Biology subjects

Watkins, P. A.

Publications and source records attributed to Watkins, P. A..

4 recordsLinked to original sources

bioRxiv2024

Role of ACSBG1 in brain lipid metabolism and X-linked adrenoleukodystrophy pathogenesis: Insights from a knockout mouse model

The "bubblegum" acyl-CoA synthetase (ACSBG1) is a pivotal player in lipid metabolism during the development of the mouse brain, facilitating the activation of long-chain fatty acids (LCFAs) and their integration into essential lipid species crucial for brain function. Through its enzymatic activity, ACSBG1 converts LCFAs into acyl-CoA derivatives, supporting vital processes like membrane formation, myelination, and energy production. Its regulatory role significantly influences neuronal growth, synaptic plasticity, and overall brain development, highlighting its importance in maintaining lipid homeostasis and proper brain function. Originally discovered in the fruit fly brain, ACSBG1 attracted attention for its potential implication in X-linked adrenoleukodystrophy (XALD) pathogenesis. Studies using Drosophila melanogaster lacking the ACSBG1 homolog, bubblegum, revealed adult neurodegeneration with elevated levels of very long-chain fatty acids (VLCFA). To explore ACSBG1s role in fatty acid (FA) metabolism and its relevance to XALD, we created an ACSBG1 knockout (Acsbg1-/-) mouse model and examined its impact on lipid metabolism during mouse brain development. Phenotypically, Acsbg1-/- mice resembled wild type (w.t.) mice. Despite its primary expression in tissues affected by XALD, brain, adrenal gland and testis, ACSBG1 depletion did not significantly reduce total ACS enzyme activity in these tissues when using LCFA or VLCFA as substrates. However, analysis unveiled intriguing developmental and compositional changes in FA levels associated with ACSBG1 deficiency. In the adult mouse brain, ACSBG1 expression peaked in the cerebellum, with lower levels observed in other brain regions. Developmentally, ACSBG1 expression in the cerebellum was initially low during the first week of life but increased dramatically thereafter. Cerebellar FA levels were assessed in both w.t. and Acsbg1-/- mouse brains throughout development, revealing notable differences. While saturated VLCFA levels were typically high in XALD tissues and in fruit flies lacking ACSBG1, cerebella from Acsbg1-/- mice displayed lower saturated VLCFA levels, especially after about 8 days of age. Additionally, monounsaturated {omega}9 FA levels exhibited a similar trend as saturated VLCFA, {omega}3 polyunsaturated FA levels werewhile elevated in Acsbg1-/- mice. Further analysis of specific FA levels provided additional insights into potential roles for ACSBG1. Notably, the decreased VLCFA levels in Acsbg1-/- mice primarily stemmed from changes in C24:0 and C26:0, while reduced {omega}9 FA levels were mainly observed in C18:1 and C24:1. ACSBG1 depletion had minimal effects on saturated long-chain FA or {omega}6 polyunsaturated FA levels but led to significant increases in specific {omega}3 FA, such as C20:5 and C22:5. Moreover, the impact of ACSBG1 deficiency on the developmental expression of several cerebellar FA metabolism enzymes, including those required for synthesis of {omega}3 polyunsaturated FA, was assessed; these FA can potentially be converted into bioactive signaling molecules like eicosanoids and docosanoids. In conclusion, despite compelling circumstantial evidence, it is unlikely that ACSBG1 directly contributes to the pathology of XALD. Instead, the effects of ACSBG1 knockout on processes regulated by eicosanoids and/or docosanoids should be further investigated.

biochemistry↗

bioRxiv2023

Depleting glioblastoma cells of very long-chain acyl-CoA synthetase 3 (ACSVL3) produces metabolic alterations in non-lipid pathways

Knockout (KO) of the fatty acid-activation enzyme very long-chain acyl-CoA synthetase 3 (ACSVL3; SLC27A3) in U87MG glioblastoma cells reduced their malignant growth properties both in vitro and in xenografts. These U87-KO glioma cells grew at a slower rate, became adherence-dependent, and were less invasive than parental U87 cells. U87-KO cells produced fewer, slower-growing subcutaneous and intracranial tumors when implanted in NOD-SCID mice. Thus, depleting U87MG cells of ACSVL3 restored these cells to a phenotype more like that of normal astrocytes. To understand the mechanisms underlying these beneficial changes, we investigated several possibilities, including the effects of ACSVL3 depletion on carbohydrate metabolism. Proteomic and metabolomic profiling indicated that ACSVL3 KO produced changes in glucose and energy metabolism. Even though protein levels of glucose transporters GLUT1 and GLUT3 were reduced by KO, cellular uptake of labeled 2-deoxyglucose was unaffected. Glucose oxidation to CO2 was reduced nearly 7-fold by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Surprisingly, lactate production and the levels of lactate dehydrogenase isozymes LDHA and LDHB were elevated by ACSVL3 KO. The activity of the pentose phosphate pathway was found to be lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria were elongated in KO cells, but had a more punctate morphology in U87 cells. The mitochondrial potential was unaffected by lack of ACSVL3. We conclude that the beneficial effects of ACSVL3 depletion in human glioblastoma cells may result in part from alterations in diverse metabolic processes that are not directly related to role(s) of this enzyme in fatty acid and/or lipid metabolism. (Supported by NIH 5R01NS062043 and KKI institutional funds.)

