Decreased Hyocholic Acid and Lysophosphatidylcholine Induce Elevated Blood Glucose in a Transgenic Porcine Model of Metabolic Disease.

(1) Background: This work aims to investigate the metabolomic changes in PIGinH11 pigs and investigate differential compounds as potential therapeutic targets for metabolic diseases. (2) Methods: PIGinH11 pigs were established with a CRISPR/Cas9 system. PNPLA3I148M, hIAPP, and GIPRdn were knocked in the H11 locus of the pig genome. The differential metabolites between and within groups were compared at baseline and two months after high-fat-high-sucrose diet induction. (3) Results: 72.02% of the 815 detected metabolites were affected by the transgenic effect. Significantly increased metabolites included isoleucine, tyrosine, methionine, oxoglutaric acid, acylcarnitine, glucose, sphinganines, ceramides, and phosphatidylserines, while fatty acids and conjugates, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins were decreased. Lower expression of GPAT3 and higher expression of PNPLA3I148M decreased the synthesis of diacylglycerol and phosphatidylcholines. Accumulated ceramides that block Akt signaling and decrease hyocholic acid and lysophosphatidylcholines might be the main reason for increased blood glucose in PIGinH11 pigs, which was consistent with metabolomic changes in patients. (4) Conclusions: Through serum metabolomics and lipidomics studies, significant changes in obesity and diabetes-related biomarkers were detected in PIGinH11 pigs. Excessive fatty acids ß-oxidation interfered with glucose and amino acids catabolism and reduced phosphatidylcholines. Decreased hyocholic acid, lysophosphatidylcholine, and increased ceramides exacerbated insulin resistance and elevated blood glucose. Phosphatidylserines were also increased, which might promote chronic inflammation by activating macrophages.

Dynamic Mitochondrial Proteome Under Polyamines Treatment in Cardiac Aging.

Age-related alteration of mitochondria causes impaired cardiac function, along with cellular and molecular changes. Polyamines can extend the life span in mice. However, whether polyamines can affect the dynamic mitochondrial proteome, thereby preventing age-related changes in cardiac function and cardiac aging, remains unclear. In this study, we found that spermine (Spm) and spermidine (Spd) injection for 6 weeks could prevent 24-month-old rats heart dysfunction, improve mitochondrial function, and downregulate apoptosis. Using iTRAQ tools, we identify 75 mitochondrial proteins of statistically significant alteration in aging hearts, which mainly participate in important mitochondrial physiological activity, such as metabolism, translation, transport, apoptosis, and oxidative phosphorylation. Moreover, four proteins of differential expression, pyruvate dehydrogenase kinase (PDK4), trifunctional enzyme subunit alpha (HADHA), nicotinamide nucleotide transhydrogenase (NNT), and Annexin6, which were significantly associated with heart aging, were validated by Western blotting. In vitro, we further demonstrated polyamines could retard cardiomyocytes aging through downregulating the expression of PDK4 and thereby inhibiting cell apoptosis. In summary, the distinct mitochondrial proteins identified in this study suggested some candidates involved in the anti-aging of the heart after polyamines treatment, and PDK4 may provide molecular clues for polyamines to inhibit apoptosis and thus retard aging-induced cardiac dysfunction.

Proteomic changes of aqueous humor in proliferative diabetic retinopathy patients treated with different intravitreal anti-VEGF agents.

Anti-VEGF-based treatment have been regularly used in recent years in proliferative diabetic retinopathy (PDR) patients. However, some of these patients fail to respond effectively to anti-VEGF. Given that VEGF is not the sole factor influencing PDR pathogenesis and that different anti-VEGF pharmaceuticals are likely to differentially impact these underlying pathophysiological processes, we performed a prospective analysis of the protein profiles of the aqueous humor (AH) in PDR patients before and after treatment with three intravitreal anti-VEGF drugs (ranibizumab, aflibercept, and conbercept) to assess and compare the short-term impacts of these agents. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic methods were used to evaluate the AH protein profiles of PDR patients using paired pre- and 7 days post-anti-VEGF treatment samples (ranibizumab [IVR]: n = 10; conbercept [IVC]: n = 10; aflibercept [IVA]: n = 5). Gene ontology (GO) annotation, KEGG pathway analyses, and protein-protein interaction (PPI) networks were then used to explore the functional relevance of proteins that were differentially expressed between groups. Here, a total of 874 proteins from 25 patients (50 AH samples) were identified in the three patient groups. Different and common clusters of regulated proteins for each group were identified. We identified RARRES1, ALDH3A1, and RBP4 as being specifically regulated following treatment with all three tested anti-VEGF agents. We further found that VEGFR1, VEGFR2, APOM, hornerin, and HSP90B1 were differentially expressed in different anti-VEGF agent groups. In summary, we discovered that ALDH3A1 was a previously unreported protein that was related to angiogenesis and was differentially expressed in the three anti-VEGF treatment groups, suggesting that it may be a new target for PDR therapy. The described proteomic changes in the AH of PDR patients treated with different anti-VEGF agents provide novel targets which may explain the heterogeneity of anti-VEGF treatment responses in these patients, providing a robust foundation for future studies of PDR pathogenesis.

Comparative proteomics analysis for identifying the lipid metabolism related pathways in patients with Klippel-Feil syndrome.

BACKGROUND: Klippel-Feil syndrome (KFS) represents the rare and complex deformity characterized by congenital defects in the formation or segmentation of the cervical vertebrae. There is a wide gap in understanding the detailed mechanisms of KFS because of its rarity, heterogeneity, small pedigrees, and the broad spectrum of anomalies. METHODS: We recruited eight patients of Chinese Han ethnicity with KFS, five patients with congenital scoliosis (CS) who presented with congenital fusion of the thoracic or lumbar spine and without known syndrome or cervical deformity, and seven healthy controls. Proteomic analysis by data-independent acquisition (DIA) was performed to identify the differential proteome among the three matched groups and the data were analyzed by bioinformatics tools including Gene Ontology (GO) categories and Ingenuity Pathway Analysis (IPA) database, to explore differentially abundant proteins (DAPs) and canonical pathways involved in the pathogenesis of KFS. RESULTS: A total of 49 DAPs were detected between KFS patients and the controls, and moreover, 192 DAPs were identified between patients with KFS and patients with CS. Fifteen DAPs that were common in both comparisons were considered as candidate biomarkers for KFS, including membrane primary amine oxidase, noelin, galectin-3-binding protein, cadherin-5, glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin-1, CD109 antigen, and eight immunoglobulins. Furthermore, the same significant canonical pathways of LXR/RXR activation and FXR/RXR activation were observed in both comparisons. Seven of DAPs were apolipoproteins related to these pathways that are involved in lipid metabolism. CONCLUSIONS: This study provides the first proteomic profile for understanding the pathogenesis and identifying predictive biomarkers of KFS. We detected 15 DAPs that were common in both comparisons as candidate predictive biomarkers of KFS. The lipid metabolism-related canonical pathways of LXR/RXR and FXR/RXR activation together with seven differentially abundant apolipoproteins may play significant roles in the etiology of KFS and provide possible pathogenesis correlation between KFS and CS.

UPLC-MS based plasma metabolomics and lipidomics reveal alterations associated with IgG4-related disease.

OBJECTIVE: The pathogenesis of IgG4-related disease (IgG4-RD) remains unclear. Metabolomic profiling of IgG4-RD patients offers an opportunity to identify novel pathophysiological targets and biomarkers. This study aims to identify potential plasma biomarkers associated with IgG4-RD. METHODS: Thirty newly diagnosed IgG4-RD patients, age-matched healthy controls and post-treated IgG4-RD patients were enrolled. Patients' clinical data, laboratory parameters and plasma were collected. Plasma was measured for ultraperformance liquid chromatography-tandem mass spectrometry based metabolomics and lipidomics profiling. Multivariate and univariate statistical analyses were conducted to identify potential biomarkers. The receiver operating characteristic and the correlations between biomarkers and clinical parameters were investigated. RESULTS: The plasma metabolites are altered among healthy controls, newly diagnosed IgG4-RD and post-treated IgG4-RD groups. Of the identified features, eight metabolites were significantly perturbed in the IgG4-RD group, including glyceric acid 1,3-biphosphate (1,3-BPG), uridine triphosphate (UTP), uridine diphosphate glucose (UDP-Glc) or uridine diphosphate galactose (UDP-Gal), lysophospholipids, linoleic acid derivatives and ceramides. Receiver operating characteristic analysis indicated that UTP, UDP-Glc/UDP-Gal and LysoPC (18:1) had high sensitivity and specificity in diagnosis of IgG4-RD. A Pearson correlation analysis showed that 1,3-BPG and UTP were strongly correlated with clinical parameters. CONCLUSION: IgG4-RD patients have a unique plasma metabolomic profile compared with healthy controls. Our study suggested that metabolomic profiling may provide important insights into pathophysiology and testable biomarkers for diagnosis of IgG4-RD.

