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Browsing by Subject "Energy Homeostasis"

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    Biochemical and functional characterization of fatty acid transport proteins.
    (2009-07) Wiczer, Brian Michael
    The adipocyte fatty acid transport proteins (FATPs), FATP1 and FATP4, have been implicated in both lipid influx and storage and understanding their role in adipose tissue would gain insight into the persistence of metabolic disorders, such as type 2 diabetes. FATP1 was previously determined to be an acyl-CoA synthetase and work described in this thesis additionally explored the acyl-CoA synthetase activity of purified FATP4. FATP4 was found to be a more robust acyl-CoA synthetase than FATP1. Through the use of RNAi in cultured adipocytes, silencing the expression of either FATP1 or FATP4 results in cellular phenotype demonstrating improved insulin responsiveness. Interestingly, silencing FATP1 abolished insulin-stimulated long-chain fatty acid (LCFA) influx, whereas silencing FATP4 had no effect on LCFA influx despite its higher activity. Furthermore, the expression of FATP1 was demonstrated to be important for the activation of the AMP-activated protein kinase during insulinstimulated LCFA influx. In addition to the cytoplasmic localization of FATP1, it was also found to exhibit mitochondrial localization. Further analysis demonstrated a novel role in the regulation of TCA cycle function and mitochondrial energy metabolism, in part, through the interaction of FATP1 with the 2-oxoglutarate dehydrogenase complex, a rate-limiting step in the TCA cycle. This work shines light on how FATPs may play broader roles in metabolism that previously appreciated and the potential implications associated with such roles.
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    Bivalent Ligands as Pharmacological Probes for The Melanocortin Receptors: The Bivalent Advantage
    (2017-05) Lensing, Cody
    Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer’s disease, social disorders, cachexia, and obesity. Of interest to our laboratory is the melanocortin system’s role in energy homeostasis that is mediated through the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R). Specifically, our laboratory focuses on the development of novel pharmacological probes to better understand the role of the melanocortin receptor system’s effects on energy homeostasis. This thesis provides the field with foundational work addressing the functional effects of melanocortin bivalent ligands both in vitro and in vivo. In Chapter 3 and Chapter 4, traditional homobivalent approaches are utilized. The synthesis and in vitro evaluation of homobivalent ligands are discussed in Chapter 3. Lead ligands (CJL-1-87 and CJL-1-31) increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts depending on the specific melanocortin receptor subtype assayed. In Chapter 4, the in vivo effects of lead ligand CJL-1-87 is characterized thoroughly. Bivalent ligand CJL-1-87 had noteworthy advantages as an anti-obesity probe over its monovalent counterpart in a fasting-refeeding in vivo paradigm. Treatment with CJL-1-87 significantly decreased food intake compared to CJL-1-14 or saline (50% less intake 2 to 8 hours after treatment). Further energy expenditure parameters are explored, and possible mechanisms are discussed. In Chapter 5 and Chapter 6, uncommon approaches are attempted to exploit melanocortin dimers to elicited undiscovered pharmacological effects. In the Chapter 5, we present melanocortin unmatched bivalent ligands (MUmBLs) as tools for studying asymmetric function of melanocortin receptor homodimers. MUmBLs contain one agonist scaffold and one antagonist scaffold designed to target a melanocortin homodimer pair such that one receptor is occupied by an agonist scaffold and the other receptor by an antagonist scaffold. Utilizing this design strategy to target the MC4R, first in class biased unmatched bivalent ligands (BUmBLs) were discovered. The BUmBLs displayed biased agonism in which they potently stimulated cAMP signaling, but resulted in minimal activation of the β-arrestin recruitment pathway. In Chapter 6, we describe two different approaches that were pursued to further study melanocortin bivalent ligands’ structure activity relationship (SAR). Homobivalent ligands were designed with 13, 16, 19, 20, and 22 atom linkers to explore the effects of linker length. Overall, these studies resulted in a “flat” SAR in which the compounds all have similar potencies and efficacies. Bivalent ligands were also designed to include the retro-inverso tetrapeptide scaffold DTrp-DArg-Phe-DHis. Although this scaffold lacked high binding affinity and potency, it was very metabolically stable. The incorporation of this scaffold into bivalent ligands yielded ligands with varying potencies and metabolic stabilities. The current discoveries may be broadly applicable to other GPCR systems. As the physiological relevancy to GPCR oligomerization is elucidated, the current medicinal chemistry strategies presented in this thesis should aid in the discovery of probes and possible therapeutics for the further understanding of GPCR pharmacology for various systems.