Screening the phytotoxicity of micro/nanoplastics through non-targeted metallomics with synchrotron radiation X-ray fluorescence and deep learning: Taking micro/nano polyethylene terephthalate as an example.

Microplastics (MPs) and nanoplastics (NPs) are global pollutants with emerging concerns. Methods to predict and screen their toxicity are crucial. Elemental dyshomeostasis can be used to assess toxicity of environmental pollutants. Non-targeted metallomics, combining synchrotron radiation X-ray fluorescence (SRXRF) and machine learning, has successfully differentiated cancer patients from healthy individuals. The whole idea of this work is to screen the phytotoxicity of nano polyethylene terephthalate (nPET) and micro polyethylene terephthalate (mPET) through non-targeted metallomics with SRXRF and deep learning algorithms. Firstly, Seed germination, seedling growth, photosynthetic changes, and antioxidant activity were used to evaluate the toxicity of mPET and nPET. It was showed that nPET, at 10 mg/L, was more toxic to rice seedlings, inhibiting growth and impairing chlorophyll content, MDA content, and SOD activity compared to mPET. Then, rice seedling leaves exposed to nPET or mPET was examined with SRXRF, and the SRXRF data was differentiated with deep learning algorithms. It was showed that the one-dimensional convolutional neural network (1D-CNN) model achieved 98.99% accuracy without data preprocessing in screening mPET and nPET exposure. In all, non-targeted metallomics with SRXRF and 1D-CNN can effectively screen the exposure and phytotoxicity of nPET/mPET and potentially other emerging pollutants. Further research is needed to assess the phytotoxicity of different types of MPs/NPs using non-targeted metallomics.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements.

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.

Arsenic and iron bioavailability in Caco-2 cells: The influence of their co-existence and concentration.

Arsenic (As) exposure in humans is primarily caused through food and drinking water. Iron (Fe) is one of the most common element of the human and can influence the toxicity and bioavailability of As. However, information on the interaction between As and Fe when present together is limited. In this study, the interaction effects of Fe(III) (0, 3, and 10 mg/L) and As (As(III) at 0, 0.05, 0.1 mg/L, and As(V) at 0, 0.1, and 2 mg/L, respectively) on their absorption and bioavailability in Caco-2 cells were analyzed. As(III) absorption significantly decreased with the addition of Fe, while Fe absorption significantly increased. Compared with 0.1 mg/L As(III) addition alone, 3 and 10 mg/L Fe(III) addition significantly reduced the As(III) absorption by 8.6 and 11 µg/L, respectively. The absorption of As and Fe(III) and the bioavailability of Fe(III) significantly increased with the addition of As(III/V). Compared with 10 mg/L Fe(III) alone, the absorption of As(III) was significantly increased by 1 and 1.3 mg/L with 0.05 and 0.1 mg/L As(III) addition, respectively. Furthermore, the absorption and bioavailability of Fe(III) were significantly increased by 1.2 mg/L and 8% and 1.2 mg/L and 8.2%, respectively, after adding 0.1 and 2 mg/L As(V).

The toxicity of nano polyethylene terephthalate to mice: Intestinal obstruction, growth retardant, gut microbiota dysbiosis and lipid metabolism disorders.

Polyethylene terephthalate (PET) are widely used in our daily life while they may be broken to smaller fractions as nano-sized PET (nPET) in the environment. The toxicity of nPET is still less studied. This work first evaluated the LD50 of different size of nPET (200 nm, S-nPET; 700 nm, B-nPET) in mice, then studied the health effects of single exposure to S/B-nPET at 200 mg/kg bw for 30 days. It was found that the LD50 was 266 mg/kg bw for S-nPET and 523 mg/kg bw for B-nPET, respectively, showing a size-dependent effect. S-nPET caused weight loss, cyst, intestinal obstruction, organ damage and mortality (40%), and perturbed gut microbiome and metabolome especially lipid metabolism, such as upregulated cholesterol, glycocholic, propionic acid, niacinamide, ectoine and xanthine, and downregulated arachidonic acid, anserine, histamine, while B-nPET did not. Serological analysis found S-nPET brought more lipid metabolic immune and neurological damage than B-nPET, confirming the size-dependent effect. To the best of our knowledge, this is the first report on the systematic toxicity of nPET to mice. Further studies are warranted for life-long effects of nPET. The protocol applied in this work may also be used for the study of the health effects of other plastics.

