Customized small-sized clinostat using 3D printing and gas-permeable polydimethylsiloxane culture dish.

Over the past few decades, research on life in space has increased. Owing to the expensive nature of and the challenges associated with conducting experiments in real space, clinostats, which continuously randomize the gravity vector by using motors, have been used to generate simulated microgravity (SMG) on Earth. Herein, by using a 3D printing method, we develop a customized small-sized clinostat (CS clinostat) that is easy to manufacture, inexpensive, and robust. Moreover, we develop and fabricate a gas-permeable polydimethylsiloxane culture dish that fits inside the CS clinostat. To validate SMG generation, ovarian cancer cells (OV- 90, TOV-21G, and Caov-3) were applied to demonstrate a significant reduction in caveolin-1 expression, a biomarker of SMG, indicating SMG generation. The proposed CS clinostat system has good accessibility for SMG research, which makes it useful as a tool for biologists, who are unfamiliar with conventional clinostat equipment, to conduct preliminary studies in the space environment.

Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

OBJECTIVE: Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue. METHODS: We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT. RESULTS: Cold exposure or treatment with a ß3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or ß3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity. CONCLUSIONS: Our study implicates NNAT in the regulation of adipocyte thermogenesis.

CFTR regulates brown adipocyte thermogenesis via the cAMP/PKA signaling pathway.

BACKGROUND: Cystic fibrosis (CF) is characterized by reduced growth and lower body weight, which are multifactorial. CF mouse models lack key disease characteristics that predispose to a negative energy balance, such as pulmonary infections or exocrine pancreatic insufficiency, and yet they still exhibit a growth defect and an abnormally increased energy expenditure. Whether adipocyte thermogenesis contributes to the elevated resting energy expenditure in CF mice is unknown. METHODS: We examined the expression of CFTR in thermogenic brown adipose tissue (BAT) and investigated a functional role for CFTR using BAT-specific CFTR null mice (CFTRBATKO). RESULTS: The CFTR protein is expressed in mouse BAT at levels comparable to those in the lungs. BAT-specific inactivation of CFTR in mice increases whole-body energy expenditure associated with sympathetic stimulation by cold exposure. Weight gain on a high-fat diet is attenuated in these mice. However, CFTR-deficient brown adipocytes themselves have impaired, rather than enhanced, thermogenic responses. These cells feature decreased lipolysis and blunted activation of the cAMP/PKA signaling pathway in response to adrenergic stimulation. This suggests that compensatory heat production in other tissues likely accounts for the increased systemic energy expenditure seen in CFTRBATKO mice. CONCLUSIONS: Our data reveal a new role for CFTR in the regulation of adipocyte thermogenesis.

The interferon-inducible protein viperin controls cancer metabolic reprogramming to enhance cancer progression.

Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we show that the interferon-inducible (IFN-inducible) protein viperin drove metabolic alteration in cancer cells. Viperin expression was observed in various types of cancer and was inversely correlated with the survival rates of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster-binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid ß-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.

Adipose Mitochondrial Complex I Deficiency Modulates Inflammation and Glucose Homeostasis in a Sex-Dependent Manner.

Mitochondrial dysfunction in adipose tissue has been associated with type 2 diabetes, but it is unclear whether it is a cause or the consequence. Mitochondrial complex I is a major site of reactive oxygen species generation and a therapeutic target. Here we report that genetic deletion of the complex I subunit Ndufs4 specifically in adipose tissue results in an increased propensity to develop diet-induced weight gain, glucose intolerance, and elevated levels of fat inflammatory genes. This outcome is apparent in young males but not in young females, suggesting that females are relatively protected from the adverse consequences of adipose mitochondrial dysfunction for metabolic health. Mutant mice of both sexes exhibit defects in brown adipose tissue thermogenesis. Fibroblast growth factor 21 (FGF21) signaling in adipose tissue is selectively blunted in male mutant mice relative to wild-type littermates, consistent with sex-dependent regulation of its autocrine/paracrine action in adipocytes. Together, these findings support that adipocyte-specific mitochondrial dysfunction is sufficient to induce tissue inflammation and can cause systemic glucose abnormalities in male mice.

DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the ß-Catenin-P-Glycoprotein Signaling Pathway.

Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan-Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while ß-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active ß-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

CD4+/CD8+ Ratio and Growth Differentiation Factor 8 Levels in Peripheral Blood of Large Canine Males Are Useful Parameters to Build an Age Prediction Model.

PURPOSE: To build an age prediction model, we measured CD4+ and CD8+ cells, and humoral components in canine peripheral blood. MATERIALS AND METHODS: Large Belgian Malinois (BGM) and German Shepherd Dog (GSD) breeds (n=27), aged from 1 to 12 years, were used for this study. Peripheral bloods were obtained by venepuncture, then plasma and peripheral blood mononuclear cells (PBMCs) were separated immediately. Six myokines, including interleukin (IL)-6, IL-8, IL-15, leukemia inhibitory factor (LIF), growth differentiation factor 8 (GDF8), and GDF11 were measured from plasma and CD4+/CD8+ T-lymphocytes ratio were measured from PBMC. These parameters were then tested with age prediction models to find the best fit model. RESULTS: We found that the T-lymphocyte ratio (CD4+/CD8+) was significantly correlated with age (r=0.46, p=0.016). Among the six myokines, only GDF8 showed a significant correlation with age (r=0.52, p=0.005). Interestingly, these two markers showed better correlations in male dogs than females, and BGM breed than GSD. Using these two age biomarkers, we could obtain the best fit in a quadratic linear mixed model (r=0.77, p=3×10-6). CONCLUSIONS: Age prediction is a challenging task because of complication with biological age. Our quadratic linear mixed model using CD4+/CD8+ ratio and GDF8 level showed a meaningful age prediction.

Defective brown adipose tissue thermogenesis and impaired glucose metabolism in mice lacking Letmd1.

Manipulation of energy-dissipating adipocytes has the potential to produce metabolic benefits. To this end, it is valuable to understand the mechanisms controlling the generation and function of thermogenic fat. Here, we identify Letm1 domain containing 1 (Letmd1) as a regulator of brown fat formation and function. The expression of Letmd1 is induced in brown fat by cold exposure and by ß-adrenergic activation. Letmd1-deficient mice exhibit severe cold intolerance concomitant with abnormal brown fat morphology, reduced thermogenic gene expression, and low mitochondrial content. The null mice exhibit impaired ß3-adrenoreceptor-dependent thermogenesis and are prone to diet-induced obesity and defective glucose disposal. Letmd1 was previously described as a mitochondrial protein, and we find that it also localizes to the nucleus and interacts with the transcriptional coregulator and chromatin remodeler Brg1/Smarca4, thus providing a way to impact thermogenic gene expression. Our study uncovers a role for Letmd1 as a key regulatory component of adaptive thermogenesis.

Disruption of nucleocytoplasmic trafficking as a cellular senescence driver.

Senescent cells exhibit a reduced response to intrinsic and extrinsic stimuli. This diminished reaction may be explained by the disrupted transmission of nuclear signals. However, this hypothesis requires more evidence before it can be accepted as a mechanism of cellular senescence. A proteomic analysis of the cytoplasmic and nuclear fractions obtained from young and senescent cells revealed disruption of nucleocytoplasmic trafficking (NCT) as an essential feature of replicative senescence (RS) at the global level. Blocking NCT either chemically or genetically induced the acquisition of an RS-like senescence phenotype, named nuclear barrier-induced senescence (NBIS). A transcriptome analysis revealed that, among various types of cellular senescence, NBIS exhibited a gene expression pattern most similar to that of RS. Core proteomic and transcriptomic patterns common to both RS and NBIS included upregulation of the endocytosis-lysosome network and downregulation of NCT in senescent cells, patterns also observed in an aging yeast model. These results imply coordinated aging-dependent reduction in the transmission of extrinsic signals to the nucleus and in the nucleus-to-cytoplasm supply of proteins/RNAs. We further showed that the aging-associated decrease in Sp1 transcription factor expression was critical for the downregulation of NCT. Our results suggest that NBIS is a modality of cellular senescence that may represent the nature of physiological aging in eukaryotes.

