Alteration of mitochondrial oxidative capacity during porcine preadipocyte differentiation and in response to leptin

Mitochondrial apparatus is a fundamental aspect in cell, serving for amino acid biosynthesis, fatty acid oxidation (FAO), and ATP production. In this article, we investigated the change of mitochondrial oxidative capacity during porcine adipocyte differentiation and in response to leptin. Rhodamine 123 staining analysis showed about 2-fold increase of mitochondrial membrane electric potential in differentiated adipocyte in comparison with preadipocyte. The mRNA expression of Cytochromes c (Cyt c), carnitine palmitoyltransferase 1 (CPT1), and malate dehydrogenases (MDH) increased markedly (P < 0.05), but that of UCP2 decreased (P < 0.05). Moreover PGC-1α and UCP3 was very low and showed no changes during the adipocyte differentiation. The protein expression of Cyt c and the enzyme activity of Cytochrome c oxidase (COX) increased with preadipocyte differentiation, but cellular ATP level decreased. Furthermore, at the level of 10 and 100 ng/ml leptin not only selectively increased the gene expression of PGC-1α, CPT1, Cyt c, UCP2, and UCP3 (P < 0.05), but also enhanced COX enzyme activity which related to mitochondrial FAO. There is no change of Mitochondrial membrane electric potential and ATP level in cell treated by leptin. These results suggested Mitochondrial is not only critical in FAO, but also play an important role in adipogenesis.     

SOCS2 inhibited mitochondria biogenesis via inhibiting p38 MAPK/ATF2 pathway in C2C12 cells

In order to investigate the mechanism of suppressor of cytokine signaling 2 (SOCS2) on mitochondrial biogenesis, RNA interference and over-expression plasmid vectors of SOCS2 were used to transfect murine skeletal muscle C2C12 cells. Results demonstrated that over-expression of SOCS2 inhibited the differentiation of C2C12 cells, and reduced the expression of MyHC, MyoD and MyoG while elevated the protein expression of MSTN. Meanwhile the expression of PGC-1α, MDH, CPT-1 were significantly elevated in the RNA interference of SOCS2 group which were decreased in SOCS2 overexpression group. However, there was no change on the expression of UCP1 in both two groups. JC-1 dyeing showed overexpression of SOCS2 decreased the mitochondrial membrane potential and results of immunofluorescence, real-time PCR and western blotting indicated the increase expression of Cyt c, while interference SOCS2 had the opposite effects in C2C12 cells. Moreover, interference of SOCS2 elevated the p38 phosphorylation level then further increased the phosphorylation of ATF2, whereas overexpression of SOCS2 alleviated this phenomenon. Taken together, our observations indicated that SOCS2 could suppress myotube formation, act as an anti-regulator of mitochondria biogenesis via inhibiting p38 MAPK signal pathway.     

Pyrroloquinoline Quinone Stimulates Mitochondrial Biogenesis through cAMP Response Element-binding Protein Phosphorylation and Increased PGC-1�� Expression

Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1–6 cells to 10–30 μm PQQ for 24–48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1α, and increased PGC-1α mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1α or CREB expression. Consistent with activation of the PGC-1α pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction.     

Enhanced lipid-but not carbohydrate-supported mitochondrial respiration in skeletal muscle of PGC-1α overexpressing mice

Skeletal muscle mitochondrial dysfunction has been linked to several disease states as well as the process of aging. A possible factor involved is the peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α), a major player in the regulation of skeletal muscle mitochondrial metabolism. However, it is currently unknown whether PGC-1α, besides stimulating mitochondrial proliferation, also affects the functional capacity per mitochondrion. Therefore, we here tested whether PGC-1α overexpression, besides increasing mitochondrial content, also leads to intrinsic mitochondrial adaptations. Skeletal muscle mitochondria from 10 male, muscle-specific PGC-1α overexpressing mice (PGC-1αTg) and 8 wild-type (WT) mice were isolated. Equal mitochondrial quantities were then analyzed for their oxidative capacity by high-resolution respirometry, fuelled by a carbohydrate-derived (pyruvate) and a lipid (palmitoyl-CoA plus carnitine) substrate. Additionally, mitochondria were tested for reactive oxygen species (superoxide) production and fatty acid (FA)-induced uncoupling. PGC-1αTg mitochondria were characterized by an improved intrinsic mitochondrial fat oxidative capacity as evidenced by pronounced increase in ADP-stimulated respiration (P < 0.001) and maximal uncoupled respiration (P < 0.001) upon palmitoyl-CoA plus carnitine. Interestingly, intrinsic mitochondrial capacity on a carbohydrate-derived substrate tended to be reduced. Furthermore, the sensitivity to FA-induced uncoupling was diminished in PGC-1αTg mitochondria (P = 0.02) and this was accompanied by a blunted reduction in mitochondrial ROS production upon FAs in PGC-1αTg versus WT mitochondria (P = 0.04). Uncoupling protein 3 (UCP3) levels were markedly reduced in PGC-1αTg mitochondria (P < 0.001). Taken together, in addition to stimulating mitochondrial proliferation in skeletal muscle, we show here that overexpression of PGC-1α leads to intrinsic mitochondrial adaptations that seem restricted to fat metabolism.     

