Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and a-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigo

Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor

Characteristics of the <i>Mcat</i> knockout mouse.

<p>A and B: mean relative body weights of 13 KO and 13 HF control male mice, initial weights 21.6±2.5 g and 22.9±2.5, respectively (A) and 10 KO and 10 HF control female mice, initial weights 20.8±3.2 g and 19.0±3.2 g, respectively (B). Asterisks indicate the earliest time points at which statistically significant differences in weight were observed. C: Mean food consumption monitored for a 2-week period 7 months after tamoxifen treatment (11–13 measurements each group). D: mean grip strength of male, 5 KO and 7 HF control, and female, 6 KO and 5 HF control mice. E: Mean residence times on the accelerating rotarod, measured 7 months post-induction, were not significantly different between KO (14 male, 18 female) and HF controls (10 male, 25 female). F: Physical activity in the Open Field Test, assessed 7–8 months after tamoxifen treatment, in KO (12 male, 7 female) and HF controls (10 male, 9 female). G: Rectal body temperatures monitored in the period 14–29 weeks after tamoxifen treatment (9 KO males and 7 KO females combined, 3 HF males and 8 HF females combined; 7 measurements for each mouse) and in the period 36–45 weeks post-treatment (sexes shown separately, 2–3 measurements each mouse). Statistical significance in the T-test, where observed, is indicated by *p>0.05, **p<0.005 or ***p<0.0005.</p

Content and enzyme activity of mitochondrial proteins.

<p>A: typical Western blots of mitochondrial proteins illustrating knockdown of Mcat (row 1), lipoylation status of the E2 subunits of PDC and BCDC (rows 2 and 3, respectively), and the content of Oxsm, as a control (row 4). B and C: Expression profiles of Mcat (B) and LipT (C) compared to controls, Oxsm and prohibitin, in mitochondria of HF control mice. The abundance of the proteins is expressed relative to that of liver, which is assigned value of 1.0 (B, n = 2–6; C, n = 1–4). D: Abundance of Mcat and control proteins in mitochondria from KO mice (n = 3–7) relative to the content in HF mice (n = 2–6). E: Lipoylation status of the E2 subunits of PDC and BCDC in KO mice (n = 3–7) relative to HF controls (n = 2–6); mean assigned value of 1.0. F: Activities of KDC and PDC in mitochondria isolated from HF (n, 3–8) and KO mice (n, 4–10). G: Overall activity of the mitochondrial fatty acid synthesis pathway, reported as production of octanoyl-ACP from malonate (n = 2, HF and KO).</p

Characterization of anemic KO mice.

<p>Approximately 20% of the KO mice were found to be anemic. A–E: blood cell analysis. A: Red blood cell distribution width. Anemic KO mice (5 males and 5 females) were distinguished from non-anemic KO (20 males and 11 females) and HF control (10 males, 9 females) on the basis of lower hematocrit (panel B), lower hemoglobin level (panel C), enlarged red cells (panel D) and elevated reticulocyte levels (panel E). **Significantly different (p<0.05) from non-anemic KO and HF control mice; *significantly different from HF controls. None of the parameters were significantly different between male and female KOs. No major differences were seen in other blood cell components. The complete blood count analysis is presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047196#pone.0047196.s005" target="_blank">Table S1</a>. F: Wright's-stained blood smears from anemic KO and HF control male mice. Green arrows indicate enlarged erythrocytes, black arrows codocytes. G: Rectal prolapse in KO female, 9 months post-induction; 9 of the 10 anemic mice exhibited rectal prolapse. H. Blood spots on cage floor blot (∼9″×6″) obtained by 5 min exposure to 3 anemic mice with rectal prolapse; no blood spots were observed on blots from cages housing non-anemic KO or HF mice. I: Red cell turnover study performed on 2 anemic KOs and 4 HF control female mice. The two anemic mice had reticulocyte levels of 13.4 and 20.2%. J: Plasma bilirubin levels in HF (n = 6), non-anemic KO (n = 5) and anemic KO (n = 2) mice; differences between groups were not statistically significant.</p

Skin Histology.

