PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.

Pharmacometabolomic Assessment of Metformin in Non-diabetic, African Americans.

Millions of individuals are diagnosed with type 2 diabetes mellitus (T2D), which increases the risk for a plethora of adverse outcomes including cardiovascular events and kidney disease. Metformin is the most widely prescribed medication for the treatment of T2D; however, its mechanism is not fully understood and individuals vary in their response to this therapy. Here, we use a non-targeted, pharmacometabolomics approach to measure 384 metabolites in 33 non-diabetic, African American subjects dosed with metformin. Three plasma samples were obtained from each subject, one before and two after metformin administration. Validation studies were performed in wildtype mice given metformin. Fifty-four metabolites (including 21 unknowns) were significantly altered upon metformin administration, and 12 metabolites (including six unknowns) were significantly associated with metformin-induced change in glucose (q < 0.2). Of note, indole-3-acetate, a metabolite produced by gut microbes, and 4-hydroxyproline were modulated following metformin exposure in both humans and mice. 2-Hydroxybutanoic acid, a metabolite previously associated with insulin resistance and an early biomarker of T2D, was positively correlated with fasting glucose levels as well as glucose levels following oral glucose tolerance tests after metformin administration. Pathway analysis revealed that metformin administration was associated with changes in a number of metabolites in the urea cycle and in purine metabolic pathways (q < 0.01). Further research is needed to validate the biomarkers of metformin exposure and response identified in this study, and to understand the role of metformin in ammonia detoxification, protein degradation and purine metabolic pathways.

AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

Profiling the reproductive toxicity of chemicals from multigeneration studies in the toxicity reference database.

Multigeneration reproduction studies are used to characterize parental and offspring systemic toxicity, as well as reproductive toxicity of pesticides, industrial chemicals and pharmaceuticals. Results from 329 multigeneration studies on 316 chemicals have been digitized into standardized and structured toxicity data within the Toxicity Reference Database (ToxRefDB). An initial assessment of data quality and consistency was performed prior to profiling these environmental chemicals based on reproductive toxicity and associated toxicity endpoints. The pattern of toxicity across 75 effects for all 316 chemicals provided sets of chemicals with similar in vivo toxicity for future predictive modeling. Comparative analysis across the 329 studies identified chemicals with sensitive reproductive effects, based on comparisons to chronic and subchronic toxicity studies, as did the cross-generational comparisons within the multigeneration study. The general pattern of toxicity across all chemicals and the more focused comparative analyses identified 19 parental, offspring and reproductive effects with a high enough incidence to serve as targets for predictive modeling that will eventually serve as a chemical prioritization tool spanning reproductive toxicities. These toxicity endpoints included specific reproductive performance indices, male and female reproductive organ pathologies, offspring viability, growth and maturation, and parental systemic toxicities. Capturing this reproductive toxicity data in ToxRefDB supports ongoing retrospective analyses, test guideline revisions, and computational toxicology research.