Elucidating cytochrome C release from mitochondria: insights from an in silico three-dimensional model

Mitochondrial regulation of apoptosis depends on the programmed release of proapoptotic proteins such as cytochrome c (Cyt c) through the outer mitochondrial membrane (OMM). Although a few key processes involved in this release have been identified, including the liberation of inner membrane-bound Cyt c and formation of diffusible pores on the OMM, other details like the transport of Cyt c within complex mitochondrial compartments, e.g., the cristae and crista junctions, are not yet fully understood (to our knowledge). In particular, a remodeling of the inner mitochondrial membrane accompanying apoptosis seen in a few studies, in which crista junctions widen, has been hypothesized to be a necessary step in the Cyt c release. Using a three-dimensional spatial modeling of mitochondrial crista and the crista junction, model simulations and analysis illustrated how the interplay among solubilization of Cyt c, fast diffusion of Cyt c, and OMM permeabilization gives rise to the observed experimental release profile. Importantly, the widening of the crista junction was found to have a negligible effect on the transport of free Cyt c from cristae. Finally, model simulations showed that increasing the fraction of free/loosely-bound Cyt c can sensitize the cell to apoptotic stimuli in a threshold manner, which may explain increased sensitivity to cell death associated with aging.     

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.     

Inactivation of glycogen synthase kinase 3β (GSK-3β) enhances mitochondrial biogenesis during myogenesis

Background

Mitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3β, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity.

PGC-1alpha over-expression promotes recovery from mitochondrial dysfunction and cell injury

Cell death from mitochondrial dysfunction and compromised bioenergetics is common after ischemia-reperfusion injury and toxicant exposure. Thus, promoting mitochondrial biogenesis is therapeutically attractive for sustaining oxidative phosphorylation and maintaining ATP-dependent cellular functions. Here, we evaluated increased mitochondrial biogenesis prior to or after oxidant exposure in primary cultures of renal proximal tubular cells (RPTC). Over-expression of the mitochondrial biogenesis regulator PPAR-gamma cofactor-1 alpha (PGC-1alpha) in control RTPC increased basal and uncoupled cellular respiration, ATP, and mitochondria. Increasing mitochondrial number/function prior to oxidant exposure did not preserve mitochondrial function, but potentiated dysfunction and cell death. However, increased mitochondrial biogenesis after oxidant injury accelerated recovery of mitochondrial function. In oxidant treated RPTC, mitochondrial protein expression was reduced by 50%. Also, ATP and cellular respiration decreased 48 h after oxidant exposure, whereas mitochondrial function in injured RPTC over-expressing PGC-1alpha returned to control values. Thus, up-regulation of mitochondrial biogenesis after oxidant exposure accelerates recovery of mitochondrial and cellular functions.     

瘦素(Leptin )对猪前体脂肪细胞分化及PGC-1α和UCPs mRNA表达的影响

 以体外培养的猪前体脂肪细胞为研究对象,分析瘦素(leptin )对前体脂肪细胞分化及能量代谢相关基因PGC- 1α和UCPs mRNA表达的影响.油红O染色提取结果显示,10～90nmol/L的leptin对猪前体脂肪细胞甘油三酯合成均无显著影响(P>0.05).RT-PCR检测结果表明,30　nmol/L和100nmol/L leptin可显著促进LPL mRNA表达,处理24 h较12 h 作用效果明显(P<0.05);两个浓度的leptin对脂肪细胞分化转录因子C/EBPα和PPARγ2在mRNA水平上均没有明显的影响(P>0.05).对能量代谢相关基因的RT-PCR检测结果表明,30 nmol/L 和100nmol/L leptin 可显著促进PGC-1α和UCP3转录 (P<0.05),30nmol/L leptin可明显提高前脂肪细胞中UCP2 mRNA水平(P<0.05),且作用24h促进效果明显优于12h处理(P<0.05).研究结果提示,leptin不影响猪前体脂肪细胞分化,但可能通过上调PGC-1α和UCPs转录,促进能量消耗.     

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0–24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6 h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24 h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.     

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX_oligo) and by a combination of monomeric BAX (BAX_mono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t^cBID). We also examined whether the effects induced by BAX_oligo or by BAX_mono activated with t^cBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t^cBID plus BAX_mono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAX_oligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAX_oligo could be at least partially suppressed by mitochondrial depolarization. The effects of t^cBID plus BAX_mono were insensitive to depolarization. BAX_oligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K⁺ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAX_oligo, but failed to influence the effects produced by t^cBID plus BAX_mono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAX_oligo and by BAX activated with t^cBID.