Abstract
BackgroundThe calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells.MethodsCellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB.ResultsCAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics.ConclusionThis study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures.FH7qJaRnRK83y_zSjUv3dhVideo
Highlights
The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases
Constitutive expression of Calcium/calmodulin-activated kinase kinase 2 (CAMKK2) differed between HEK293 and HepG2 cells Previously, alternatively spliced CAMKK2 isoforms were detected as two distinct protein bands in the range of p70-75 kDa [17]
CAMKK2 deficiency dampened cellular respiration in HEK293 and HepG2 cells Cellular glucose metabolism was interrogated by simultaneous measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse flux analyzer
Summary
The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. Activated CAMKK2 directly phosphorylates multiple downstream effectors including Ca2+/CAM-dependent protein kinase I and 4 (CAMKI and CAMK4), AMP-activated protein kinase (AMPK), and the NAD-dependent protein deacetylase—Sirtuin 1 (SIRT1), each have their distinct signaling effects on cellular metabolism [2]. AMPK is a cellular energy sensor activated by metabolic stresses that inhibits mitochondrial ATP production or accelerates ATP consumption [3, 4]. The mechanistic basis of CAMKK2-mediated glucose oxidation and mitochondrial bioenergetics is not well characterized despite a wealth of knowledge accumulated over the years regarding CAMKK2-downstream effector-mediated regulation of cellular glucose homeostasis and the maintenance of energy balance
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