Kv1.1 protein levels dramatically drop. In addition, we found that miR-129-5p, the negative regulator of Kv1.1 mRNA translation increases by 21 days post status epilepticus (SE) to sustain Kv1.1 mRNA translational repression. Thus, long-term changes in Kv1.1 protein levels result in a hyperpolarized threshold for action potential firing. Our results suggest that increased mTOR activity following SE results in two phases of Kv1.1 repression (1) in an initial repression of Kv1.1 mRNA translation by mTOR activity that is followed by (2) an onset of elevated miR-129-5p expression that sustains Kv1.1 repression. These studies suggest that dynamic changes in miR- 129-5p provide potential novel targets for epilepsy interventions. mTOR is a protein kinase that promotes CaMKIIα mRNA translation (Sosanya et al., 2013; Chapter 2); however, the mechanism and site of dendritic expression are unknown. Herein (Chapter 4), we show that mTOR activity mediates the dendritic branch specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. Notably, reduction in mTOR activity decreases the overall dendritic expression of CaMKIIα protein and RNA through the shortening of its poly(A) tail. Overexpression of HuD both increases total CaMKIIα levels and rescues the selective expression of CaMKIIα in one daughter branch over the other. These results suggest that differential branch targeting of HuD may mediate the branch specific expression of CaMKIIα in neuronal dendrites during mTOR activity. Furthermore, when mTOR activity is reduced HuD releases CaMKIIα mRNA and thus exposes its poly(A) tail to be deadenylated, reducing its overall expression and eliminating its branch specific expression.Item The function of mTORC1 in craniofacial morphogenesis and ethanol teratogenesis(2023-04-21) Tucker, Scott K.; Eberhart, Johann K.; Dalby, Kevin; Fischer, Janice; Gray, Ryan; Vokes, StevenThe mTOR pathway is a critical regulator of cell growth, cell survival, and autophagy. Over the last decade, there has been growing evidence suggesting that the mTOR pathway plays an essential role in craniofacial development. My research seeks to characterize the precise mechanisms by which this pathway functions during craniofacial development, as well as the downstream effects of its dysregulation. At the same time, prenatal ethanol exposure has long been known to cause a range of birth defects, including those affecting the craniofacial region. While the precise mechanisms by which ethanol exerts its teratogenic effects are still not fully understood, nearly all of the known mechanisms are regulated by the mTOR pathway. My research aims to characterize the role of the mTOR pathway in craniofacial development and to gain insight into the teratogenic effects of ethanol. Here, I utilized a zebrafish tp53 mutant which is deficient in inducing cell death and normally functions downstream of mTOR. I demonstrated that alcohol-induced reductions of eye size and trabecula length were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. This work, along with that of our collaborators using a mouse model, established that Tp53 plays a conserved role in prenatal ethanol exposure-induced cell death. I followed up this work by characterizing the role of mTORC1 in craniofacial development, by generating a zebrafish raptor mutant, an essential component of the complex. The raptor mutants exhibited a severe reduction in the size of craniofacial elements. I observed that this size reduction was attributed to reduced cell size and elevated cell death. Furthermore, I demonstrated that autophagy was elevated in raptor mutants, and that inhibition of autophagy reduced cell death and increased craniofacial element size. The findings of these studies have important implications for understanding the molecular and cellular processes that underlie craniofacial development and for developing new strategies to prevent and treat craniofacial abnormalities caused by the combinatorial effects of genetic mutations and environmental toxicants such as ethanol exposure.