Abstract
Citrate synthase (CS) is a key mitochondrial enzyme. The aim of this study was to test the hypothesis that low CS activity impairs the metabolic health of mice fed a high fat diet (HFD) and promotes palmitate-induced lipotoxicity in muscle cells. C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A), a strain which carries the A/J allele of CS on the B6 strain background, were fed HFD (45% kcal from fat) for 12 weeks. C2C12 mouse muscle cells were used to investigate effects of CS knockdown on cell viability and signalling after incubation in 0.8 mM palmitate. CS activity, but not that of β-hydroxyacyl-coenzyme-A dehydrogenase was lower in the gastrocnemius muscle and heart of B6.A mice compared to B6 mice (P < 0.001). During HFD feeding, glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Body weight and fat gain did not differ between B6.A and B6 mice. After an 18 h incubation in 0.8 mM palmitate C2C12 muscle cells with ∼50% shRNA mediated reduction in CS activity showed lower (P < 0.001) viability and increased (P < 0.001) levels of cleaved caspase-3 compared to the scramble shRNA treated C2C12 cells. A/J strain variant of CS is associated with low enzyme activity and impaired metabolic health. This could be due to impaired lipid metabolism in muscle cells.
Highlights
Mitochondria play a key role in functioning of skeletal muscles and metabolic health [1, 2]
As neither abundance of the citrate synthase (CS) protein nor other mitochondrial markers could explain this reduction, we attributed this phenomenon to the missense mutation in exon 3 of Cs, i.e., H55N substitution (A for C, rs29358506) in the A/J mice. us, mice carrying the A/J allele of Cs could be a prudent model for studying the effects of reduced CS activity on such conditions as obesity and diabetes. is is important in view of the evidence suggesting that innate impairment in CS functioning might be responsible for low rates of fatty acid oxidation and insulin resistance in skeletal muscles of diabetic patients [8, 9]
We examined effects of reduced Cs expression on muscle cell viability; ATP levels and production of reactive oxygen species (ROS) were measured before and after incubation in the media supplemented with high concentrations of palmitate which induces apoptosis [10]
Summary
Mitochondria play a key role in functioning of skeletal muscles and metabolic health [1, 2]. A congenic strain carrying the A/J allele in the telomeric region of chromosome 10, where the Cs gene resides, showed higher resistance to obesity than B6 mice [15, 16] All these findings prompted us to investigate the role of the H55N polymorphism in physiological adaptations to HFD. We have tested the hypothesis about the link between low CS activity and impairment in lipid metabolism and lipotoxicity in muscle cells In these experiments, we examined effects of reduced Cs expression on muscle cell viability; ATP levels and production of reactive oxygen species (ROS) were measured before and after incubation in the media supplemented with high concentrations of palmitate which induces apoptosis [10]
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