<p>Dorsal skin sections from HF control (A) and KO (B) mice were fixed, paraffin embedded, sectioned at 10 µm and stained with Hematoxylin and Eosin; bar, 100 µm.</p

Genotype and phenotype of the <i>Mcat</i> knockout mouse.

<p>A–D: genotyping by PCR. Mice were screened by PCR analysis of tail DNA, using the primer pair p5:pEx2, which generates 900 and 300 bp products from the floxed and wild-type alleles, respectively (panels A and D), and those carrying the floxed <i>Mcat</i> allele were bred to homozygosity (HF). HF mice were mated with <i>Cre</i> mice and bred to homozygosity for the HF <i>Mcat</i> allele and hemizygosity for the <i>Cre</i> gene, to give the KO genotype. The <i>Cre</i> gene was detected by the unique 100 bp product amplified in the <i>Cre</i>-PCR reaction, in addition to the 300 bp internal positive control (panel B). After treatment with tamoxifen, deletion of exon 2 was detected in various tissues from the KO mice, as judged by two independent PCR reactions (panels C and D). Primer pair p5:pEx2 detects the HF allele (900 bp product) and primer pair p5:p3 generates an 1800 bp product from the HF allele and an 800 bp product from the exon-2-deleted KO allele. Similar results, confirming exon 2 deletion, were observed in skin, bone marrow and lung from KO mice. E–O: phenotyping. E–H, mouse photographs. E and F, Female HF control and KO, respectively, 4 months after tamoxifen treatment. G: Three KO and one HF control female mice 9 months after tamoxifen treatment. H: Male KO mouse 9 months after tamoxifen treatment.</p

Analysis of fecal, plasma and tissue composition.

<p>Values for plasma glucose and lactate and for fecal compositions were pooled from males and females; there was no significant difference in these values between the sexes. All other values were obtained from male mice.</p>§<p>Two of the seven KO mice were anemic with prolapsed rectums and had bilirubin levels of 0.073±0.056. The five non-anemic KO mice had bilirubin levels of 0.12±0.027. Statistically significant differences are noted:</p>*<p>p<0.05,</p>**<p><0.005.</p

Respiratory properties of skeletal muscle mitochondria.

<p>A: Oxygen consumption by mitochondria isolated from mice 8–9 months following treatment with tamoxifen. Values are means ± SEM (n = 4). “State 3” indicates respiration rates during ATP synthesis, “State 4” is in the presence oligomycin to inhibit ATP synthesis, and “uncoupled” is in the presence of carbonyl cyanide p-[trifluoromethoxy]phenylhydrazone to measure maximal respiration rates. The uncoupled rate with α-ketoglutarate is low because the enzyme was depleted of ADP, its allosteric activator; additional data (not shown) demonstrated that addition of ADP increased the uncoupled rate. B: Cytochrome content: values are means ± SEM (n = 5). C: Respiration of mitochondria treated with NaCN. NaCN (15 µM) was added to control mitochondria to mimic the 40% reduction in cytochrome oxidase observed in mitochondria from KO compared to control mice. Respiration, measured in the presence of ketoglutarate, pyruvate+malate, and succinate+rotenone, was compared with that of untreated mitochondria from KO mice. D: Abundance of subunits of the five respiratory chain complexes in mitochondria from KO (n = 3) relative to HF control (n = 4) mice. Western blotting was used to detect subunits of complexes I (NDUFB8), II (30 kDa subunit), III (core protein 2), IV (subunit 1) and V (alpha subunit), employing primary antibodies obtained from Mitosciences. The antigen targets represent labile subunits that are degraded when not assembled into the appropriate complex and thus are representative of the abundance of the fully assembled complexes. Mitochondria were isolated from two groups of mice 8–9 months and 3–4 months following treatment with tamoxifen. Statistical significance in unpaired <i>t</i>-tests is indicated by *p>0.05 or **p<0.005; for p values of marginal significance actual values are shown.</p