Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.     

An integrated disease/pharmacokinetic/pharmacodynamic model suggests improved interleukin-21 regimens validated prospectively for mouse solid cancers

Interleukin (IL)-21 is an attractive antitumor agent with potent immunomodulatory functions. Yet thus far, the cytokine has yielded only partial responses in solid cancer patients, and conditions for beneficial IL-21 immunotherapy remain elusive. The current work aims to identify clinically-relevant IL-21 regimens with enhanced efficacy, based on mathematical modeling of long-term antitumor responses. For this purpose, pharmacokinetic (PK) and pharmacodynamic (PD) data were acquired from a preclinical study applying systemic IL-21 therapy in murine solid cancers. We developed an integrated disease/PK/PD model for the IL-21 anticancer response, and calibrated it using selected "training" data. The accuracy of the model was verified retrospectively under diverse IL-21 treatment settings, by comparing its predictions to independent "validation" data in melanoma and renal cell carcinoma-challenged mice (R(2)>0.90). Simulations of the verified model surfaced important therapeutic insights: (1) Fractionating the standard daily regimen (50 μg/dose) into a twice daily schedule (25 μg/dose) is advantageous, yielding a significantly lower tumor mass (45% decrease); (2) A low-dose (12 μg/day) regimen exerts a response similar to that obtained under the 50 μg/day treatment, suggestive of an equally efficacious dose with potentially reduced toxicity. Subsequent experiments in melanoma-bearing mice corroborated both of these predictions with high precision (R(2)>0.89), thus validating the model also prospectively in vivo. Thus, the confirmed PK/PD model rationalizes IL-21 therapy, and pinpoints improved clinically-feasible treatment schedules. Our analysis demonstrates the value of employing mathematical modeling and in silico-guided design of solid tumor immunotherapy in the clinic.     

A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.     

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.     

SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number

In the field of breast biology, there is a growing appreciation for the "gatekeeping function" of basal cells during development and disease processes yet mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-β1 upstream of Robo1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer alone results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of β-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.     

Quantitative phospho-proteomic profiling of hepatocyte growth factor (HGF)-MET signaling in colorectal cancer

Colorectal cancer (CRC) is the second leading cause of death from cancer. The MET receptor tyrosine kinase and/or its ligand HGF are frequently amplified or overexpressed in CRC. It is known that tyrosine phosphorylated proteins are involved in progression and metastasis of colorectal cancer; however, little is known about the MET phospho-proteome in CRC. High resolution mass spectrometry was used to characterize immunoaffinity-purified, phosphotyrosine (pY)-containing tryptic peptides of the MET-expressing CRC cell model, DLD1. A total of 266 unambiguously identified pY sites spanning 168 proteins were identified. Quantification of mass spectrometry ion currents identified 161 pY sites, including many not previously linked to MET signaling, that were modulated in abundance by HGF stimulation. Overlay of these data with protein-protein interaction data sets suggested that many of the identified HGF-modulated phospho-proteins may be directly or indirectly associated with MET. Analysis of pY sequence motifs indicated a prevalence of Src family kinase consensus sequences, and reciprocal signaling between Src and MET was confirmed by using selective small molecule inhibitors of these kinases. Therefore, using quantitative phospho-proteomics profiling, kinase modulation by ligand and inhibitors, and data integration, an outline of the MET signaling network was generated for the CRC model.     

Increased adipose protein carbonylation in human obesity

Insulin resistance is associated with obesity but mechanisms controlling this relationship in humans are not fully understood. Studies in animal models suggest a linkage between adipose reactive oxygen species (ROS) and insulin resistance. ROS oxidize cellular lipids to produce a variety of lipid hydroperoxides that in turn generate reactive lipid aldehydes that covalently modify cellular proteins in a process termed carbonylation. Mammalian cells defend against reactive lipid aldehydes and protein carbonylation by glutathionylation using glutathione-S-transferase A4 (GSTA4) or carbonyl reduction/oxidation via reductases and/or dehydrogenases. Insulin resistance in mice is linked to ROS production and increased level of protein carbonylation, mitochondrial dysfunction, decreased insulin-stimulated glucose transport, and altered adipokine secretion. To assess protein carbonylation and insulin resistance in humans, eight healthy participants underwent subcutaneous fat biopsy from the periumbilical region for protein analysis and frequently sampled intravenous glucose tolerance testing to measure insulin sensitivity. Soluble proteins from adipose tissue were analyzed using two-dimensional gel electrophoresis and the major carbonylated proteins identified as the adipocyte and epithelial fatty acid-binding proteins. The level of protein carbonylation was directly correlated with adiposity and serum free fatty acids (FFAs). These results suggest that in human obesity oxidative stress is linked to protein carbonylation and such events may contribute to the development of insulin resistance.     

Obesity modifies the relations between serum markers of dairy fats and inflammation and oxidative stress among adolescents

Pentadecanoic acid (15:0) and heptadecanoic acid (17:0), the dairy-specific saturated fatty acids have been inversely, while inflammation and oxidative stress have been positively related to the risk of cardiovascular disease (CVD). Both fatty acid metabolism and inflammation and oxidative stress may be influenced by adiposity. In the current cross-sectional analyses among adolescents (mean age 15 years), we determined whether overweight status modified the associations between dairy fatty acids (pentadecanoic acid (15:0) and heptadecanoic acid (17:0)) represented in serum phospholipids (PL) and markers of inflammation and oxidative stress. Six biomarkers for inflammation and oxidative stress were analyzed, including circulating adiponectin, C-reactive protein (CRP), cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and urinary 15-keto-dihydro-PGF2α (15-keto) and 8-iso-PGF2α (F2-iso). Generalized linear regression analyses, adjusted for age, gender, race, tanner score, total energy intake and physical activity, revealed that PL dairy fatty acids were inversely associated with CRP, F2-iso and 15-keto in overweight, but not in normal weight adolescents (all P(interaction) < 0.05). However, higher level of PL dairy fatty acids was associated with lower IL-6 among all adolescents. Further adjustment for dietary intake of calcium, vitamin D, protein, total flavonoids, and ω-3 fatty acids did not materially change the findings. Dairy-specific saturated fats, i.e., 15:0 and 17:0 fatty acids, may contribute to the potential health benefits of dairy products, especially for overweight adolescents.     

Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.     

A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.