Supplementary Figure 1 from The Cyclooxygenase Inhibitor Sulindac Sulfide Inhibits EP4 Expression and Suppresses the Growth of Glioblastoma Cells

The loss of functional insulin-producing β-cells is a hallmark of type 1 diabetes mellitus (T1DM). Previously, we reported that the non-steroidal anti-inflammatory drug activated gene-1, or growth differentiation factor-15 (NAG-1/GDF15) inhibits obesity and improves insulin sensitivity in both genetic and dietary-induced obese mice. However, the regulatory role of NAG-1/GDF15 in the structure and function of β-cells and the prevention of T1DM is largely unknown. In the current study, we reported that NAG-1/GDF15 transgenic (Tg) mice are resistant to diabetogenesis induced by multiple low-dose streptozotocin (MLD-STZ) treatment. NAG-1/GDF15 overexpression significantly reduced diabetes incidence, alleviated symptoms of T1DM, and improved MLD-STZ-induced glucose intolerance and insulin resistance. Both the mass and function of pancreatic β cells were preserved in the NAG-1/GDF15 Tg mice as evidenced by significantly increased islet area and insulin production. The mechanistic study revealed that NAG-1/GDF15 significantly inhibited STZ-induced apoptosis and preserved the reduction of proliferation in the islets of the Tg mice as compared to the wild-type (WT) mice upon MLD-STZ treatment. Additionally, NAG-1/GDF15 significantly reduced both the serum and islet levels of the inflammatory cytokines (IL-1β, IL-6, and TNFα), and reduced the expression of NF-κB expression and immune cells infiltration in the islets. Collectively, these results indicate that NAG-1/GDF15 is effective in improving STZ-induced glucose intolerance, probably was mediated via suppressing inflammation, inhibiting apoptosis, and preserving β-cell mass and function.

Aims: Our previous studies showed that the nonsteroidal anti-inflammatory drug-activated gene-1, or growth differentiation factor-15 (NAG-1/GDF15) inhibits obesity and diabetes in mice. The current study aimed to examine the role and molecular mechanisms of NAG-1/GDF15 in diabetic nephropathy (DN), which is largely unknown. Main methods: Both male and female wild-type (Wt) C57BL/6 mice and mice overexpressing human NAG-1/GDF15 (transgenic, Tg) were used, which were induced by high-fat diet (HFD)/streptozotocin (STZ) to establish the mouse model of DN. Transcriptome study was performed to identify the underlying molecular mechanisms of NAG-1/GDF15 against DN. In addition, human renal tubular epithelial cells (HK-2) were cultured with high glucose (HG) to establish a DN cellular model and were treated with NAG-1/GDF15 plasmid or the recombinant NAG-1/GDF15 protein for mechanism studies. Key findings: Overexpression of NAG-1/GDF15 in Tg mice significantly alleviated HFD/STZ-induced typical symptoms of DN, improved lipid homeostasis, glucose intolerance, and insulin sensitivity. Histopathology of renal tissues revealed that NAG-1/GDF15 mice had significantly reduced renal injury, glycogen deposition, and renal fibrosis. Transcriptome study uncovered inflammation, cell adhesion, and the inflammation-related signaling pathways as major pathways suppressed in the NAG-1/GDF15 mice. Further studies demonstrated that NAG-1/GDF15 overexpression inhibited renal and systematic inflammation, inhibited the AGE/RAGE axis and its associated downstream inflammatory molecules and adhesion molecules, and inhibited the upregulation of TLR4/MyD88/NF-kappa B signaling pathway in mice. These results were further confirmed in HG-induced HK-2 cells. Significance: NAG-1/GDF15 plays an important role in the inhibition of the development and progression of DN via targeting AGE/RAGE-mediated inflammation pathways.

