Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans

© 2015 Elsevier B.V. Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans

Impaired Mitochondrial Fat Oxidation Induces FGF21 in Muscle

SummaryFatty acids are the primary fuel source for skeletal muscle during most of our daily activities, and impaired fatty acid oxidation (FAO) is associated with insulin resistance. We have developed a mouse model of impaired FAO by deleting carnitine palmitoyltransferase-1b specifically in skeletal muscle (Cpt1bm−/−). Cpt1bm−/− mice have increased glucose utilization and are resistant to diet-induced obesity. Here, we show that inhibition of mitochondrial FAO induces FGF21 expression specifically in skeletal muscle. The induction of FGF21 in Cpt1b-deficient muscle is dependent on AMPK and Akt1 signaling but independent of the stress signaling pathways. FGF21 appears to act in a paracrine manner to increase glucose uptake under low insulin conditions, but it does not contribute to the resistance to diet-induced obesity

Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism

© 2015, National Academy of Sciences. All rights reserved. The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity

Examination of carnitine palmitoyltransferase 1 abundance in white adipose tissue: implications in obesity research

Carnitine palmitoyltransferase 1 (CPT1) is essential for the transport of long-chain fatty acids into the mitochondria for oxidation. Recently, it was reported that decreased CPT1b mRNA in adipose tissue was a contributing factor for obesity in rats. We therefore closely examined the expression level of in adipose tissue from mice, rats, and humans. is the predominate isoform in adipose tissue from all three species. Rat white adipose tissue has a moderate amount of mRNA, but it is very minor compared with expression in muscle. Total CPT1 activity in adipose tissue is also minor relative to other tissues. Both and mRNA were increased in gonadal fat but not inguinal fat by diet-induced obesity in mice. We also measured and expression in subcutaneous adipose tissue from human subjects with a wide range of body mass indexes (BMIs). Interestingly, expression positively correlated with BMI ( = 0.46), but there was no correlation with ( = 0.04). Our findings indicate that white adipose tissue fatty acid oxidation capacity is minor compared with that of metabolically active tissues. Furthermore, given the already low abundance of in white adipose tissue, it is unlikely that decreases in its expression can quantitatively decrease whole body energy expenditure enough to contribute to an obese phenotype

Female Mice Are Protected from Metabolic Decline Associated with Lack of Skeletal Muscle HuR

Male mice lacking HuR in skeletal muscle (HuRm−/−) have been shown to have decreased gastrocnemius lipid oxidation and increased adiposity and insulin resistance. The same consequences have not been documented in female HuRm−/− mice. Here we examine this sexually dimorphic phenotype. HuRm−/− mice have an increased fat mass to lean mass ratio (FM/LM) relative to controls where food intake is similar. Increased body weight for male mice correlates with increased blood glucose during glucose tolerance tests (GTT), suggesting increased fat mass in male HuRm−/− mice as a driver of decreased glucose clearance. However, HuRm−/− female mice show decreased blood glucose levels during GTT relative to controls. HuRm−/− mice display decreased palmitate oxidation in skeletal muscle relative to controls. This difference is more robust for male HuRm−/− mice and more exaggerated for both sexes at high dietary fat. A high-fat diet stimulates expression of Pgc1α in HuRm−/− male skeletal muscle, but not in females. However, the lipid oxidation Pparα pathway remains decreased in HuRm−/− male mice relative to controls regardless of diet. This pathway is only decreased in female HuRm−/− mice fed high fat diet. A decreased capacity for lipid oxidation in skeletal muscle in the absence of HuR may thus be linked to decreased glucose clearance in male but not female mice

A low fat diet ameliorates pathology but retains beneficial effects associated with CPT1b knockout in skeletal muscle