Pathological features of COVID-19-associated lung injury: a preliminary proteomics report based on clinical samples.

The COVID-19 pandemic has emerged as a global health emergency due to its association with severe pneumonia and relative high mortality. However, the molecular characteristics and pathological features underlying COVID-19 pneumonia remain largely unknown. To characterize molecular mechanisms underlying COVID-19 pathogenesis in the lung tissue using a proteomic approach, fresh lung tissues were obtained from newly deceased patients with COVID-19 pneumonia. After virus inactivation, a quantitative proteomic approach combined with bioinformatics analysis was used to detect proteomic changes in the SARS-CoV-2-infected lung tissues. We identified significant differentially expressed proteins involved in a variety of fundamental biological processes including cellular metabolism, blood coagulation, immune response, angiogenesis, and cell microenvironment regulation. Several inflammatory factors were upregulated, which was possibly caused by the activation of NF-κB signaling. Extensive dysregulation of the lung proteome in response to SARS-CoV-2 infection was discovered. Our results systematically outlined the molecular pathological features in terms of the lung response to SARS-CoV-2 infection, and provided the scientific basis for the therapeutic target that is urgently needed to control the COVID-19 pandemic.

A Metabolic Insight Into the Neuroprotective Effect of Jin-Mai-Tong (JMT) Decoction on Diabetic Rats With Peripheral Neuropathy Using Untargeted Metabolomics Strategy.

Jin-Mai-Tong (JMT) decoction is a traditional Chinese compound prescription for treating diabetic peripheral neuropathy (DPN). The aim of this study is to investigate the neuroprotective effect of JMT decoction on diabetic rats with peripheral neuropathy and to elucidate the potential mechanism based on a metabolomics approach. Sprague-Dawley (SD) rats were randomly divided into four groups: control group, Streptozotocin (STZ) induced model group, JMT low dose (JMT-L) treated group and JMT high dose (JMT-H) treated group. After 12 weeks of treatment, behavioral changes, small fiber loss, and histopathological damages of sciatic nerves were estimated. Serum samples were collected for untargeted metabolomics analysis based on UPLC/QTOF-MS and multivariate statistics. As a result, JMT treatment at two dosages (13.9 and 27.8 g/kg⋅d) evidently improved the mechanical pain threshold (P < 0.05), increased the intraepidermal nerve fiber density (IENFD) and subepidermal nerve fiber density (SNFD) (P < 0.05), and renovated the demyelination and axonal atrophy of sciatic nerves on DPN rats. Furthermore, metabolomics study revealed that the serum metabolic profiles altered significantly among the control group and the STZ-induced model group. A total of 21 metabolites were identified as potential biomarkers related to the therapeutic effect of JMT decoction. Among them, 16 biomarkers were found in both JMT-H and JMT-L treated groups, while the five others were specific to JMT-H group. These metabolites mainly involved in lipid metabolism, tricarboxylic acid (TCA) cycle, amino acid metabolism, and so on. Besides, correlation analysis indicated that both mechanical pain threshold and distal nerve fiber density were negatively correlated with the serum levels of metabolites from lipid metabolism and TCA cycle. In conclusion, the results demonstrated that JMT decoction has an obvious protective effect against DPN, which could be mediated via ameliorating the metabolic disorders in diabetic rats with peripheral neuropathy.

Genetic and molecular mechanism for distinct clinical phenotypes conveyed by allelic truncating mutations implicated in FBN1.

BACKGROUND: The molecular and genetic mechanisms by which different single nucleotide variant alleles in specific genes, or at the same genetic locus, cause distinct disease phenotypes often remain unclear. Allelic truncating mutations of FBN1 could cause either classical Marfan syndrome (MFS) or a more complicated phenotype associated with Marfanoid-progeroid-lipodystrophy syndrome (MPLS). METHODS: We investigated a small cohort, encompassing two classical MFS and one MPLS subjects from China, whose clinical presentation included scoliosis potentially requiring surgical intervention. Targeted next generation sequencing was performed on all the participants. We analyzed the molecular diagnosis, clinical features, and the potential molecular mechanism involved in the MPLS subject in our cohort. RESULTS: We report a novel de novo FBN1 mutation for the first Chinese subject with MPLS, a more complicated fibrillinopathy, and two subjects with more classical MFS. We further predict that the MPLS truncating mutation, and others previously reported, is prone to escape the nonsense-mediated decay (NMD), while MFS mutations are predicted to be subjected to NMD. Also, the MPLS mutation occurs within the glucogenic hormone asprosin domain of FBN1. In vitro experiments showed that the single MPLS mutation p.Glu2759Cysfs*9 appears to perturb proper FBN1 protein aggregation as compared with the classical MFS mutation p.Tyr2596Thrfs*86. Both mutations appear to upregulate SMAD2 phosphorylation in vitro. CONCLUSION: We provide direct evidence that a dominant-negative interaction of FBN1 potentially explains the complex MPLS phenotypes through genetic and functional analysis. Our study expands the mutation spectrum of FBN1 and highlights the potential molecular mechanism for MPLS.