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) plays a role in various diseases. Here, the anti-diabetic effects of NAG-1 were evaluated using a high-fat diet/streptozotocin-induced diabetic mouse model. NAG-1-overexpressing transgenic (NAG-1 Tg) mice exhibited lower body weight, fasting blood glucose levels, and serum insulin levels than wild-type (WT) mice. The homeostatic model assessment of insulin resistance scores of NAG-1 Tg mice were lower than those of WT mice. Hematoxylin and eosin staining revealed a smaller lipid droplet size in the adipose tissues, lower lipid accumulation in the hepatocytes, and larger beta cell area in the pancreas of NAG-1 Tg mice than in those of WT mice. Immunohistochemical analysis revealed downregulated expression of cleaved caspase-3, an apoptosis marker, in the beta cells of NAG-1 Tg mice. Adiponectin and leptin mRNA levels were upregulated and downregulated in NAG-1 Tg mice, respectively. Additionally, the expression of IRS1/PI3K/AKT signaling pathway components, especially Foxo1, which regulates gluconeogenesis in the muscle and white adipose tissue, was downregulated in NAG-1 Tg mice. Furthermore, NAG-1 overexpression promoted the expression of As160 in both muscles and adipocytes, and the mRNA levels of the NLRP3 pathway members were downregulated in NAG-1 Tg mice. Our findings suggest that NAG-1 expression alleviates diabetes in mice.

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) has a role in various diseases. Here, the anti-diabetic effects of NAG-1 were evaluated using a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mouse model. NAG-1-overexpressing transgenic (NAG-1 Tg) mice exhibited lower bodyweight and fasting blood glucose and insulin levels than wildtype (WT) mice. The homeostatic model assessment of insulin resistance scores of NAG-1 Tg mice were three times lower than those of WT mice. Hematoxylin and eosin staining demonstrated that NAG-1 Tg mice exhibited a smaller lipid droplet size in the adipose tissues, lower lipid accumulation in the hepatocytes, and larger beta cell area in the pancreas, compared to WT mice. Immunohistochemical analysis revealed downregulated expression of cleaved caspase-3, an apoptosis marker, in the beta cells of NAG-1 Tg mice. The adiponectin and leptin mRNA levels were upregulated and downregulated in NAG-1 Tg mice, respectively. Additionally, the IRS1/PI3K/AKT signaling pathway, especially Foxo1 , which regulates gluconeogenesis in the muscle and WAT, was downregulated in NAG-1 Tg mice. Furthermore, NAG-1 overexpression promoted As160 (AKT substrate of 160 kDa) expression in both muscles and adipocytes and the mRNA levels of the Nlrp3 pathway members were downregulated in NAG-1 Tg mice. Our findings suggest that NAG-1 expression alleviates diabetes in mice.

Growth Differentiation Factor 15 (GDF15), also known as NSAID activated gene-1 (NAG-1), is associated with a large number of biological processes and diseases, including cancer and obesity. GDF15 is synthesized as pro-GDF15, is dimerized, and is cleaved and secreted into the circulation as a mature dimer GDF15. Both the intracellular GDF15 and the circulating mature GDF15 are implicated in biological processes, such as energy homeostasis and body weight regulation. Although there have been many studies on GDF15, GFRAL, a member of the glial-derived neurotropic factor receptor α family, has only been recently identified as a receptor for mature GDF15. In this review, we focused on cancer and energy homeostasis along with obesity and body weight, and the effect of the identification of the GDF15 receptor in these investigations. In addition, the therapeutic potential of GDF15 as a pharmacological agent in obesity and other metabolic diseases was discussed.

Many volatile organic chemicals (VOCs) have not been tested for sensory pulmonary irritation. Development of in vitro non-animal sensory irritation assay suitable for a large number of chemicals is needed to replace the mouse assay. An adverse outcome pathway (AOP) is designed to provide a clear description of the biochemical and cellular processes leading to toxicological effects or an adverse outcome. The AOP for chemical sensory pulmonary irritation was developed according to the Organization for Economic Co-operation and Development guidance including the Bradford Hill criteria for a weight of evidence to determine the confidence of the AOP. The proposed AOP is based on an in-depth review of the relevant scientific literature to identify the initial molecular event for respiratory irritation. The activation of TRPA1 receptor (transient receptor potential cation channel, subfamily A, member 1) is the molecular initial event (MIE) leading to sensory irritation. A direct measure of TRPA1 activation in vitro should identify chemical sensory irritants and provide an estimate of potency. Fibroblasts expressing TRPA1 are used to determine TRPA1 activation and irritant potency. We report a linear relationship between the in vivo RD₅₀ and the in vitro pEC₅₀ values (R=0.81) to support this hypothesis. We propose that this in vitro assay after additional analysis and validation could serve as a suitable candidate to replace the mouse sensory irritation assay.

Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to altered milk quality and quantity.

Oxidative stress is elevated in numerous environmental exposures and diseases. Millions of dollars have been spent to try to ameliorate this damaging process using anti-oxidant therapies. Currently, the best accepted biomarker of oxidative stress is the lipid oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α), which has been measured in over a thousand human and animal studies. 8-iso-PGF2α generation has been exclusively attributed to nonenzymatic chemical lipid peroxidation (CLP). However, 8-iso-PGF2α can also be produced enzymatically by prostaglandin-endoperoxide synthases (PGHS) in vivo. When failing to account for PGHS-dependent generation, 8-iso-PGF2α cannot be interpreted as a selective biomarker of oxidative stress. We investigated the formation of 8-iso-PGF2α in rats exposed to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2α/PGF2α ratio to quantitatively determine the source(s) of 8-iso-PGF2α. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6±19.4% of measured 8-iso-PGF2α, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2α (86.6±8.0% of total). In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS (59.5±7.0) and CLP (40.5±14.0%). In conclusion, significant generation of 8-iso-PGF2α occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The 8-iso-PGF2α/PGF2α ratio accurately determines the source of 8-iso-PGF2α and provides an absolute measure of oxidative stress in vivo.

The effects of exposure to 100 % oxygen (O2) on the release of thromboxane B2 (TXB2) and prostaglandins (PG) from guinea pig lungs during anaphylaxis were studied. Lungs from sensitized guinea pigs were isolated, perfused, and challenged. Serologic examination of the perfusate showed that larger amounts of TXB2, 6-keto-PGF1α, PGE2, and PGF2α were released from the lungs of animals previously exposed to 100 % O2 for 72 h. Conversely, these lungs released smaller amounts of 13,14-dihydro-15-keto-PGF2α. Histamine release, measured spectrofluorometrically, was unchanged. 15-hydroxyprostaglandin dehydrogenase (PGDH) was inhibited by 83 % after O2 exposure, as measured by an in vitro enzyme assay. These results suggested that the reported enhancement of systemic anaphylaxis in the guinea pig in vivo after exposure to oxidant gases may be due in part to inhibition of pulmonary PGDH resulting in increased half-lives of primary PG and TX released from the lung.

The biomarker 8-iso-prostaglandin F2α (8-iso-PGF2α) is regarded as the gold standard for detection of excessive chemical lipid peroxidation in humans. However, biosynthesis of 8-iso-PGF2α via enzymatic lipid peroxidation by prostaglandin-endoperoxide synthases (PGHSs), which are significantly induced in inflammation, could lead to incorrect biomarker interpretation. To resolve the ambiguity with this biomarker, the ratio of 8-iso-PGF2α to prostaglandin F2α (PGF2α) is established as a quantitative measure to distinguish enzymatic from chemical lipid peroxidation in vitro, in animal models, and in humans. Using this method, we find that chemical lipid peroxidation contributes only 3% to the total 8-iso-PGF2α in the plasma of rats. In contrast, the 8-iso-PGF2α levels in plasma of human males are generated >99% by chemical lipid peroxidation. This establishes the potential for an alternate pathway of biomarker synthesis, and draws into question the source of increases in 8-iso-PGF2α seen in many human diseases. In conclusion, increases in 8-iso-PGF2α do not necessarily reflect increases in oxidative stress; therefore, past studies using 8-iso-PGF2α as a marker of oxidative stress may have been misinterpreted. The 8-iso-PGF2α/PGF2α ratio can be used to distinguish biomarker synthesis pathways and thus confirm the potential change in oxidative stress in the myriad of disease and chemical exposures known to induce 8-iso-PGF2α.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively for analgesic and antipyretic treatments. In addition, NSAIDs reduce the risk and mortality to several cancers. Their mechanisms in anti-tumorigenesis are not fully understood, but both cyclooxygenase (COX)-dependent and -independent pathways play a role. We and others have been interested in elucidating molecular targets of NSAID-induced apoptosis. In this review, we summarize updated literature regarding cellular and molecular targets modulated by NSAIDs. Among those NSAIDs, sulindac sulfide and tolfenamic acid are emphasized in this review because these two drugs have been well investigated for their anti-tumorigenic activity in many different types of cancer.

ObjectiveThe NLRP3 inflammasome plays an important regulatory role in obesity‐induced insulin resistance. NSAID activated gene‐1 (NAG‐1) is a divergent member of the TGF‐β superfamily. NAG‐1 Tg mice are resistant to dietary‐ and genetic‐induced obesity and have improved insulin sensitivity. The objective was to examine whether NLRP3 inflammasome activity is associated with this observed phenotype in NAG‐1 Tg mice.MethodsKey components of the NLRP3 inflammasome were examined in NAG‐1 Tg mice on both regular and high fat diet (HFD) conditions.ResultsThe expression of caspase‐1 and ASC, key components of the NLRP3 inflammasome, is significantly reduced at mRNA and protein levels in white adipose tissue (WAT) of NAG‐1 Tg mice. HFD increases the expression of caspase‐1 and ASC in WT mice, but their expression is reduced in NAG‐1 Tg mice. Furthermore, there is reduced IL‐18, IL‐1β, and TNF‐α expression in the WAT of NAG‐1 Tg mice. NAG‐1 Tg mice have significantly lower serum leptin and insulin levels and reduced expression of macrophage infiltration markers (F4/80, CD11b, and CD11c) in WAT.ConclusionsThe study suggests the lower NLRP3 inflammasome activity may play a role in the resistance of NAG‐1 Tg mice to diet‐induced obesity and improved insulin sensitivity.

While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.

Transhumance sheep and goat production have been a common and traditional practice in Greece, with its origins dating back to ancient times. Despite the diminishing number of transhumance farms, it remains an essential activity in less-favoured and mountainous areas of the country. This article applies DEA (Data Envelopment Analysis) in a sample of transhumance farms in Greece in order to assess the technical efficiency of sheep and goat transhumance flocks and determine the factors that affect their performance. The effect of EU subsidies on the technical efficiency of transhumance farms is assessed and the type of farms that benefit most is investigated. Results accrued reveal that the overall technical efficiency of transhumance farms in Greece is quite low and it is affected by herd size. EU subsidies have a significant impact on the technical efficiency of only the low-efficiency, small-sized farms.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) or GDF15 is a divergent member of the transforming growth factor beta (TGF-β) superfamily and mice expressing hNAG-1/hGDF15 have been shown to be resistant to HFD-induced obesity and inflammation. This study investigated if hNAG-1 increases lifespan in mice and its potential mechanisms. Here we report that female hNAG-1 mice had significantly increased both mean and median life spans in two transgenic lines, with a larger difference in life spans in mice on a HFD than on low fat diet. hNAG-1 mice displayed significantly reduced body and adipose tissue weight, lowered serum IGF-1, insulin and glucose levels, improved insulin sensitivity, and increased oxygen utilization, oxidative metabolism and energy expenditure. Gene expression analysis revealed significant differences in conserved gene pathways that are important regulators of longevity, including IGF-1, p70S6K, and PI3K/Akt signaling cascades. Phosphorylation of major components of IGF-1/mTOR signaling pathway was significantly lower in hNAG-1mice. Collectively, hNAG-1 is an important regulator of mammalian longevity and may act as a survival factor. Our study suggests that hNAG-1 has potential therapeutic uses in obesity-related diseases where life span is frequently shorter.