The regulation of AMP-activated protein kinase (AMPK) in an insulinoma cell line, adipocytes, and muscle.

摘要

AMP-activated protein kinase (AMPK) is an important metabolite-sensing protein kinase with numerous substrate targets aimed at the conservation and regeneration of cellular energy levels. The goals of this thesis were to identify novel substrate targets and cellular processes in which AMPK is involved.; In light of these goals, we first investigated the role of AMPK in glucose transport. In these studies, we found that activation of both α1 and α2 isoforms of AMPK, via 5-Aminoimidazole-4-carboxamide-1-β-D-ribofurnaoside (AICAR), uncouplers of oxidative phosphorylation and hyperosmolarity, resulted in increased rates of GLUT4-mediated glucose transport in skeletal muscle. In addition, AMPK activation via AICAR resulted in increased rates of GLUT1-mediated glucose transport in the liver-like cell line, Clone 9.; The role of AMPK activation via AICAR was also investigated in the process of adipocyte differentiation. We found that treatment of 3T3-L1 cells with AICAR prevented differentiation if added early in the differentiation process, between day 0 and day 2. AICAR treatment caused the downregulation of the regulatory transcription factors PPARγ and C/EBPα. In addition, expression levels of C/EBPβ were also affected, such that levels of this transcription factor remained elevated during the time in which the endogenous protein was beginning to degrade. This suggests that perhaps AICAR may function by inhibiting the activity, but not the expression of, C/EBPβ. However, it was determined that this inhibition is not happening through increased expression of the dominant negative CHOP-10 protein.; In order to further our studies, recombinant adenoviruses containing the AMPK-α subunit were created in order to have a more specific method in which to modulate AMPK activity. Recombinant adenoviruses containing both the catalytically active (Adα1, Ad-α2, Ad-α312) and inactive forms of AMPK-α (Ad-αK45R, Ad-αT172D) were successfully constructed and expressed.; These viruses served as a useful tool in the study of the regulation of AMPK activity in islet β-cell intermediary metabolism in the islet cell line, 832/13. Using a two-way approach, we modulated AMPK activity both through AICAR treatment and infection with a recombinant adenovirus containing the catalytic subunit of AMPK (Ad-α1). Although AMPK activation by these two methods had no effect on insulin secretion, both methods increased rates of fatty acid oxidation. However, only treatment with AICAR resulted in changes in acetyl-CoA carboxylase (ACC) enzyme activity, thus suggesting Ad-α1 can effect fatty acid oxidation rates in an ACC-independent manner. This data demonstrates the first dissociation between increases in AMPK activity and AICAR treatment.; Lastly, this thesis examined the hypothesis that malonyl-CoA decarboxylase (MCD) was a target of AMPK. Using a three-way approach, we found that AMPK activation had no effect on MCD activity in either exercising muscle, in vitro, or in the islet cell line, 832/13.