<div><p>Inhibiting fatty acid oxidation is one approach to lowering glucose levels in diabetes. Skeletal muscle specific Carnitine Palmitoyltransferase 1b knockout mice (Cpt1b^m-/-) comprise a model of impaired fat oxidation; and have decreased fat mass and enhanced glucose disposal and muscle oxidative capacity compared to controls. However, unfavorable effects occur relative to controls when Cpt1b^m-/- mice are fed a 25% fat diet, including decreased activity and fat free mass and increased intramuscular lipid and serum myoglobin. In this study we explore if a low fat, high carbohydrate diet can ablate the unfavorable effects while maintaining the favorable phenotype in Cpt1b^m-/- mice. Mice were fed either 10% fat (low fat) or 25% fat (chow) diet. Body composition was measured biweekly and indirect calorimetry was performed. Low fat diet abolishes the decreased activity, fat, and fat free mass seen in Cpt1b^m-/- mice fed chow diet. Low fat diet also reduces serum myoglobin levels in Cpt1b^m-/- mice and diminishes differences in IGF-1 seen between Cpt1b^m-/- mice and control mice fed chow diet. Glucose tolerance tests reveal that glucose clearance is improved in Cpt1b^m-/- mice relative to controls regardless of diet, and serum analysis shows increased levels of muscle derived FGF21. Electron microscopic analyses and measurements of mRNA transcripts show increased intramuscular lipids, FGF21, mitochondrial and oxidative capacity markers regardless of diet. The favorable metabolic phenotype of Cpt1b^m-/- mice therefore remains consistent regardless of diet; and a combination of a low fat diet and pharmacological inhibition of CPT1b may offer remedies to reduce blood glucose.</p></div

Diet effects on Cpt1b^m-/- adiposity.

<p>Control mice (red dashed lines, N = 10 animals per time point on chow and N = 8 animals per time point on low fat diet) and Cpt1b^m-/- mice (black lines, N = 9 animals per time point on chow and N = 12 animals per time point on low fat diet) were monitored for 24 weeks to assess gain of Weight, Fat, and FFM. Results are compared between groups fed low fat diet (A-C) and chow diet (D-F). Asterisks indicate significance with P ≤ 0.05.</p

Diet has little effect on glucose clearance and muscle physiology of Cpt1b^m-/- mice.

<p>(A-B) GTT are shown for control (white circles) and Cpt1b^m-/- (black squares) mice that were fed (A) low fat diet (N = 8 animals per genotype) or (B) chow diet (N = 15 animals per genotype). (C) Levels in control (white) and Cpt1b^m-/- mice (black) of FGF21 mRNA in red quadriceps muscle (left panel), and FGF21 protein in serum; (right panel) (N = 8 animals per genotype for low fat diet and N = 10 animals per genotype for chow diet). (D) EM showing increased IMCL in soleus of Cpt1b^m-/- (right) mice relative to controls (left). (E-F) (N = 4 animals per genotype for low fat diet and N = 6 animals per genotype for chow diet) Relative levels of markers of mitochondrial biogenesis and lipid usage in control (white) and Cpt1b^m-/- (black) mouse red quadriceps muscle from mice fed (E) low fat diet or (F) chow diet. Asterisks indicate significance with P ≤ 0.05.</p

Low fat diet restores levels of IGF-1, myoglobin, activity, and serum lipids in Cpt1b^m-/- mice.

<p>(A) Serum levels of IGF-1 are plotted for control (white circles) and Cpt1b^m-/- (black squares) mice for each of the diets tested at 4–6 months (N = 10 animals per genotype for low fat diet and N = 14 animals per genotype for chow diet). (B) Serum myoglobin levels in animals fed either low fat or chow diets (N = 9 animals per genotype for low fat diet; and N = 10 control and 8 Cpt1b^m-/- for chow diet). (C-D) Activity variation is plotted for control (white circles) and Cpt1b^m-/- (black squares) mice fed chow diet (C) (N = 9 animals per genotype) or low fat diet (D) (N = 12 animals per genotype). Grey sections indicate periods of darkness, while white sections indicate periods of light. (E-F) Serum levels of NEFA (E) and serum levels of 3-hydroxybutyrate (F) in control (white) and Cpt1b^m-/- (black) mice fed chow or low fat diet (N = 9 for control animals and N = 10 for Cpt1b^m-/- animals). Asterisks indicate significance with P ≤ 0.05.</p

Changes in Cpt1b^m-/- relative to controls fed chow and low fat diets.

<p>Changes in Cpt1b^m-/- relative to controls fed chow and low fat diets.</p