Intrinsic expression of viperin regulates thermogenesis in adipose tissues.

Viperin is an interferon (IFN)-inducible multifunctional protein. Recent evidence from high-throughput analyses indicates that most IFN-inducible proteins, including viperin, are intrinsically expressed in specific tissues; however, the respective intrinsic functions are unknown. Here we show that the intrinsic expression of viperin regulates adipose tissue thermogenesis, which is known to counter metabolic disease and contribute to the febrile response to pathogen invasion. Viperin knockout mice exhibit increased heat production, resulting in a reduction of fat mass, improvement of high-fat diet (HFD)-induced glucose tolerance, and enhancement of cold tolerance. These thermogenic phenotypes are attributed to an adipocyte-autonomous mechanism that regulates fatty acid ß-oxidation. Under an HFD, viperin expression is increased, and its function is enhanced. Our findings reveal the intrinsic function of viperin as a novel mechanism regulating thermogenesis in adipose tissues, suggesting that viperin represents a molecular target for thermoregulation in clinical contexts.

Sulfate assimilation regulates hydrogen sulfide production independent of lifespan and reactive oxygen species under methionine restriction condition in yeast.

Endogenously produced hydrogen sulfide was proposed to be an underlying mechanism of lifespan extension via methionine restriction. However, hydrogen sulfide regulation and its beneficial effects via methionine restriction remain elusive. Here, we identified the genes required to increase hydrogen sulfide production under methionine restriction condition using genome-wide high-throughput screening in yeast strains with single-gene deletions. Sulfate assimilation-related genes, such as MET1, MET3, MET5, and MET10, were found to be particularly crucial for hydrogen sulfide production. Interestingly, methionine restriction failed to increase hydrogen sulfide production in mutant strains; however, it successfully extended chronological lifespan and reduced reactive oxygen species levels. Altogether, our observations suggested that increased hydrogen sulfide production via methionine restriction is not the mechanism underlying extended yeast lifespan, even though increased hydrogen sulfide production occurred simultaneously with yeast lifespan extension under methionine restriction condition.

Clusterin is highly expressed in tubular complexes during spontaneous pancreatitis of spontaneous hypertensive rats.

Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.

Caloric Restriction and Rapamycin Differentially Alter Energy Metabolism in Yeast.

Rapamycin (RM), a drug that inhibits the mechanistic target of rapamycin (mTOR) pathway and responds to nutrient availability, seemingly mimics the effects of caloric restriction (CR) on healthy life span. However, the extent of the mechanistic overlap between RM and CR remains incompletely understood. Here, we compared the impact of CR and RM on cellular metabolic status. Both regimens maintained intracellular ATP through the chronological aging process and showed enhanced mitochondrial capacity. Comparative transcriptome analysis showed that CR had a stronger impact on global gene expression than RM. We observed a like impact on the metabolome and identified distinct metabolites affected by CR and RM. CR severely reduced the level of energy storage molecules including glycogen and lipid droplets, whereas RM did not. RM boosted the production of enzymes responsible for the breakdown of glycogen and lipid droplets. Collectively, these results provide insights into the distinct energy metabolism mechanisms induced by CR and RM, suggesting that these two anti-aging regimens might extend life span through distinctive pathways.

Targeting ODC1 inhibits tumor growth through reduction of lipid metabolism in human hepatocellular carcinoma.

Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions.

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F1F0-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.

Disruption of Snf3/Rgt2 glucose sensors decreases lifespan and caloric restriction effectiveness through Mth1/Std1 by adjusting mitochondrial efficiency in yeast.

Down-regulation of intracellular nutrient signal pathways was proposed to be a primary mechanism of caloric restriction (CR)-mediated lifespan extension. However, the link between lifespan and glucose sensors in the plasma membrane was poorly understood in yeast. Herein, a mutant that lacked glucose sensors (snf3Δrgt2Δ) had impaired glucose fermentation, showed decreased chronological lifespan (CLS), and reduced CLS extension by CR. The mutant also had reduced mitochondrial efficiency, as inferred by increased mitochondrial superoxide and decreased ATP levels. Mth1 and Std1, which are downstream effectors of the Snf3/Rgt2 pathway, were required for viability through mitochondrial function but not fermentative metabolism.