MnO2-melittin nanoparticles serve as an effective anti-tumor immunotherapy by enhancing systemic immune response.

Cancer vaccines are viewed as a promising immunotherapy to eradicate malignant tumors and aim to elicit the patients' own tumor-specific immune response against tumor cells. However, few cancer vaccines have been applied due to the low immunogenicity of antigen and invalidation of adjuvant. Herein, we designed a tumor microenvironment (TME) responsive MnO2-melittin nanoparticles (M-M NPs). The M-M NPs consumed glutathione and produced •OH via Fenton-like reaction in the mimic TME, specifically caused tumor cell death in vitro, activated cGAS-STING pathway in vitro and promoted the maturation of antigen-presenting cells in vitro and in vivo to elicit systemic anti-tumor immune response including the augmentation of tumor-specific T cells and more productions of pro-inflammatory cytokines and chemokines, which all were stronger than MnO2 NPs and melittin. The anti-tumor effects of M-M NPs were evaluated in three subcutaneous tumor models and the B16-F10 lung metastasis model and the tumor growth and lung metastasis were more obviously inhibited in the M-M NPs treated mice, compared with MnO2 NPs and melittin treatments. More importantly, only M-M NPs promoted the MHC-I cross-dressing by dendritic cells to prime tumor-specific CD8+ T cells and remarkably suppressed the growth of left tumors if express cognate antigen while treating on the right in the bilateral tumor model. Our findings proposed a strategy to enhance the cancer vaccine efficiency which showed great therapeutic effect on tumor immunotherapy.

Ceria nanoparticles prophylactic used for renal ischemia-reperfusion injury treatment by attenuating oxidative stress and inflammatory response.

Renal ischemia-reperfusion (IR) injury (RIRI) is the leading cause of acute kidney injury (AKI), a common disease with high morbidity and mortality. However, due to the lack of effective diagnostic and therapeutic tools, patients have to resort to conservative treatment. To address this issue, we have developed a novel prophylactic strategy that involves the pre-treatment use of ceria nanoparticles (CNPs) before surgery. Based on our careful study of the three different sizes of CNPs that we synthesized, 46 nm (NP46), 81 nm (NP81), and 118 nm (NP118), we have found that NP118 can be used as effective prophylactic agents against RIRI and subsequent renal fibrosis. In our experiments, the CNPs exhibited excellent antioxidant and anti-inflammatory activities in vitro and effectively protected the kidney against RIRI and renal fibrosis in vivo, as proved by the decreases in renal lesions, serum creatinine, blood urea nitrogen, apoptotic cell, KIM-1 expression, and fibrotic area in CNPs treated samples relative to RIRI group. Mechanistically, not only did the CNPs reduce oxidative stress by regulating the Nrf2 pathway, but they also attenuated RIRI induced inflammatory response by decreasing macrophage infiltration and polarization to M1 phenotype, and reducing pro-inflammatory cytokine and chemokine production. In vitro results further confirmed that CNPs pre-treatment not only dramatically decreased intracellular ROS production in renal tubular epithelial cells and vascular endothelial cells, but also effectively attenuated lipopolysaccharide-induced inflammation in RAW264.7 cells. In addition, we found that one fourth of the NP118 persisted for more than 21 days in IR kidneys, and that out of the three sizes of CNPs, NP118 achieved the best results in all our experiments. Our study provides new insights into the usage and majorization of CNPs as a potential therapy to treat or prevent RIRI and renal fibrosis.

Controlling microbial arsenite oxidation and mobilization in arsenite-adsorbed iron minerals: The Influence of pH conditions and mineralogical composition.