biochemistry↗

bioRxiv2023

VERY LONG-CHAIN ACYL-CoA SYNTHETASE-3 (ACSVL3) PROMOTES THE MALIGNANT GROWTH BEHAVIOR OF U87 GLIOMA CELLS VIA CHANGES IN CELL CYCLE WITHOUT AFFECTING APOPTOSIS

Decreasing the expression of very long-chain acyl-CoA synthetase 3 (ACSVL3) in U87MG glioblastoma cells by either RNA interference or genomic knockout (KO) significantly decreased their growth rate in culture, as well as their ability to form rapidly growing tumors in mice. U87-KO cells grew at a 9-fold slower rate than U87MG cells. When injected subcutaneously in nude mice, the tumor initiation frequency of U87-KO cells was 70% of that of U87MG cells, and the average growth rate of tumors that did form was decreased by 9-fold. Two hypotheses to explain the decreased growth rate of KO cells were investigated. Lack of ACSVL3 could reduce cell growth either by increasing apoptosis, or via effects on the cell cycle. We examined intrinsic, extrinsic, and caspase-independent apoptosis pathways; none were affected by lack of ACSVL3. However, significant differences in the cell cycle were seen in KO cells, suggesting arrest in S-phase. Levels of cyclin-dependent kinases 1, 2, and 4 were elevated in U87-KO cells, as were regulatory proteins p21 and p53 that promote cell cycle arrest. In contrast, lack of ACSVL3 reduced the level of the inhibitory regulatory protein p27. {gamma}-H2AX, a marker of DNA double strand breaks, was elevated in U87-KO cells, while pH3, a mitotic index marker, was reduced. Previously reported alterations in sphingolipid metabolism in ACSVL3-depleted U87 cells may explain the effect of KO on cell cycle. These studies reinforce the notion that ACSVL3 is a promising therapeutic target in glioblastoma.

biochemistry↗

bioRxiv2022

ACSF2: A MEDIUM-CHAIN ACYL-CoA SYNTHETASE WITH A POTENTIAL ROLE IN NEURONAL DIFFERENTIATION

By activating fatty acids to their CoA derivatives, acyl-CoA synthetases (ACS) play an essential role in fatty acid metabolism. We previously identified ACSF2 as an ACS that was phylogenetically distinct from known families of short-chain, medium-chain, long-chain, very long-chain, and bubblegum ACSs. Functionally, ACSF2 preferentially activated medium-chain fatty acids. In this work, we provide further characterization of this unique ACS. ACSF2 mRNA expression was found in most tissues, although immunohistochemical analysis revealed differences in protein expression between various cell types in each tissue. Endogenous ACSF2 was found in the Golgi region in Neuro2a and P19 cells, and disruption of the Golgi in Neuro2a cells with nocodazole disrupted ACSF2 localization. In contrast, MA-10, HepG2, and skin fibroblasts had a mitochondrial ACSF2 localization. ACSF2 activated saturated fatty acids containing 6 to 10 carbons when overexpressed in COS-1 cells. The Kmapp for C10:0 was 24.4 M and Vmaxapp was 385 nmol/20min/mg COS cell protein. Knockdown by RNA interference revealed that ACSF2 was responsible for most of the medium-chain ACS activity in Neuro2a cells. A lysine residue critical for activity in bacterial short-chain ACSs was found in ACSF2, and mutation of this residue to alanine abolished enzyme activity. Neurite outgrowth results when Neuro2a cells are induced to differentiate with retinoic acid, and ACSF2 migrated to nodes and points of neurite-neurite contact along with the presynaptic marker, synaptophysin. ACSF2-deficient Neuro2a cells showed significantly blunted neurite outgrowth in response to retinoic acid. These results suggest that this medium-chain ACS may play an important role in neuronal development.

biochemistry↗