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Mechanisms Underlying the Altered Bioenergetics of Aged Brain Microvasculature
Author(s) -
Sakamuri Siva S.V.P.,
Sure Venkata,
Kolli Lahari,
Evans Wesley,
Murfee Walter,
Mostany Ricardo,
Katakam Prasad
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04572
Subject(s) - bioenergetics , glycolysis , oxidative phosphorylation , glut1 , medicine , endocrinology , citrate synthase , lactate dehydrogenase , glucose transporter , phosphofructokinase , mitochondrion , anaerobic glycolysis , pyruvate kinase , chemistry , fructose , extracellular , biology , biochemistry , metabolism , enzyme , insulin
Objective Aging impairs the cerebral microvascular function leading to increased risk of stroke and cognitive decline. The objective of this study was to identify the mechanisms underlying the aging‐induced alterations in cellular energetics of brain microvessels (BMVs). Methods BMVs were isolated from young (2‐4 months) and aged (24 months) male mice (C57Bl/6) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured from real time ATP rate assay utilizing Seahorse XF e 24 analyzer. Subsequently, total ATP production rate, GlycoATP and MitoATP levels were calculated. Mitochondrial fuel dependency of BMVs was assayed. Immunoblotting measured the protein levels of glucose transporter 1 (GLUT1), carnitine palmitoyltransferases (CPTs), 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐biphosphatase 3 (PKKFB3), and ETC complexes (ETCs). Enzyme assays measured the activity of lactate dehydrogenase (LDH), citrate synthase (CS), and glutamate dehydrogenase (GDH). Results We found that oxidative phosphorylation (OxPhos) is the major contributor of cellular energy in the BMVs instead of glycolysis (67.0±2.1% vs 32.9±2.1%, n=7‐10 mice/age group). Aged BMVs exhibited decreased total ATP production (49.7%, 8.8±2.0 vs 17.6±6.7 pico moles of ATP/µg/min, p=0.004, n=7‐10 mice/age group). ATP generation from the glycolysis was more diminished in aged BMVs (74.7%, 1.5±0.3 vs 5.9±1.1 pico moles of ATP/µg/min, p=0.004, n=7‐10 mice/group) than OxPhos (55.6%, 5.2±0.53 vs 11.7±1.8 pico moles of ATP/µg/min, p=0.01, n=7‐10mice/group) when compared with young BMVs. GLUT1 levels were decreased in the aged BMVs when compared with the young BMVs (32%, 0.19±0.01 vs 0.28±0.02 A.U., p=0.01, n=12 mice/group). Glycolysis‐stimulating enzyme, PFKFB3 expression was diminished (51.4%, 0.69±0.08 vs 1.42±0.13 A.U., n=6 mice/group) in the aged BMVs. Neither CPT nor ETC protein levels were affected by aging. Activity of Krebs cycle enzyme, CS, was reduced in the aged versus young BMVs (25.7%, 74.3±6.1% vs 100.0±9.9% nanomoles of substrate/min/mg, p=0.04, n=11‐12 mice/group). Although relative utilizations of glucose and fatty acids were not affected by aging, glutamine utilization was enhanced in aged BMVs compared to young BMVs which was accompanied by increased activity of GDH, an enzyme involved in the mobilization of glutamine through Krebs cycle (24.7%, 116.8±4.3% vs 93.6±7.3% pico moles of NADH/min/mg, p=0.01, n=11‐12 mice/group). Conclusions The results suggest that 1). diminished energy production in the aged BMVs is related to impaired glycolysis and OxPhos, and 2). multiple defects involving the expression of proteins and activity of enzymes related to glycolysis, OxPhos, and energy substrate utilization mediate the aging‐induced impaired microvascular bioenergetics.