The oxidation of aqueous arsenite (As(III)) by As(III)-oxidizing bacteria is known to attenuate the mobilization and toxicity of arsenic, and is regarded as potential method for As(III)-pollution remediation. However, during the interactions between As(III)-oxidizing bacteria and different As(III)-adsorbed soil Fe-minerals, the oxidation and partitioning of solid-phase As(III), as well as the controlling mechanisms, remain unclear. In this study, we therefore incubated three As(III)-adsorbed Fe-minerals with a typical As(III)-oxidizing bacteria (Pseudomonas sp. HN-1) at different pH conditions. After microbial oxidation, the percentage of arsenate (As(V)) was significantly higher at pH 7 (15-94%) and 9 (12-89%) than at pH 4 (6-50%) in all Fe-minerals. Incubation of As(III)-oxidizing bacteria promoted As-immobilization under acidic-conditions but As-mobilization under alkaline-conditions. Arsenic-X-ray adsorption spectroscopy results showed that solid-phase As(V) fraction in goethite, hematite and magnetite was 27-64%, 5-12% and 50-91%, respectively. Compared with the corner-sharing As(III)-adsorption complexes formed on magnetite, the edge-sharing complexes on hematite were significantly more stable towards microbial-oxidation. Additionally, the strong adhesion between strain HN-1 and hematite probably limit bacterial-activity and mobility, thereby inhibiting microbial As(III)-oxidation. Our findings elucidate the controlling mechanisms of microbial As(III)-oxidation in different As(III)-adsorbed Fe-minerals and demonstrate strain HN-1 is an excellent candidate for As(III)-remediation in soils containing goethite and magnetite.

Ginsenoside Rb3 Alleviates CSE-induced TROP2 Upregulation through p38 MAPK and NF-κB Pathways in Basal Cells.

Smoking-mediated reprogramming of the phenotype and function of airway basal cells (BCs) disrupts airway homeostasis and is an early event in chronic obstructive pulmonary disease (COPD)-associated airway remodeling. Here, we examined the expression and regulation of the transmembrane glycoprotein TROP2 (trophoblast antigen 2), a putative stem cell marker in airway BCs, in lung tissue samples from healthy smokers and healthy nonsmokers and in models in culture to identify therapeutic targets. TROP2 expression was upregulated in the airway epithelia of smokers and positively correlated with the smoking index. In vitro, cigarette smoke extract (CSE) induced TROP2 expression in airway BCs in a time- and dose-dependent manner. The p38 MAPK and NF-κB pathways were also activated by CSE, and their specific antagonists inhibited CSE-induced TROP2 expression. A therapeutic component derived from traditional Chinese medicine, ginsenoside Rb3, inhibited CSE-induced TROP2 expression as well as activation of the p38 MAPK and NF-κB pathways in BCs in monolayer culture. Furthermore, ginsenoside Rb3 prevented the increase in TROP2 expression and antagonized CSE-induced BC hyperplasia and expression of inflammatory factors and epithelial-mesenchymal transition changes in an air-liquid culture model. Thus, CSE-induced TROP2 is a possible biomarker for early changes in the epithelium of smokers, and ginsenoside Rb3 may serve as a therapeutic molecule, preventing the disruption of epithelial homeostasis in COPD.

Impact of COPD on prognosis of lung cancer: from a perspective on disease heterogeneity.

BACKGROUND: COPD is an important comorbidity of lung cancer, but the impact of COPD on the outcomes of lung cancer remains uncertain. Because both COPD and lung cancer are heterogeneous diseases, we evaluated the link between COPD phenotypes and the prognosis of different histological subtypes of lung cancer. METHODS: In this retrospective study, subjects with a newly and pathologically confirmed diagnosis of lung cancer were enrolled from patients preparing for lung cancer surgery. All participants underwent pulmonary function test (PFT). The diagnosis of COPD was based on GOLD criteria. Lung cancer subtypes and COPD phenotypes were categorized by WHO classification of lung tumors and computer quantitative analysis of PFT. The HRs were estimated by Cox regression analysis. RESULTS: Among 2,222 lung cancer patients, 32.6% coexisted with COPD. After adjustment for age, sex, body mass index (BMI), smoking status, and therapy method, COPD was significantly associated with the decreased overall survival (OS) of lung cancer (HR 1.28, 95% CI 1.05-1.57). With the increased severity of COPD, the OS of lung cancer was gradually worsened (HR 1.23, 95% CI 1.08-1.39). But surgical treatment and high BMI were independent prognostic protective factors (HR 0.46, 95% CI 0.37-0.56; HR 0.96, 95% CI 0.94-0.99). Moreover, in terms of disease heterogeneity, emphysema-predominant phenotype of COPD was an independent prognostic risk factor for squamous carcinoma (HR 2.53, 95% CI 1.49-4.30). No significant relationship between COPD phenotype and lung cancer prognosis was observed among adenocarcinoma, small cell lung cancer, large cell lung cancer, and other subtype patients. CONCLUSION: These findings suggest that COPD, especially emphysema-predominant phenotype, is an independent prognostic risk factor for squamous carcinoma only.

