Enhanced Differentiation of Three-Gene-Reprogrammed Induced Pluripotent Stem Cells into Adipocytes via Adenoviral-Mediated PGC-1α Overexpression

Induced pluripotent stem cells formed by the introduction of only three factors, Oct4/Sox2/Klf4 (3-gene iPSCs), may provide a safer option for stem cell-based therapy than iPSCs conventionally introduced with four-gene iPSCs. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) plays an important role during brown fat development. However, the potential roles of PGC-1α in regulating mitochondrial biogenesis and the differentiation of iPSCs are still unclear. Here, we investigated the effects of adenovirus-mediated PGC-1α overexpression in 3-gene iPSCs. PGC-1α overexpression resulted in increased mitochondrial mass, reactive oxygen species production, and oxygen consumption. Microarray-based bioinformatics showed that the gene expression pattern of PGC-1α-overexpressing 3-gene iPSCs resembled the expression pattern observed in adipocytes. Furthermore, PGC-1α overexpression enhanced adipogenic differentiation and the expression of several brown fat markers, including uncoupling protein-1, cytochrome C, and nuclear respiratory factor-1, whereas it inhibited the expression of the white fat marker uncoupling protein-2. Furthermore, PGC-1α overexpression significantly suppressed osteogenic differentiation. These data demonstrate that PGC-1α directs the differentiation of 3-gene iPSCs into adipocyte-like cells with features of brown fat cells. This may provide a therapeutic strategy for the treatment of mitochondrial disorders and obesity

Effects of olive oil and its minor phenolic constituents on obesity-induced cardiac metabolic changes

<p>Abstract</p> <p>Background</p> <p>Olive oil and its minor constituents have been recommended as important dietary therapeutic interventions in preventive medicine. However, a question remains to be addressed: what are the effects of olive oil and its phenolic compounds on obesity-induced cardiac metabolic changes?</p> <p>Methods</p> <p>Male Wistar rats were divided into two groups (<it>n </it>= 24/group): (C) receiving standard-chow; (Ob) receiving hypercaloric-chow. After 21 days C and Ob groups were divided into four subgroups (<it>n </it>= 6/group):(C) standard-chow and saline; (C-Olive)standard-chow and olive-oil (3.0 g/kg.day); (C-Oleuropein)standard-chow and oleuropein (0.023 mg/kg/day); (C-Cafeic) standard-chow and cafeic-acid (2.66 mg/kg/day); (Ob)receiving hypercaloric-chow and saline;(Ob-Olive) hypercaloric-chow and olive-oil;(Ob-Oleuropein) hypercaloric-chow and oleuropein;(Ob-Cafeic) hypercaloric-chow and cafeic-acid. Treatments were given twice a week during 21 days.</p> <p>Results</p> <p>After 42 days, obesity was evidenced in Ob rats from enhanced body-weight, surface-area, and body-mass-index. Energy-expenditure, oxygen consumption(VO₂) and fat-oxidation were lower in Ob-group than in C. Despite no morphometric changes, Ob-Olive, Ob-Oleuropein and Ob-Cafeic groups had higher VO₂, fat-oxidation, myocardial beta-hydroxyacyl coenzyme-A dehydrogenase and lower respiratory-quotient than Ob. Citrate-synthase was highest in Ob-Olive group. Myocardial lipid-hydroperoxide(LH) and antioxidant enzymes were unaffected by olive-oil and its compounds in obesity condition, whereas LH was lower and total-antioxidant-substances were higher in C-Olive and C-Oleuropein than in C.</p> <p>Conclusions</p> <p>The present study demonstrated for the first time that olive-oil, oleuropein and cafeic-acid enhanced fat-oxidation and optimized cardiac energy metabolism in obesity conditions. Olive oil and its phenolic compounds improved myocardial oxidative stress in standard-fed conditions.</p

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

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a ρ0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor