Browsing by Subject "Glucose uptake"

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    CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle
    (Elsevier, 2023) Negoita, Florentina; Addinsall, Alex B.; Hellberg, Kristina; Bringas, Conchita Fraguas; Hafen, Paul S.; Sermersheim, Tyler J.; Agerholm, Marianne; Lewis, Christopher T. A.; Ahwazi, Danial; Ling, Naomi X. Y.; Larsen, Jeppe K.; Deshmukh, Atul S.; Hossain, Mohammad A.; Oakhill, Jonathan S.; Ochala, Julien; Brault, Jeffrey J.; Sankar, Uma; Drewry, David H.; Scott, John W.; Witczak, Carol A.; Sakamoto, Kei; Anatomy, Cell Biology and Physiology, School of Medicine
    Objective: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. Methods: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. Results: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. Conclusions: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.
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    Inflammation-induced DNA methylation of DNA polymerase gamma alters the metabolic profile of colon tumors
    (BMC, 2018-07-10) Maiuri, Ashley R.; Li, Hongde; Stein, Barry D.; Tennessen, Jason M.; O’Hagan, Heather M.; Medicine, School of Medicine
    Background: Inflammation, metabolism, and epigenetic modulation are highly interconnected processes that can be altered during tumorigenesis. However, because of the complexity of these interactions, direct cause and effect during tumorigenesis have been difficult to prove. Previously, using a murine model of inflammation-induced colon tumorigenesis, we determined that the promoter of the catalytic subunit of DNA polymerase gamma (Polg) is DNA hypermethylated and silenced in inflammation-induced tumors, but not in non-inflammation-induced (mock) tumors, suggesting that inflammation can induce silencing of Polg through promoting DNA methylation during tumorigenesis. Polg is the only mitochondrial DNA polymerase and mutations in Polg cause mitochondrial diseases in humans. Because of the role of mitochondria in metabolism, we hypothesized that silencing of Polg in inflammation-induced tumors would result in these tumors having altered metabolism in comparison to mock tumors. Methods: Inflammation-induced and mock colon tumors and colon epithelium from a mouse model of inflammation-induced colon tumorigenesis were assayed for alterations in Polg expression, mitochondria, and metabolism. Organoids derived from these tissues were used to study the direct effect of loss of Polg on mitochondria and metabolism. Results: We demonstrate that inflammation-induced tumors with reduced Polg expression have decreased mitochondrial DNA content and numbers of mitochondria compared to normal epithelium or mock tumors. Tumoroids derived from mock and inflammation-induced tumors retained key characteristics of the original tumors. Inflammation-induced tumoroids had increased glucose uptake and lactate secretion relative to mock tumoroids. shRNA-mediated knockdown of Polg in mock tumoroids reduced mtDNA content, increased glucose uptake and lactate secretion, and made the tumoroids more resistant to oxidative stress. Conclusions: These results suggest that inflammation-induced DNA methylation and silencing of Polg plays an important role in the tumorigenesis process by resulting in reduced mitochondria levels and altered metabolism. An enhanced understanding of how metabolism is altered in and drives inflammation-induced tumorigenesis will provide potential therapeutic targets.
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    Regulation of skeletal muscle insulin sensitivity by PAK1
    (2016-012) Tunduguru, Ragadeepthi; Thurmond, Debbie C.; Elmendorf, Jeffrey S.; Atkinson, Simon J.; Brozinick, Joseph T.; Gunst, Susan J.
    Insulin-stimulated glucose uptake into skeletal muscle cells requires translocation of the glucose transporter-4 (GLUT4) from the cell interior to the plasma membrane. Insulin-stimulated GLUT4 vesicle translocation is dysregulated in Type 2 diabetes (T2D). The Group I p21–activated kinase (PAK1) is a required element in insulin-stimulated GLUT4 vesicle translocation in mouse skeletal muscle in vivo, although its placement and function(s) in the canonical insulin signaling cascade in skeletal muscle cells, remain undetermined. Therefore, the objective of my project is to determine the molecular mechanism(s) underlying the requirement for PAK1 in the process of insulin-stimulated GLUT4 vesicle translocation and subsequent glucose uptake by skeletal muscle cells. Toward this, my studies demonstrate that the pharmacological inhibition of PAK1 activation blunts insulin-stimulated GLUT4 translocation and subsequent glucose uptake into L6-GLUT4myc skeletal myotubes. Inhibition of PAK1 activation also ablates insulin-stimulated F-actin cytoskeletal remodeling, a process known to be required for mobilizing GLUT4 vesicles to the plasma membrane. Consistent with this mechanism, PAK1 activation was also required for the activation of cofilin, another protein implicated in F-actin remodeling. Interestingly, my studies reveal a novel molecular mechanism involving PAK1 signaling to p41-ARC, a regulatory subunit of the cytoskeletal Arp2/3 complex, and its interactions with another cytoskeletal factor, N-WASP, to elicit the insulin-stimulated F-actin remodeling in skeletal muscle cells. Pharmacological inactivation of N-WASP fully abrogated insulin-stimulated GLUT4 vesicle translocation to the cell surface, coordinate with blunted F-actin remodeling. Furthermore, my studies revealed new insulin-induced interactions amongst N WASP, actin, p41-ARC and PAK1; inactivation of PAK1 signaling blocked these dynamic interactions. Taken together, the above studies demonstrate the significance of PAK1 and its downstream signaling to F-actin remodeling in insulin-stimulated GLUT4 vesicle translocation and glucose uptake, revealing new signaling elements that may prove to be promising targets for future therapeutic design.