High prevalence of bronchiectasis in emphysema-predominant COPD patients.

Background: COPD has been identified as an etiology or related disease of bronchiectasis, and bronchiectasis has been classified as a comorbidity of COPD. In this study, we investigated the prevalence of bronchiectasis in different phenotypes of COPD subjects and the correlation between bronchiectasis and different phenotypes, especially emphysema. Methods: COPD patients were recruited from April 2012 to December 2015. The presence of bronchiectasis and related information were statistically analyzed. COPD subjects were separated into subgroups in two ways: COPD with and without bronchiectasis groups and emphysema-predominant (emphysema index, EI≥9.9%) and non-emphysema-predominant (EI<9.9%) groups. Results: In total, 1,739 COPD patients were incorporated into the study, among which 140 cases (8.1%) were accompanied with radiological bronchiectasis. COPD patients with concomitant bronchiectasis presented worse pulmonary function (FEV1% predicted, P<0.001), higher EI (15.0% vs 13.4%, P<0.001), and higher proportion of pulmonary hypertension and cor pulmonale (6.4% vs 2.4%, P=0.005 and 23.6% vs 16.1%, P=0.022) than patients without bronchiectasis. Of all the COPD patients, 787 with EI data were divided into emphysema-predominant (n=369) and non-emphysema-predominant groups (n=418). The proportion of bronchiectasis was 16.5% and 10.3% (P=0.01), respectively. Severity of bronchiectasis increased as the degree of airflow limitation (r=-0.371, P<0.001) and emphysema increased (r=0.226, P=0.021). After adjusting confounding factors, FEV1% predicted (OR, 1.636; 95% CI, 1.219-2.197; P=0.001) and EI (OR, 1.993; 95% CI, 1.199-3.313; P=0.008) were significantly related with the presence of bronchiectasis in COPD patients. Conclusion: The proportion of bronchiectasis is higher in emphysema-predominant COPD subjects. Emphysema measured by EI and FEV1% predicted are independent predictors for bronchiectasis in COPD subjects, while the underlying mechanism deserves further investigation.

The link between chronic obstructive pulmonary disease phenotypes and histological subtypes of lung cancer: a case-control study.

BACKGROUND: COPD is considered an independent risk factor for lung cancer. COPD and lung cancer are both very heterogeneous diseases, and the study herein investigates the link between COPD phenotypes and specific histological subtypes of lung cancer. METHODS: This case-control study comprised 2,283 patients with newly diagnosed pathological lung cancer and 2,323 non-lung cancer controls. All participants underwent pulmonary function tests. The diagnosis of COPD was based on Global Initiative for Chronic Obstructive Lung Disease criteria. Subtypes of the two diseases were categorized according to 2015 World Health Organization classification of lung cancer and computer quantification of airway collapse on maximum expiratory flow volume. ORs were estimated using logistic regression analysis. RESULTS: The prevalence of COPD was higher (32.8%) in lung cancer patients compared to controls (16.0%). After adjustment for age, sex, body-mass index, and smoking status, the presence of COPD significantly increased the risk of lung cancer (OR 2.88, 95% CI 2.48-3.34) and all common histological subtypes (ORs 2.04-5.26). Both emphysema-predominant and non-emphysema-predominant phenotypes of COPD significantly increased the risk of lung cancer (OR 4.43, 95% CI 2.85-6.88; OR 2.82, 95% CI 2.40-3.31). Higher risk of squamous-cell carcinoma and small-cell lung cancer was observed in patients with the emphysema-predominant than the non-emphysema-predominant phenotype (OR 1.73, 95% CI 1.03-2.89; OR 3.74, 95% CI 1.64-8.53). CONCLUSION: COPD was an independent risk factor for lung cancer and all common histological subtypes. Both emphysema-predominant and non-emphysema-predominant phenotypes of COPD significantly increased the risk of lung cancer. Relative to non-emphysema-predominant phenotype of COPD, emphysema-predominant phenotype had a higher risk of squamous-cell carcinoma and small-cell lung cancer.

Co-existence of COPD and bronchiectasis: a risk factor for a high ratio of main pulmonary artery to aorta diameter (PA:A) from computed tomography in COPD patients.