Isobaric tag for relative and absolute quantitation based quantitative proteomics reveals unique urinary protein profiles in patients with preeclampsia.

Preeclampsia (PE) is one of the most significant pregnancy-related hypertensive disorders. Currently, there are no useful markers to predict the onset of the condition in pregnant women. To provide further insights into the pathogenesis of PE and identify biomarkers of the condition, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics coupled with 2-D LC-MS/MS, to analyze urinary protein profiles from 7 PE patients and 7 normotensive pregnant women. A total of 294 proteins were abnormally expressed in PE patients. Of these, 233 were significantly down-regulated and 61 proteins were significantly up-regulated. Bioinformatics analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, found that the most differentially expressed proteins (DEPs) were involved in coagulation and complement pathways, the renin-angiotensin system and cell adhesion molecules (CAMs) pathways. We further validated three of the DEPs, including serotransferrin (TF) and complement factor B (CFB) by immunoblottingand serum paraoxonase/arylesterase 1 (PON1) by ELISA using 14 pairs of urine samples from PE patients and normal pregnant women. Taken together, our results provide the basis for further understanding the pathogenesis of PE and identifying predictive biomarkers.

Alterations in the gut microbiome and metabolism with coronary artery disease severity.

BACKGROUND: Coronary artery disease (CAD) is associated with gut microbiota alterations in different populations. Gut microbe-derived metabolites have been proposed as markers of major adverse cardiac events. However, the relationship between the gut microbiome and the different stages of CAD pathophysiology remains to be established by a systematic study. RESULTS: Based on multi-omic analyses (sequencing of the V3-V4 regions of the 16S rRNA gene and metabolomics) of 161 CAD patients and 40 healthy controls, we found that the composition of both the gut microbiota and metabolites changed significantly with CAD severity. We identified 29 metabolite modules that were separately classified as being positively or negatively correlated with CAD phenotypes, and the bacterial co-abundance group (CAG) with characteristic changes at different stages of CAD was represented by Roseburia, Klebsiella, Clostridium IV and Ruminococcaceae. The result revealed that certain bacteria might affect atherosclerosis by modulating the metabolic pathways of the host, such as taurine, sphingolipid and ceramide, and benzene metabolism. Moreover, a disease classifier based on differential levels of microbes and metabolites was constructed to discriminate cases from controls and was even able to distinguish stable coronary artery disease from acute coronary syndrome accurately. CONCLUSION: Overall, the composition and functions of the gut microbial community differed from healthy controls to diverse coronary artery disease subtypes. Our study identified the relationships between the features of the gut microbiota and circulating metabolites, providing a new direction for future studies aiming to understand the host-gut microbiota interplay in atherosclerotic pathogenesis.

ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia.

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice.

Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.

Proteomic Analysis Reveals Dab2 Mediated Receptor Endocytosis Promotes Liver Sinusoidal Endothelial Cell Dedifferentiation.

Sinusoidal dedifferentiation is a complicated process induced by several factors, and exists in early stage of diverse liver diseases. The mechanism of sinusoidal dedifferentiation is poorly unknown. In this study, we established a NaAsO2-induced sinusoidal dedifferentiation mice model. Liver sinusoidal endothelial cells were isolated and isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic approach was adopted to globally examine the effects of arsenic on liver sinusoidal endothelial cells (LSECs) during the progression of sinusoidal dedifferentiation. In all, 4205 proteins were identified and quantified by iTRAQ combined with LC-MS/MS analysis, of which 310 proteins were significantly changed in NaAsO2 group, compared with the normal control. Validation by western blot showed increased level of clathrin-associated sorting protein Disabled 2 (Dab2) in NaAsO2 group, indicating that it may regulate receptor endocytosis, which served as a mechanism to augment intracellular VEGF signaling. Moreover, we found that knockdown of Dab2 reduced the uptake of VEGF in LSECs, furthermore blocking VEGF-mediated LSEC dedifferentiation and angiogenesis.