Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells

Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies

Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages

Abstract Background A majority of primary ovarian neoplasms arise from cell surface epithelium of the ovaries. Although old age and a positive family history are associated risk factors, the etiology of the epithelial ovarian tumors is not completely understood. Additionally, knowledge of factors involved in the histogenesis of the various subtypes of this tumor as well as those factors that promote progression to advanced stages of ovarian malignancy are largely unknown. Current evidence suggests that mitochondrial alterations involved in cellular signaling pathways may be associated with tumorigenesis. Methods In this study, we determined the presence of polymorphisms and other sequence variants of mitochondrial DNA (mtDNA) in 102 epithelial ovarian tumors including 10 matched normal tissues that paired with some of the tumors. High-resolution restriction endonucleases and PCR-based sequencing were used to assess the mtDNA variants spanning 3.3 kb fragment that comprised the D-Loop and 12S rRNA-tRNAphe, tRNAval, tRNAser, tRNAasp, tRNAlys, ATPase 6, ATPase 8, cytochrome oxidase I and II genes. Results Three hundred and fifty-two (352) mtDNA sequence variants were identified, of which 238 of 352 (68%) have not been previously reported. There were relatively high frequencies of three mutations in the 12S rRNA gene at np 772, 773, and 780 in stage IIIC endometrioid tumors, two of which are novel (773delT and 780delC), and occurred with a frequency of 100% (7/7). Furthermore, two mutations were observed in serous tumors only at np 1657 in stage IV (10/10), and at np 8221delA in benign cystadenomas (3/3) and borderline tumors (4/4). A high frequency, 81% (13/16) of TC insertion at np 310 was found only in early stages of serous subtype (benign cystadenomas, 3/3; borderline tumors, 4/4; stage I tumors, 2/5 and matched normal tissues 4/4). Conclusion Our findings indicate that certain mtDNA mutations can reliably distinguish the different histologic subtypes of epithelial ovarian tumors. In addition, these data raise the possibility that certain mtDNA mutations may be useful biomarkers for predicting tumor aggressiveness and may play a potential role in tumorigenesis.</p

Content of mitochondrial PHBs and mtDNA in 3T3-L1 cells pre- and post-adipogenesis.

<p><b>A, B and C.</b> Over-confluent 3T3-L1 cells (Day 0) were induced for adipocyte differentiation for 7 days. <b>A,</b> PHB1 (green) or <b>B,</b> PHB2 (green) was detected by using immunocytochemistry. CytC (red) was used as the mitochondrial marker. Bar = 10 µm. <b>C.</b> The levels of PHB1 and PHB2 in isolated mitochondria were detected using the immunoblotting analysis. The mitochondrial marker porin, also called the voltage dependent anion channel (VDAC), was used as a loading control. <b>D.</b> 3T3-L1 cells were transfected with indicated siRNAs and cultured for three days. The relative mtDNA content was evaluated by a ratio of the DNA level of mitochondrial Complex I to the DNA level of nuclear 18SrRNA. The relative mtDNA content in 3T3-L1 cells transfected with siControl was set to 1. *p<0.05, **p<0.01 compared to siControl (Day 0). ^#p<0.05, ^##p<0.01 compared to siControl (Day 7).</p

Effect of PHB knockdown on the morphology of mitochondria in 3T3-L1 cells.

<p>3T3-L1 cells were transfected with siRNA targeting PHB1 or PHB2, and cultured for three days. siControl was used as the control. <b>A.</b> The cristae morphology of mitochondria (arrows) in 3T3-L1 cells, transfected with the indicated siRNAs, was assessed by transmission electron microscopy (TEM) at 80 kV acceleration voltage and 50 k magnification. Scale bar, 100 nm. <b>B.</b> The siRNA-transfected 3T3-L1 cells were subject to adipogenic induction for 7 days. The cells at day 0 and day 7 were stained with MitoTracker Red. The mitochondrial morphology was analyzed with a Confocal Microscopy System. Scale bar, 10 µm. High-power magnification insets are included to emphasize the comparison of tubular mitochondria to fragmented ones. The bar graph represents the percentage of cell populations with fragmented mitochondria. **<i>p</i><0.01 compared to siControl at day 0; ^##<i>p</i><0.01 compared to siControl at day 7.</p

Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells.

<p><b>A, B and C.</b> 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. <b>A and B.</b> Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (<b>A</b>) and PHB2 (<b>B</b>) three days post transfection. 18S rRNA was used as an internal control. ** <i>p</i><0.01 compared to siControl. <b>C.</b> Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. β-actin was used as a loading control. <b>D, E and F.</b> 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. <b>D.</b> The levels of C/EBPβ at day1, and the levels of PHB1, PHB2, PPARγ and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. <b>E.</b> The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **<i>p</i><0.01 compared to siControl. <b>F.</b> Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control.</p