Low Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity

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.

Figure 1

Genotyping chromosome 10 in congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) strain. Black, white, and gray colours mark alleles of C57BL/6J (B6), A/J genotype, and unknown genotype, respectively. The chromosomal markers are also indicated. The data are also presented for congenic (10C1) strain in [16].

Figure 2

Body mass and composition of mice. Body mass (a), food intake (b), body fat, (c) and lean body mass (d) in C57BL/6J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice (M, males; F, females) during the baseline period of high carbohydrate diet (HCD) followed by high fat diet (HFD) feeding. Values (n=16 each) are means in A and B as well as means ± SEM in (c) and (d) ^∗∗P < 0.01, B6 vs B6.A strains.

Figure 3

Energy balance and resting metabolic rate of mice. Metabolisable energy intake (MEI) (n=8 each) (a), daily energy expenditure (DEE) (n=8 each) (b), and resting metabolic rate (RMR) (n=16 each) (c) in C57BL/6J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice (M, males; F, females) fed high carbohydrate diet (HCD) and high fat diet (HFD). Values are means ± S.D. ^∗∗P < 0.05, B6 vs B6.A strains.

Figure 4

Physical activity and body temperature. Average daily physical activity (a) and body temperature (b) in C57BL/6J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice (M, males; F, females). The mean values for physical activity during the light phase are marked by white colour. The data are presented for the baseline period of high carbohydrate diet (HCD) feeding followed by HFD feeding for 2 weeks (HFD2), 6 weeks (HFD6), and 12 weeks (HFD12). Values are means ± SD (n=16 each).

Figure 5

Glucose tolerance. Blood glucose concentrations (mM) and area under curve (AUC) during glucose tolerance tests consisting of the peritoneal bolus injection of glucose (2 g glucose/kg body weight) in C57BL/6J (B6) and B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice fed high carbohydrate diet (a, b) as well as after for 2 weeks (c, d), 6 weeks (e, f), and 12 weeks (g, h) of high fat diet feeding. Values are means (a c, e, and g) and means ± SD (b, d, f, and h). ^†P < 0.001 males vs females; ^∗∗P < 0.001, B6 vs B6.A strains.

Figure 6

Palmitate-induced lipotoxicity in C2C12 muscle cells. C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted Cs mRNA. Afterwards Con shRNA and Cs shRNA cells were incubated in the media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate (P). (a) Cell proliferation was assessed using cell counting assay kit-8 (96992, Sigma, UK) and verified by (b) crystal violet staining; (c) cellular ATP concentrations were assessed using ATP calorimetric assay (ab83355, Abcam, Cambridge, UK); (d) levels of cleaved caspase-3 were assessed using immunoblotting; (e) production of reactive oxygen species was assessed by measuring 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence (values are means ± SEM). ^∗∗P < 0.01, B6 vs B6.A strains. ^†p < 0.001 G vs G + P; ^∗∗∗P < 0.001, Con shRNA vs Cs shRNA.

Figure 7

Palmitate-induced lipotoxicity in C2C12 muscle cells (Cont). C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted Cs mRNA. Afterwards Con shRNA and Cs shRNA cells were incubated in the media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate (P). (a) Effect of palmitate exposure on the phosphorylation of AMPK (Thr 172) in Con shRNA and Cs shRNA cells using western blot analysis. Quantification of immunoblots from six experiments. P.AMPK was normalized to AMPK. (b) Effect of palmitate exposure on the phosphorylation of PKB (Ser473) in Con shRNA and Cs shRNA cells. Quantification of immunoblots from six experiments. P.PKB was normalized to PKB. Control cells with 5.5 mM glucose were used as reference. Protein load: 25 µg expression. Values are means ± SEM.