Faculty Bibliography

There is an unmet need and urgency to find safe and effective anti-obesity interventions. Our recent study in mice fed on obesogenic diet found that treatment with the alcohol aversive drug disulfiram reduced feeding efficiency and led to a decrease in body weight and an increase in energy expenditure. The intervention with disulfiram improved glucose tolerance and insulin sensitivity, and mitigated metabolic dysfunctions in various organs through poorly defined mechanisms. Here, integrated analysis of transcriptomic and proteomic data from mouse and rat livers unveiled comparable signatures in response to disulfiram, revealing pathways associated with lipid and energy metabolism, redox, and detoxification. In cell culture, disulfiram was found to be a potent activator of autophagy, the malfunctioning of which has negative consequences on metabolic regulation. Thus, repurposing disulfiram may represent a potent strategy to combat obesity.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3'-untranslated region (3'-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3'-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr^+/- mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

Mitochondria and their associated membranes actively participate in biosynthesis, trafficking, and degradation of cellular phospholipids. Two crucial lipid biosynthetic activities of mitochondria include (i) the decarboxylation of phosphatidylserine to phosphatidylethanolamine and (ii) the de novo synthesis of cardiolipin. Here we describe protocols to measure these two activities, applying isotope-labeled or exogenous substrates in combination with thin-layer chromatography or mass spectrometry.

DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.

Background/UNASSIGNED:Cartilage defects account for substantial economic and humanistic burdens and pose a significant clinical problem. The efficacy of clinical approaches to cartilage repair is often inadequate, in part, owing to the restricted proliferative capacity of chondrocytes. Molecules have the capacity to promote the differentiation of multipotent mesenchymal stem cells into chondrocytes and may also gain the ability to repair the damaged cartilage. Objective/UNASSIGNED:This study aimed to investigate the role of Atsttrin (progranulin-derived engineered protein) in cartilage repair as well as the signaling pathway involved. Methods/UNASSIGNED:. Real-time polymerase chain reaction and Western blot analysis were used to monitor the effect of Atsttrin on the transcriptional and protein levels, respectively, of key anabolic and catabolic signaling molecules. Results/UNASSIGNED:In addition, Atsttrin-mediated cartilage repair occurred primarily through tumor necrosis factor receptor 2-initiated Akt signaling and downstream JunB transcription factor. Conclusion/UNASSIGNED:Atsttrin might serve as a promising therapeutic modality for cartilage regeneration.