Background: Pulmonary vascular disease, especially pulmonary hypertension, is an important complication of COPD. Bronchiectasis is considered not only a comorbidity of COPD, but also a risk factor for vascular diseases. The main pulmonary artery to aorta diameter ratio (PA:A ratio) has been found to be a reliable indicator of pulmonary vascular disease. It is hypothesized that the co-existence of COPD and bronchiectasis may be associated with relative pulmonary artery enlargement (PA:A ratio >1). Methods: This retrospective study enrolled COPD patients from 2012 through 2016. Demographic and clinical data were collected. Bhalla score was used to determine the severity of bronchiectasis. Patient characteristics were analyzed in two ways: the high (PA:A >1) and low (PA:A ≤1) ratio groups; and COPD with and without bronchiectasis groups. Logistic regression analysis was used to assess risk factors for high PA:A ratios. Results: In this study, 480 COPD patients were included, of whom 168 had radiographic bronchiectasis. Patients with pulmonary artery enlargement presented with poorer nutrition (albumin, 35.6±5.1 vs 38.3±4.9, P<0.001), lower oxygen partial pressure (74.4±34.5 vs 81.3±25.4, P<0.001), more severe airflow obstruction (FEV1.0, 0.9±0.5 vs 1.1±0.6, P=0.004), and a higher frequency of bronchiectasis (60% vs 28.8%, P<0.001) than patients in the low ratio group. Patients with both COPD and bronchiectasis had higher levels of systemic inflammation (erythrocyte sedimentation rate, P<0.001 and fibrinogen, P=0.006) and PA:A ratios (P<0.001). A higher PA:A ratio was significantly closely correlated with a higher Bhalla score (r=0.412, P<0.001). Patients with both COPD and bronchiectasis with high ratios presented higher levels of NT-proBNP (P<0.001) and systolic pulmonary artery pressure (P<0.001). Multiple logistic analyses have indicated that bronchiectasis is an independent risk factor for high PA:A ratios in COPD patients (OR =3.707; 95% CI =1.888-7.278; P<0.001). Conclusion: Bronchiectasis in COPD has been demonstrated to be independently associated with relative pulmonary artery enlargement.

Quantitative computed tomography measurements of emphysema for diagnosing asthma-chronic obstructive pulmonary disease overlap syndrome.

BACKGROUND: The diagnostic criteria of asthma-COPD overlap syndrome (ACOS) are controversial. Emphysema is characteristic of COPD and usually does not exist in typical asthma patients. Emphysema in patients with asthma suggests the coexistence of COPD. Quantitative computed tomography (CT) allows repeated evaluation of emphysema noninvasively. We investigated the value of quantitative CT measurements of emphysema in the diagnosis of ACOS. METHODS: This study included 404 participants; 151 asthma patients, 125 COPD patients, and 128 normal control subjects. All the participants underwent pulmonary function tests and a high-resolution CT scan. Emphysema measurements were taken with an Airway Inspector software. The asthma patients were divided into high and low emphysema index (EI) groups based on the percentage of low attenuation areas less than -950 Hounsfield units. The characteristics of asthma patients with high EI were compared with those having low EI or COPD. RESULTS: The normal value of percentage of low attenuation areas less than -950 Hounsfield units in Chinese aged >40 years was 2.79%±2.37%. COPD patients indicated more severe emphysema and more upper-zone-predominant distribution of emphysema than asthma patients or controls. Thirty-two (21.2%) of the 151 asthma patients had high EI. Compared with asthma patients with low EI, those with high EI were significantly older, more likely to be male, had more pack-years of smoking, had more upper-zone-predominant distribution of emphysema, and had greater airflow limitation. There were no significant differences in sex ratios, pack-years of smoking, airflow limitation, or emphysema distribution between asthma patients with high EI and COPD patients. A greater number of acute exacerbations were seen in asthma patients with high EI compared with those with low EI or COPD. CONCLUSION: Asthma patients with high EI fulfill the features of ACOS, as described in the Global Initiative for Asthma and Global Initiative for Chronic Obstructive Lung Disease guidelines. Quantitative CT measurements of emphysema may help in diagnosing ACOS.

Continuous parenteral and enteral nutrition induces metabolic dysfunction in neonatal pigs.

