Adiponectin and Liver Figure 5. Mitochondrial content of hnRNP K protein is decreased in AKO mice. Mitochondria were isolated from C57 mice and AKO mice (A), or AKO mice subjected to chronic or acute treatment with adiponectin (B). hnRNP K protein content in mitochondrial fractions was measured by Western blotting (A and B). RNA was extracted from the mitochondrial fractions and used for QPCR analysis. The PCR products were visualized in agarose gel for confirming the specific products (C). The QPCR results were compared and presented as fold changes against C57 samples (D). The gene encoding COXII was used as the internal control. *, P,0.05 vs corresponding controls, n = 3. doi:10.1371/journal.pone.0032349.g005 significantly different between C57 and AKO mice (data not shown), the abundance of this protein was much lower in the mitochondria fractions of AKO mice liver compared to those of C57 mice (Figure 5A). Adenovirus-mediated chronic over- expression of adiponectin significantly elevated hnRNP K levels in mitochondria isolated from AKO mice (Figure 5B, top panel). Similarly, injection of recombinant adiponectin, via portal vein, also increased the mitochondrial hnRNP K protein content (Figure 5B, bottom panel). UCP2 mRNA is transported to mitochondria-associated polysomes for protein synthesis. A 123 bp specific fragment of UCP2 mRNA was amplified from mitochondrial RNA samples (Figure 5C). The mitochondrial contents of UCP2 mRNA were quantified by QPCR using the mitochondrial gene cytochrome C oxidase subunit II (COX II) as the internal control (Figure 5D). It was found that UCP2 mRNA content in the mitochondria-associated polysome fraction of AKO mice was only ,29.1% of that in the C57 group. Adiponectin replacement by either adenovirus administration or portal vein injection restored the UCP2 mRNA contents (Figure 5D). Treatment with rotenone, a MRC complex I inhibitor, abolished adiponectin-mediated induction of UCP2 and hnRNP K translocation Western blotting was performed on mitochondria protein lysates of AKO mice treated with recombinant adiponectin in the presence or absence of of MRC complexes inhibitors, including rotenone (Rot, complex I inhibitor), antimycin A (AntA, complex III inhibitor) and sodium azide (NaN3, complex IV inhibitor). The stimulatory effect of adiponectin on mitochondrial UCP2 protein expression was significantly attenuated by rotenone, but not by antimycin A or NaN3 (Figure 6A). Adiponectin-induced elevation of UCP2 mRNA level was also attenuated by rotenone treatment (Figure 6B). This inhibitor mainly blocked the expression of UCP2 in NPCs (Figure 6C). Rotenone treatment also prevented adiponectin-induced mitochondria translocation of hnRNP K in NPCs (Figure 6D). MRC is a major source of reactive oxygen species (ROS), which stimulate UCP2 expression [33,34,35]. By applying a fluorescent probe for mitochondrial superoxide, MitoSOX, it was found that PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32349