Biology subjects

Humphries, K. M.

Publications and source records attributed to Humphries, K. M..

2 recordsLinked to original sources

bioRxiv2020

Sirt5 deficiency causes post-translational protein malonylation and dysregulated cellular metabolism in chondrocytes under obesity conditions

ObjectiveObesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. The objective of this study was to investigate the role of sirtuin 5 (SIRT5) in regulating chondrocyte protein lysine malonylation (MaK) and cellular metabolism under obesity-related conditions. MethodsMaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus (DMM) surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5-/- mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by Western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling. ResultsMaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (GluhiInshi). Sirt5 protein was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under GluhiInshi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis. ConclusionThis study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in Sirt5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

cell biology↗

bioRxiv2020

Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin

The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. Akita ACMs also had decreased PKA signaling in the absence of insulin and this was not rescued by insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were therefore used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.

cell biology↗