Aims/hypothesis: The aim of this study was to confirm a link between mitochondrial dysfunction and type 2 diabetes. Materials and methods: Cellular levels of mitochondrial proteins, cellular mitochondrial DNA content, and mitochondrial function and morphology were assessed by MitoTracker staining and electron microscopy, in white adipose tissue of 12-week-old male wild-type, obese (ob/ob), and diabetic (db/db) mice. Results: Levels of mitochondrial proteins were found to be very similar in the livers and muscles of all the mice studied. However, levels were greatly decreased in the adipocytes of db/db mice, but not in those of the wild-type and ob/ob mice. Levels of mitochondrial DNA were also found to be considerably reduced in the adipocytes of db/db mice. MitoTracker staining and under electron microscopy revealed that the number of mitochondria was reduced in adipocytes of db/db mice. Respiration and fatty acid oxidation studies indicated mitochondrial dysfunction in adipocytes of db/db mice. Interestingly, there was an increase in mitochondria and mitochondrial protein production in adipocytes of db/db mice treated with rosiglitazone, an agent that enhances insulin sensitivity. Conclusions/interpretation: Taken together, these data indicate that mitochondrial loss in adipose tissue is correlated with the development of type 2 diabetes.

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H 2 O 2 (200 mM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF b1-or H 2 O 2 -induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.

Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMPresponsive element binding (CREB)-mediated induction of Krupplelike factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages downregulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system.U nder obese conditions, macrophage infiltration and activation in adipose tissue leads to a chronic inflammatory state with increased secretion of proinflammatory cytokines (1). The activation of IkB and Jun N-terminal kinases impairs insulin signaling in metabolic tissues and thereby contributes to insulin resistance (2-4). Classically activated M1 macrophages secrete proinflammatory cytokines, such as TNF-α and IL-12, which promote insulin resistance. Alternatively activated M2 macrophages are thought to protect adipocytes from the development of insulin resistance in response to IL-4 signaling (5, 6). Increases in STAT6 activity stimulate the expression of Krupplelike factor 4 (KLF4), which in turn promotes expression of the M2 program. Obesity causes an M2-to-M1 shift in adipose tissue that leads to insulin resistance (7).The eicosanoid prostaglandin E2 (PGE2) has been found to promote M2 macrophage polarization in part via induction of the cAMP pathway. Indeed, circulating catecholamines also exert potent anti-inflammatory effects on macrophage function via cAMP signaling (8). In this regard, a number of bacteria appear to evade the innate immune system by producing toxins that enhance cAMP production. cAMP stimulates the expression of cellular genes in part via the phosphorylation of CREB at Ser133 and via the dephosphorylation of the cAMP regulated transcriptional coactivators (CRTC) family of coactivators (9). Following its activation, the cyclic AMP-responsive element binding (CREB) pathway appears to block M1 macrophage function in part via the induction of the anti-inflammatory cytokine IL-10 (10, 11). Superimposed on these effects, cAMP also inhibits the expression of proinflammatory cytokines via the induction of class IIa histone deacetylases (HDACs) and subsequent deacetylation of NF-κB.Here we explore the potential roles of the class IIa HDAC and CREB/CRTC pathways in M2 macrophage...

Transforming growth factor b1 (TGF b1) is a wellcharacterized cytokine that suppresses epithelial cell growth. We report here that TGF b1 arrested lung epithelial Mv1Lu cells at G1 phase of the cell cycle with acquisition of senescent phenotypes in the presence of 10% serum, whereas it gradually induced apoptosis with lower concentrations of serum. The senescent arrest was accompanied by prolonged generation of reactive oxygen species (ROS) and persistent disruption of mitochondrial membrane potential (DWm). We demonstrated that the sustained ROS overproduction was derived from mitochondrial respiratory defect via decreased complex IV activity and was involved in the arrest. Moreover, we verified that hepatocyte growth factor released Mv1Lu cells from the arrest by protecting mitochondrial respiration, thereby preventing both the DWm disruption and the ROS generation. Our present results suggest the TGF b1-induced senescent arrest as another plausible mechanism to suppress cellular growth in vivo and provide a new biochemical association between the mitochondrial functional defects and the cytokine-induced senescent arrest, emphasizing the importance of maintenance of mitochondrial function in cellular protection from the arrest.

Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (⌬⌿m), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The ⌬⌿m disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.