Tumor-specific delivery of siRNA using supramolecular assembly of hyaluronic acid nanoparticles and 2b RNA-binding protein/siRNA complexes.

Anticancer therapeutics delivering exogenous siRNA have been explored to suppress the tumor-associated genes, but several limitations of siRNA delivery such as tumor-targeted delivery, controlled siRNA release at the sites of interest, or instabilities of siRNA in physiological fluids should be preferentially addressed for its clinical applications. As an attempt to meet these criteria, we designed a supramolecular assembly, which was composed of cholesterol-bearing hyaluronic acid (HA-Chol) conjugates and 2b RNA-binding protein (2b)/siRNA complexes. In contrast to the traditional siRNA polyplexes using electrostatic interactions, HA-Chol nanoparticles, as a results of self-assembly of HA-Chol conjugates, provide the hydrophobic core that acts as the container for 2b protein/siRNA complexes, where a high affinity of 2b protein for siRNA could neutralize the negative-charged siRNA. Here, we investigated the potential of HA-Chol/2b/siRNA complexes as the siRNA carriers that provide encapsulation, protection, and targeted delivery of siRNA. The HA-Chol nanoparticles could selectively deliver 2b protein/siRNA complexes to the tumor cells with up-regulated CD44 receptors and suppress the expression of target gene. The pH-associated binding properties of siRNA for 2b proteins allowed the controlled release of siRNA in the endocytic compartments, and ultimately the released siRNA could obtain the RNAi acitivities in the cells, whereas the encapsulated 2b proteins still stayed within the HA-Chol nanoparticles. Our delivery systems demonstrate the promising potential of the efficient siRNA carriers in the anticancer therapeutic applications.

Characterization of global gene expression during assurance of lifespan extension by caloric restriction in budding yeast.

Caloric restriction (CR) is the best-studied intervention known to delay aging and extend lifespan in evolutionarily distant organisms ranging from yeast to mammals in the laboratory. Although the effect of CR on lifespan extension has been investigated for nearly 80years, the molecular mechanisms of CR are still elusive. Consequently, it is important to understand the fundamental mechanisms of when and how lifespan is affected by CR. In this study, we first identified the time-windows during which CR assured cellular longevity by switching cells from culture media containing 2% or 0.5% glucose to water, which allows us to observe CR and non-calorically-restricted cells under the same conditions. We also constructed time-dependent gene expression profiles and selected 646 genes that showed significant changes and correlations with the lifespan-extending effect of CR. The positively correlated genes participated in transcriptional regulation, ribosomal RNA processing and nuclear genome stability, while the negatively correlated genes were involved in the regulation of several metabolic pathways, endoplasmic reticulum function, stress response and cell cycle progression. Furthermore, we discovered major upstream regulators of those significantly changed genes, including AZF1 (YOR113W), HSF1 (YGL073W) and XBP1 (YIL101C). Deletions of two genes, AZF1 and XBP1 (HSF1 is essential and was thus not tested), were confirmed to lessen the lifespan extension mediated by CR. The absence of these genes in the tor1Δ and ras2Δ backgrounds did show non-overlapping effects with regard to CLS, suggesting differences between the CR mechanism for Tor and Ras signaling.

Enhancement of mitochondrial function correlates with the extension of lifespan by caloric restriction and caloric restriction mimetics in yeast.

Caloric restriction mimetics (CRMs) have been developed to mimic the effects of caloric restriction (CR). However, research reports for the effects of CRMs are often times inconsistent across different research groups. Therefore, in this study, we compared seven identified CRMs which extend the lifespans of various organisms including caffeine, curcumin, dapsone, metformin, rapamycin, resveratrol, and spermidine to CR for mitochondrial function in a single model, Saccharomyces cerevisiae. In this organism, rapamycin extended chronological lifespan (CLS), but other CRMs failed to extend CLS. Rapamycin enhanced mitochondrial function like CR did, but other CRMs did not. Both CR and rapamycin worked on mitochondrial function, but they worked at different windows of time during the chronological aging process.