Rapid exercise-induced changes in PGC-1alpha mRNA and protein in human skeletal muscle

The mRNA of the nuclear coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) increases during prolonged exercise and is influenced by carbohydrate availability. It is unknown if the increases in mRNA reflect the PGC-1alpha protein or if glycogen stores are an important regulator. Seven male subjects [23 +/- 1.3 yr old, maximum oxygen uptake (Vo(2 max)) 48.4 +/- 0.8 ml.kg(-1).min(-1)] exercised to exhaustion ( approximately 2 h) at 65% Vo(2 max) followed by ingestion of either a high-carbohydrate (HC) or low-carbohydrate (LC) diet (7 or 2.9 g.kg(-1).day(-1), respectively) for 52 h of recovery. Glycogen remained depressed in LC (P < 0.05) while returning to resting levels by 24 h in HC. PGC-1alpha mRNA increased both at exhaustion (3-fold) and 2 h later (6.2-fold) (P < 0.05) but returned to rest levels by 24 h. PGC-1alpha protein increased (P < 0.05) 23% at exhaustion and remained elevated for at least 24 h (P < 0.05). While there was no direct treatment effect (HC vs. LC) for PGC-1alpha mRNA or protein, there was a linear relationship between the changes in glycogen and those in PGC-1alpha protein during exercise and recovery (r = -0.68, P < 0.05). In contrast, PGC-1beta did not increase with exercise but rather decreased (P < 0.05) below rest level at 24 and 52 h, and the decrease was greater (P < 0.05) in LC. PGC-1alpha protein content increased in prolonged exercise and remained upregulated for 24 h, but this could not have been predicted by the changes in mRNA. The beta-isoform declined rather than increasing, and this was greater when glycogen was not resynthesized to rest levels.     

Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1alpha or PGC-1beta coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1alpha and PGC-1beta were deficient. These data show that PGC-1alpha is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Activation of the PPAR/PGC-1alpha pathway prevents a bioenergetic deficit and effectively improves a mitochondrial myopathy phenotype

Neuromuscular disorders with defects in the mitochondrial ATP-generating system affect a large number of children and adults worldwide, but remain without treatment. We used a mouse model of mitochondrial myopathy, caused by a cytochrome c oxidase deficiency, to evaluate the effect of induced mitochondrial biogenesis on the course of the disease. Mitochondrial biogenesis was induced either by transgenic expression of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator alpha (PGC-1alpha) in skeletal muscle or by administration of bezafibrate, a PPAR panagonist. Both strategies successfully stimulated residual respiratory capacity in muscle tissue. Mitochondrial proliferation resulted in an enhanced OXPHOS capacity per muscle mass. As a consequence, ATP levels were conserved resulting in a delayed onset of the myopathy and a markedly prolonged life span. Thus, induction of mitochondrial biogenesis through pharmacological or metabolic modulation of the PPAR/PGC-1alpha pathway promises to be an effective therapeutic approach for mitochondrial disorders.     

Endurance training increases skeletal muscle LKB1 and PGC-1alpha protein abundance: effects of time and intensity

Recent research suggests that LKB1 is the major AMP-activated protein kinase kinase (AMPKK). Peroxisome-proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a master coordinator of mitochondrial biogenesis. Previously we reported that skeletal muscle LKB1 protein increases with endurance training. The purpose of this study was to determine whether training-induced increases in skeletal muscle LKB1 and PGC-1alpha protein exhibit a time course and intensity-dependent response similar to that of citrate synthase. Male Sprague-Dawley rats completed endurance- and interval-training protocols. For endurance training, rats trained for 4, 11, 25, or 53 days. Interval-training rats trained identically to endurance-trained rats, except that after 25 days interval training was combined with endurance training. Time course data were collected from endurance-trained red quadriceps (RQ) after each time point. Interval training data were collected from soleus, RQ, and white quadriceps (WQ) muscle after 53 days only. Mouse protein 25 (MO25) and PGC-1alpha protein increased significantly after 4 days. Increased citrate synthase activity, increased LKB1 protein, and decreased AMPKK activity were found after 11 days. Maximal increases occurred after 4 days for hexokinase II, 25 days for MO25, and 53 days for citrate synthase, LKB1, and PGC-1alpha. In WQ, but not RQ or soleus, interval training had an additive effect to endurance training and induced significant increases in all proteins measured. These results demonstrate that LKB1 and PGC-1alpha protein abundances increase with endurance and interval training similarly to citrate synthase. The increase in LKB1 and PGC-1alpha with endurance and interval training may function to maintain the training-induced increases in mitochondrial mass.     

Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a complex process involving more than 20 components. So far, functional investigations have mainly been performed in Saccharomyces cerevisiae. Here, we have analyzed the role of the human cysteine desulfurase Nfs1 (huNfs1), which serves as a sulfur donor in biogenesis. The protein is located predominantly in mitochondria, but small amounts are present in the cytosol/nucleus. huNfs1 was depleted efficiently in HeLa cells by a small interfering RNA (siRNA) approach, resulting in a drastic growth retardation and striking morphological changes of mitochondria. The activities of both mitochondrial and cytosolic Fe/S proteins were strongly impaired, demonstrating that huNfs1 performs an essential function in Fe/S protein biogenesis in human cells. Expression of murine Nfs1 (muNfs1) in huNfs1-depleted cells restored both growth and Fe/S protein activities to wild-type levels, indicating the specificity of the siRNA depletion approach. No complementation of the growth retardation was observed, when muNfs1 was synthesized without its mitochondrial presequence. This extramitochondrial muNfs1 did not support maintenance of Fe/S protein activities, neither in the cytosol nor in mitochondria. In conclusion, our study shows that the essential huNfs1 is required inside mitochondria for efficient maturation of cellular Fe/S proteins. The results have implications for the regulation of iron homeostasis by cytosolic iron regulatory protein 1.     

Insulin therapy modulates mitochondrial dynamics and biogenesis,autophagy and tau protein phosphorylation in the brain of type 1 diabetic rats

The main purpose of this study was to examine whether streptozotocin (STZ)-induced type 1 diabetes (T1D) and insulin (INS) treatment affect mitochondrial function, fission/fusion and biogenesis, autophagy and tau protein phosphorylation in cerebral cortex from diabetic rats treated or not with INS. No significant alterations were observed in mitochondrial function as well as pyruvate levels, despite the significant increase in glucose levels observed in INS-treated diabetic rats. A significant increase in DRP1 protein phosphorylated at Ser616 residue was observed in the brain cortex of STZ rats. Also an increase in NRF2 protein levels and in the number of copies of mtDNA were observed in STZ diabetic rats, these alterations being normalized by INS. A slight decrease in LC3-II levels was observed in INS-treated rats when compared to STZ diabetic animals. An increase in tau protein phosphorylation at Ser396 residue was observed in STZ diabetic rats while INS treatment partially reversed that effect. Accordingly, a modest reduction in the activation of GSK3β and a significant increase in the activity of phosphatase 2A were found in INS-treated rats when compared to STZ diabetic animals. No significant alterations were observed in caspases 9 and 3 activity and synaptophysin and PSD95 levels. Altogether our results show that mitochondrial alterations induced by T1D seem to involve compensation mechanisms since no significant changes in mitochondrial function and synaptic integrity were observed in diabetic animals. In addition, INS treatment is able to normalize the alterations induced by T1D supporting the importance of INS signaling in the brain.     

Purification and characterization of yeast Sco1p, a mitochondrial copper protein

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains approximately 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.     

Biochemical lesions in copper-deficient rats caused by secondary iron deficiency. Derangement of protein synthesis and impairment of energy metabolism

Severe copper deficiency was induced in rats by rearing nursing dams and their offsprings on a semisynthetic diet comprising all the requisite nutrients and trace metals except copper. The copper-deprived rats exhibited growth retardation, severe anaemia, loss of caeruloplasmin, decrease of cytochrome oxidase, accumulation of salt-soluble collagen and a drastic decrease in iron in plasma and liver. Apart from these characteristic signs of deficiency, a marked inhibition of protein synthesis was found to occur both in vivo and in cell-free liver preparations. The curtailed ability to carry out endogenously coded amino acid incorporation into protein contrasted with the unimpaired poly(U)-acid-directed phenylalanine polymerization. This inhibition pattern, as well as the attendant disaggregation of the liver polyribosomes, suggested that the primary biosynthetic lesion was located at the stage of peptide-chain initiation. Concurrently with this alteration there was a pronounced depletion of the hepatic ATP content, associated with a parallel depression of mitochondrial respiration and an enhancement of ATPase activity. Supplementation of the copper-deficient diet with a 2–4-fold excess of iron (relative to the standard diet) prevented growth retardation and anaemia and restored normal energy metabolism, as well as unimpaired protein-synthesizing capacity. The conclusion that these disturbances were primarily determined by the secondary iron deficiency was also borne out by the finding that similar alterations occurred in rats maintained on a copper-sufficient but iron-deficient diet. On the other hand, the iron-fortified diet failed to reverse the other signs of copper deficiency, namely the loss of caeruloplasmin, the diminished rate of cytochrome oxidase and the increase of soluble collagen. The interrelations between the various biochemical lesions induced by deprivation of copper or iron are discussed and the possible role of ATP depletion in determining the derangement of protein synthesis is considered.