Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells.

Transgene expression in eHAP cells, a haploid cell line commonly used to generate gene knockouts, is difficult due to its low transfection efficiency and poor expression of integrated transgenes. To enable simple and reliable transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells.

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel.

Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells.

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals.

Genome-wide analysis in Drosophila reveals diet-by-gene interactions and uncovers diet-responsive genes.

Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individual's genotype. Here, we sought to understand how such gene-diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease.

A Genome-Wide Screen of Deletion Mutants in the Filamentous Saccharomyces cerevisiae Background Identifies Ergosterol as a Direct Trigger of Macrophage Pyroptosis.

Phagocytic cells such as macrophages play an important role in the host defense mechanisms mounted in response to the common human fungal pathogen , triggers macrophage NLRP3--mediated pyroptosis, an inflammatory programmed cell death pathway. Here, we provide evidence that -dependent pyroptosis occurs in the kidney of infected mice during the early stages of infection. We have also used a genome-wide screen of nonessential Σ1278b genes to identify genes required for yeast-triggered macrophage pyroptosis.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice.

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD).

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes.

Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy.

mTORC1 Is a Major Regulatory Node in the FGF21 Signaling Network in Adipocytes.

FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins.