Browsing by Author "Scott, John W."

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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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    Factors associated with optimal patient outcomes after operative repair of isolated hip fractures in the elderly
    (BMJ, 2020-12-22) deMeireles, Alirio J.; Gerhardinger, Laura; Oliphant, Bryant W.; Jenkins, Peter C.; Cain-Nielsen, Anne H.; Scott, John W.; Hemmila, Mark R.; Sangji, Naveen F.; Surgery, School of Medicine
    Background: Increased time to operative intervention is associated with a greater risk of mortality and complications in adults with a hip fracture. This study sought to determine factors associated with timeliness of operation in elderly patients presenting with an isolated hip fracture and the influence of surgical delay on outcomes. Methods: Trauma quality collaborative data (July 2016 to June 2019) were analyzed. Inclusion criteria were patients ≥65 years with an injury mechanism of fall, Abbreviated Injury Scale (AIS) 2005 diagnosis of hip fracture, and AIS extremity ≤3. Exclusion criteria included AIS in other body regions >1 and non-operative management. We examined the association of demographic, hospital, injury presentation, and comorbidity factors on a surgical delay >48 hours and patient outcomes using multivariable regression analysis. Results: 10 182 patients fit our study criteria out of 212 620 patients. Mean age was 82.7±8.6 years and 68.7% were female. Delay in operation >48 hours occurred in 965 (9.5%) of patients. Factors that significantly increased mortality or discharge to hospice were increased age, male gender, emergency department hypotension, functionally dependent health status (FDHS), advanced directive, liver disease, angina, and congestive heart failure (CHF). Delay >48 hours was associated with increased mortality or discharge to hospice (OR 1.52; 95% CI 1.13 to 2.06; p<0.01). Trauma center verification level, admission service, and hip fracture volume were not associated with mortality or discharge to hospice. Factors associated with operative delay >48 hours were male gender, FDHS, CHF, chronic renal failure, and advanced directive. Admission to the orthopedic surgery service was associated with less incidence of delay >48 hours (OR 0.43; 95% CI 0.29 to 0.64; p<0.001). Discussion: Hospital verification level, admission service, and patient volume did not impact the outcome of mortality/discharge to hospice. Delay to operation >48 hours was associated with increased mortality. The only measured modifiable characteristic that reduced delay to operative intervention was admission to the orthopedic surgery service. Level of evidence: III.