OBJECTIVE-Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation-related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance. RESEARCH DESIGN AND METHODS-We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of dietinduced obese (DIO) mice were also measured.RESULTS-Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by ϳ2-to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P Ͻ 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency.CONCLUSIONS-These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes. Diabetes

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

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BackgroundMyocardial infarction (MI) is an acute and fatal condition that threatens human health. Dl-3-n-butylphthalide (NBP) has been used for the treatment of acute ischemic stroke. Mitochondria may play a protective role in MI injury. However, there are few reports on the cardioprotective effect of NBP or the potential mitochondrial mechanism for the NBP-induced protection against cardiac ischemia injury. We investigated the therapeutic effects of NBP in an in vivo MI model and an in vitro oxidative stress model, as well as the potential mitochondrial mechanism.MethodsThis study comprised two different experiments. The aim of experiment 1 was to determine the protective effects of NBP on MI and the underlying mechanisms in vivo. In part 1, myocardial infarct size was measured by staining with 2,3,5-triphenyltetrazoliumchloride (TTC). Myocardial enzymes and mitochondrial enzymes were assayed. The aim of experiment 2 was to investigate the role of NBP in H2O2-induced myocardial ischemic injury in H9c2 cells and to determine the potential mechanism. In part 2, H9c2 cell viability was evaluated. ROS levels, mitochondrial morphology, and mitochondrial membrane potential of H9c2 cells were measured. ATP levels were evaluated using an assay kit; mitochondrial DNA (mtDNA), the expressions of NRF-1 and TFAM, and mitochondrial biogenesis factors were determined.ResultsNBP treatment significantly reduced the infarct ratio, as observed by TTC staining, decreased serum myocardial enzymes in MI, and restored heart mitochondrial enzymes (isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and a-ketoglutarate dehydrogenase (a-KGDH) activities after MI. Moreover, in in vitro studies, NBP significantly increased the viability of H9c2 cells in a dose-dependent manner, reduced cell apoptosis, protected mitochondrial functions, elevated the cellular ATP levels, and promoted H2O2-induced mitochondrial biogenesis in H9c2 cardiomyoblasts.ConclusionCollectively, the results from both the in vivo and in vitro experiments suggested that NBP exerted a cardioprotective effect on cardiac ischemic injury via the regulation of mitochondrial function and biogenesis.