BACKGROUND: We previously showed that parenteral nutrition (PN) compared with formula feeding results in hepatic insulin resistance and steatosis in neonatal pigs. The current aim was to test whether the route of feeding (intravenous [IV] vs enteral) rather than other feeding modalities (diet, pattern) had contributed to the outcome. METHODS: Neonatal pigs were fed enterally or parenterally for 14 days with 1 of 4 feeding modalities as follows: (1) enteral polymeric formula intermittently (FORM), (2) enteral elemental diet (ED) intermittently (IEN), (3) enteral ED continuously (CEN), and (4) parenteral ED continuously (PN). Subgroups of pigs underwent IV glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP). Following CLAMP, pigs were euthanized and tissues collected for further analysis. RESULTS: Insulin secretion during IVGTT was significantly higher and glucose infusion rates during CLAMP were lower in CEN and PN than in FORM and IEN. Endogenous glucose production rate was suppressed to zero in all groups during CLAMP. In the fed state, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide (GLP)-1, and GLP-2 were different between feeding modalities. Insulin receptor phosphorylation in liver and muscle was decreased in IEN, CEN, and PN compared with FORM. Liver weight was highest in PN. Steatosis and myeloperoxidase (MPO) activity tended to be highest in PN and CEN. Enterally fed groups had higher plasma GLP-2 and jejunum weight compared with PN. CONCLUSIONS: PN and enteral nutrition (EN) when given continuously as an elemental diet reduces insulin sensitivity and the secretion of key gut incretins. The intermittent vs continuous pattern of EN produced the optimal effect on metabolic function.

Chronic parenteral nutrition induces hepatic inflammation, steatosis, and insulin resistance in neonatal pigs.

Prematurity and overfeeding in infants are associated with insulin resistance in childhood and may increase the risk of adult disease. Total parenteral nutrition (TPN) is a major source of infant nutritional support and may influence neonatal metabolic function. Our aim was to test the hypothesis that TPN induces increased adiposity and insulin resistance compared with enteral nutrition (EN) in neonatal pigs. Neonatal pigs were either fed enteral formula orally or i.v. administered a TPN mixture for 17 d; macronutrient intake was similar in both groups. During the 17-d period, we measured body composition by dual-energy X-ray absorptiometry scanning; fasting i.v. glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP) were performed to quantify insulin resistance. On d 17, tissue was collected after 1-h, low-dose CLAMP for tissue insulin signaling assays. TPN pigs gained less lean and more body fat and developed hepatic steatosis compared with EN pigs. After 7 and 13 d, IVGTT showed evidence of insulin resistance in the TPN compared with the EN group. Fasting plasma glucose and insulin also were higher in TPN pigs. CLAMP showed that insulin sensitivity was markedly lower in TPN pigs than in EN pigs. TPN also reduced the abundance of the insulin receptor, insulin receptor substrate 1, and phosphatidylinositol 3 kinase in skeletal muscle and liver and the proliferation of total pancreatic cells and ß-cells. Hepatic proinflammatory genes as well as c-Jun-N-terminal kinase 1 phosphorylation, plasma interleukin 6, and tumor necrosis factor-α were all higher in TPN pigs than in EN pigs. The results demonstrate that chronic TPN induces a hepatic inflammatory response that is associated with significant insulin resistance, hepatic steatosis, and fat deposition compared with EN in neonatal pigs. Further studies are warranted to establish the mechanism of TPN-induced insulin resistance and hepatic metabolic dysfunction and whether there are persistent metabolic consequences of this lifesaving form of infant nutritional support.

Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide.

Arginine is an indispensable amino acid in neonates and is required for growth. Neonatal intestinal epithelial cells (IEC) are capable of arginine transport, catabolism, and synthesis and express nitric oxide (NO) synthase to produce NO from arginine. Our aim was to determine whether arginine directly stimulates IEC growth and protein synthesis and whether this effect is mediated via mammalian target of rapamycin (mTOR) and is NO-dependent. We studied neonatal porcine IEC (IPEC-J2) cultured in serum- and arginine-free medium with increasing arginine concentrations for 4 or 48 h. Our results show that arginine enhances IPEC-J2 cell survival and protein synthesis, with a maximal response at a physiological concentration (0.1-0.5 mM). Addition of arginine increased the activation of mTOR, p70 ribosomal protein S6 (p70 S6) kinase, and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in a time- and dose-dependent manner. The arginine-induced protein synthesis response was not inhibited by the NO inhibitors nitro-l-arginine methyl ester (l-NAME) and aminoguanidine, despite inducible NO synthase expression in IPEC-J2 cells. Moreover, protein synthesis was not increased or decreased in some cases by addition of an NO donor (S-nitroso-N-acetylpenicillamine), arginine precursors (proline and citrulline) in the absence of arginine, or insulin; S-nitroso-N-acetylpenicillamine suppressed phosphorylation of mTOR, p70 S6 kinase, and 4E-BP1. We found a markedly higher arginase activity in IPEC-J2 cells than in primary pig IEC. Furthermore, mTOR inhibition by rapamycin partially (42%) reduced the arginine-induced protein synthesis response and phosphorylation of mTOR and 4E-BP1. We conclude that arginine-dependent cell survival and protein synthesis signaling in IPEC-J2 cells are mediated by mTOR, but not by NO.

GLP-2 rapidly activates divergent intracellular signaling pathways involved in intestinal cell survival and proliferation in neonatal piglets.

We previously demonstrated the dose-dependent glucagon-like peptide (GLP)-2 activation of intracellular signals associated with increased epithelial cell survival and proliferation in the neonatal intestine. Our current aim was to quantify the acute, temporal GLP-2 activation of these key intracellular signals and relate this to changes in epithelial cell survival and proliferation in the neonatal intestine. We studied 29 total parenteral nutrition-fed neonatal piglets infused intravenously with either saline (control) or human GLP-2 (420 micromol.kg(-1).h(-1)) for 1, 4, or 48 h. GLP-2 infusion increased small intestinal weight, DNA and protein content, and villus height at 48 h, but not at 1 or 4 h. Intestinal crypt and villus apoptosis decreased and crypt cell proliferation and protein synthesis increased linearly with duration of GLP-2 infusion, but were statistically different from controls only after 48 h. Before the morphological and cellular kinetic changes, GLP-2 rapidly activated putative GLP-2 receptor downstream signals within 1-4 h, including phosphorylation of protein kinase A, protein kinase B, extracellular signal-regulated kinase 1/2, and the transcription factors cAMP response element-binding protein and c-Fos. GLP-2 rapidly suppressed caspase-3 activation and upregulated Bcl-2 abundance within 1 h, whereas there was an increase in apoptosis inhibitors X-linked inhibitor of apoptosis at 1 h and cellular inhibitor of apoptosis-2 at 4 and 48 h. We also show that the increased c-Fos and reduced active caspase-3 immunostaining after GLP-2 infusion was localized in epithelial cells. We conclude that GLP-2-induced activation of intracellular signals involved in both cell survival and proliferation occurs rapidly and precedes the trophic cellular kinetic effects that occur later in intestinal epithelial cells.

Glucagon-like peptide 2 dose-dependently activates intestinal cell survival and proliferation in neonatal piglets.

Glucagon-like peptide 2 (GLP-2) is a gut hormone that stimulates mucosal growth in total parenteral nutrition (TPN)-fed piglets; however, the dose-dependent effects on apoptosis, cell proliferation, and protein synthesis are unknown. We studied 38 TPN-fed neonatal piglets infused iv with either saline or GLP-2 at three rates (2.5, 5.0, and 10.0 nmol.kg(-1).d(-1)) for 7 d. Plasma GLP-2 concentrations ranged from 177 +/- 27 to 692 +/- 85 pM in the low- and high-infusion groups, respectively. GLP-2 infusion dose-dependently increased small intestinal weight, DNA and protein content, and villus height; however, stomach protein synthesis was decreased by GLP-2. Intestinal crypt and villus apoptosis decreased and crypt cell number increased linearly with GLP-2 infusion rates, whereas cell proliferation and protein synthesis were stimulated only at the high GLP-2 dose. The intestinal activities of caspase-3 and -6 and active caspase-3 abundance decreased, yet procaspase-3 abundance increased markedly with increasing infusion rate and plasma concentration of GLP-2. The GLP-2-dose-dependent suppression of intestinal apoptosis and caspase-3 activity was associated with increased protein kinase B and glycogen-synthase kinase-3 phosphorylation, yet the expression phosphatidylinositol 3-kinase was unaffected by GLP-2. Intestinal endothelial nitric oxide synthase mRNA and protein expression was increased, but only at the high GLP-2 dose. We conclude that the stimulation of intestinal epithelial survival is concentration dependent at physiological GLP-2 concentrations; however, induction of cell proliferation and protein synthesis is a pharmacological response. Moreover, we show that GLP-2 stimulates intestinal cell survival and proliferation in association with induction of protein kinase B and glycogen-synthase kinase-3 phosphorylation and Bcl-2 expression.